Department of Biological Science and Technology

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					                 Department of Biological Science and Technology

Title:Intrinstically Disordered Protein, Folding, Aggregation and Intermolecular


Principal Investigator:CHIA-CHING CHANG

Sponsor:National Science Council

Keywords:Intrinsically Disordered Protein, First Order-Like State Transition Model,
                Folding Intermediates, Diffusion Limited Aggregation, Self-Assembly

     Intrinsically disordered protein plays a vital role in the networks of
protein-protein interactions. However, their stabilities rely on the process of folding.
The first order-like state transition model is the proper model to describe the global
reaction of protein folding. However, the folding reaction of intrinsically disordered
protein remains unknown. Therefore, one of the aims of this project is focusing on the
mechanisms study of intrinsically disordered proteins folding and their interactions.
Meanwhile, the ―first order-like state transition model‖ will be examined in this
project. By using Raman and fluorescence spectroscopy analysis of their folding
intermediates, the folding cores of intrinsically disordered protein can be revealed.
Meanwhile, the major affecting factors of folding transition region/boundary can be
determined by systematically folding studies. These affecting factors may help us to
reveal the protein aggregation mechanism. Meanwhile, we will test the competitive
model of protein folding and diffusion limited aggregation in this project. In order to
reveal the self-assembly mechanism of intrinsically disordered protein, in this project,
we will use the molecular dielectrophoresis driven self-assembly method, which
developed my laboratory, and make the protein aggregated in the nano-pattern of
silicon substrates. Furthermore, the conformation of aggregated protein will be
revealed by atomic force microsopy and cryo-transmitted electron microscopy.
Meanwhile, the dynamics of this protein aggregation and conformation transition of
the loop region during protein-protein interactions can be revealed by optical
combining single molecular manipulation methods which development by my
NSC97-2112-M-009-009-MY3 (97R043)

Title:Mechanisms of Cytoskeleton Alteration and Cell Death by PT-262
Principle Investigator:Jui-I chao
Sponsor:National Science Council
Keywords: Anticancer Drug, Cytoskeleton, Apoptosis, Actin, RhoGTPases, MAP

    Cancer has become the major mortality in Taiwan. Development of the more
efficient anticancer drugs is highly desirable. Recently, we found a brand-new
compound derived from 5,8-quinolinedione (designated as PT-262), which
dramatically induced the cytoskeleton alteration and death in cancer cells. However,
the mechanisms of PT-262 actions are still unclear. In this project, we will investigate
the mechanisms of cytoskeleton alteration and cell death by PT-262. The model of
human lung cancer cells will be established, and the effect of PT-262 on normal lung
cells will be compared. We will determine whether PT-262 is through direct binding
to actin filaments or indirect effect on RhoGTPase actin-remodeling signaling
pathways for causing abnormal actin polymerization. Actin polymerization assay,
MALDI-TOF MS analysis, and confocal bio-atomic force microscopy are adopted to
examine PT-262 binding to actin. Furthermore, we will study the blockade of
RhoGTPases (RhoA, Rac1, and Cdc42) and MAP kinases (JNK, p38, and ERK) by
using gene knockdowns or inhibitors on the effects of PT-262-induced cytoskeleton
alteration and cell death. Additionally, the cells will be transfected with expressed
vectors of RhoGTPases for testing on the cytoskeleton alteration, survival, apoptosis,
and cell cycle in the PT-262-exposed cells. The inhibition of migration and invasion
will be evaluated for the anti-metastasis ability by PT-262. Finally, the interaction and
regulation between RhoGTPases and MAP kinases will be investigated by Western
blot, immunoprecipitation, immunofluorescence, and confocal microscopy for
analysis of protein expression, interaction, and location in the cells. The final goal of
this project is to delineate the mechanisms of PT-262-induced cytoskeleton alteration
and cell death and offers applications on cancer therapy.
NSC96-2311-B-009-003-MY3 (96N598)

Title:Molecular Mechanism and Biosynthetic Study of Antibiotic C-Methylations
       and Their Applications in Research and Developments of Novel Anticancer
Principle Investigator:Hsien-Tai Chiu
Sponsor:National Science Council
Keywords: Biosynthesis, C-Methylation, Enzymology, Antibiotics, NDP-sugar,
         Indolocarbazole Glycosides, Drug Development
     Methylation of biologically active molecules has been an important mechanism
of nature to control cellular processes and to monitor biological activity of
biomolecules, including cell differentiation, gene expression regulation,
posttranslational processes, tRNA maturation, antibiotic resistance and natural
product biosynthesis. Many important bioactive natural product glycosides bear
structurally diverse sugars indispensible for their biological potency and specificity.
Notably, many of these sugar moieties were found to be C-methylated by various
C-methyltransferases (C-MTases), thereby leading to control of structural
conformation and global hydrophobic properties of the sugar moieties. Therefore, it
would be an interesting and important task to study the biosynthetic origin and
mechanism of the C-methylation in these biologically important glycosylated natural
products, serving as antibiotics, antitumor and anticancer agents. In this study, we
have chosen four valuable natural product glycosides of this kind, i.e. coumermycin
A1, novobiocin, erythromycin and nogalamycin, to investigate the molecular
mechanism and substrate specificity of the four C-MTases involved in their
biosyntheses. To this aim, a series of TDP-4-keto-sugar analogs will be used as
structural and mechanistic probes to explore the active-site cavities of the C-MTases.
The study may be realized by extensive steady-state and inhibition kinetic
experiments using the C-MTases cloned, expressed and purified from the
drug-producing organisms. The resulting systematic kinetic information will be very
useful to resolve the structure-activity relationship of the enzymes. The C-MTases
will also be subjected to intensive biochemical characterization, e.g., metal ion
requirements and pH-dependent activity profiles. In addition, site-directed
mutagenesis will be conducted to identify the important active-site residues involved
in the chemical catalysis of the C-MTases.
     Another interesting study is to utilize these four C-MTases as a synthetic tool for
combinatorial biosyntheses of a variety of methylated nucleotide diphosphate sugars
(NDP-sugars). The synthesis may be accomplished by catalytic actions of the
C-MTases on the TDP-4-keto-sugar analogs, further coupled with NDP-sugar
reductases or chemical reduction. The resulting methylated NDP-sugar library can be
valuable for various glycosylation events in glycobiology and natural product
biosynthesis. In this study, the methylated analog pool of NDP-sugars will be utilized
for molecular engineering of the glycosylated indolocarbazole family of antitumor
antibiotics by N-glycosyltransferases, which may decorate the indolocarbazole
aglycones with various methylated sugar molecules. The indolocarbazole glycosides
generated in this study may thus serve as a valuable source of drug leads for
anticancer drug developments and therapeutic applications.
NSC98-2113-M-009-003 (98N055)

Title:Eeffects and Mechanisms of Neonatal Sound Exposure on Neurite
       Development at the Auditory Mibrain of Rats
Principle Investigator:Tzai-Wen Chiu
Sponsor:National Science Council
Keywords: Eeffects and Mechanisms

     The brain is plastic after birth. Previous studies showed that the central auditory
neurons, particularly at the cortical and midbrain levels, can be altered in response
property after early sound exposure. Furthermore, functional changes are highly
dependent on the type of stimuli and the onset time of the sound exposure.
Activity-driven neural changes are also reflected in neural structures. Two models
have been proposed to explain how the activity influences the neural wiring in the
brain. The first is that the activity might regulate the formation of new synapses and
the second is that the input signals control the elimination or destabilization of
existing synapses. In the auditory system, both in vivo and in vitro experiments
showed that the synaptic density and morphology are altered by reduced inputs.
Whether the increased activities, particularly during the neonatal stages, have similar
effects on altering the neural structures is undetermined. Furthermore, whether the
experience-driven plastic changes on the neural structures are local or global remains
unexplored. The aim of this study is to determine at the auditory midbrain
activity-driven plastic changes following early sound exposure: (a) whether neonatal
sound exposures have effects on remodeling the dendritic or axonal morphology at the
midbrain; (b) if sound exposures induce reorganized neurites locally or beyond the
affected frequency laminae; (c) the role of dendritic and pre-synaptic machinery like
MAP2 and the GAP-43. A total of 16 batches of animals will be used in this two-year
project. The animals will be randomly divided into control (n=8 batches) and
experimental groups (n=8 batches). The neonatal rats will be exposed from week-2 to
week-5 to a moderate tone of 4 kHz and 65 dB SPL. Control animals will be raised in
the same environment without sound exposure. In first year, the response properties of
midbrain units from 12 batches animals (control=6, experimental =6) will be
characterized by extracellular single unit recording after sound exposure ends. Then,
neurons with best frequencies at 4 kHz, 8 kHz and 32 kHz will be dye-labeled using
juxtacellularly-injected neurobiotin. In the second year, 4 batches of animals will be
processed with immunohistochemistry for MAP2 and GAP-43 regarding their roles in
activity-driven dendritic remodeling. Results would be important for the
understanding of how the long-term increased input signals would induce the neural
plasticity at the functional and structural domains as well as their underlying
mechanisms. Results would have important contribution on designing clinical
treatment of cochlear implants and stroke, like combined treatment with drugs, which
can modulate the expression of MAP2 and GAP-43 together with maximizing sensory
inputs therapy and motor rehabilitation.
NSC98-2320-B-009-001-MY2 (98N027)

Title:Developing Computer-Aided Vaccine Design Systems Using Bio-Inspired
      Optimization Methods
Principal:Investigator:Shinn-Ying Ho
Sponsor:National Science Council
Keywords:Data Mining, Vaccine Design, Evolutionary Computation, Genetic
         Algorithm, Parameter Optimization

     This is a three-year project: Developing Computer-aided Vaccine Design
Systems Using Bio-inspired Optimization Methods. The objectives are to develop
computer-aided systems to help immunologist for vaccine design. The core project is
two-fold: 1) to develop various high-performance optimization algorithms for solving
large-scale parameter optimization problems of bio informatics to mine informative
physicochemical properties from known experimental data; and 2) to integrate
immune knowledge base and application system for analysis and annotation of
informative features. Developing these algorithms involves three important phase: (a)
collection of various features including physicochemical properties, evolutionary
information and structure information; (b) design of optimization problems by
identifying system parameters to be optimized from combining bio-knowledge and
computing techniques, and (c) design of powerful optimization algorithms for
obtaining near-optimal solutions. The integrated feature mining systems can extract
potentially good features from a large number of physicochemical properties in the
biological database and apply them to vaccine design systems.
     The individual projects of the three year focus on both study of vaccine design
and algorithm development, which are fully cooperated, described below.
Year 1 : Develop the core high-performance feature mining system, and study
cytotoxic T lymphocyte related immune response by mining informative features.
Year 2 : Study the immune responses of T helper cell and B cell, and their prediction
Year 3 : Establish integrated computer-aided vaccine design systems, and develop an
informative feature database of immune systems.
The goals of this project are 1) identify informative features to further understand
immune systems; 2) construct computer-aided vaccine design systems, and 3) develop
an informative feature database of immune systems.
NSC96-2628-E-009-141-MY3 (96N452)
Title:Reconstructing MicroRNA Regulatory Networks in Human Genome: A
       Database and Data Mining Approach
Principle Investigator:Hsien Da Huang
Sponsor:National Science Council
Keywords: MicroRNA

     MicroRNAs (miRNAs), which are 21~22 nts RNA molecules,can negatively
regulate gene expression and thus control numerous cellular mechanisms.Previous
investigations suggest that approximate 50% of miRNAs can function as tumor
suppressors or oncogenes via reduction or overexpression to regulate downstream
genes and result in oncogenesis. Recent investigations also suggest that the majority
of intergenic miRNAs are transcribed by RNA polymerase II. The investigation of
transcriptional regulation of miRNAs is as important as the investigation of miRNA
functions. However, during last five years, researcher both in wet and dry works
majorly focused on the annotation of miRNA genes and the identification of their
target genes. Less attention was spent on the transcriptional regulation of miRNA
genes. In this proposal, our interests are in the transcriptional regulation of miRNA
genes and the regulatory networks involved by miRNAs.
     The Aims and Methods: The major aim of this proposal is to reconstruct
microRNA regulatory networks in human genome using a database‐based and data
mining approach. The microRNA regulatory networks defined here include
transcriptional regulation of miRNAs and down‐regulation of miRNA target genes.
The proposed methods are summarized as follows:
(1) miRNA Information Repository: This work plans to develop an information
repository to collect biological information concerning miRNAs, as well as the
information required in the proposed analysis in this work. The gene annotation of
miRNAs, known miRNA targets, expression profiles of miRNAs, genomic locations,
cross‐species information, data of transcription factor binding sites, gene ontology and
other effective information will be integrated.
(2) Identifying miRNA target genes: We have good experiences (Nucl. Acids Res,
2006, 2007, 2008, 2009) for identifying miRNA target genes. In this work, we plan to
extend our method to filter out false positive predictions to improve prediction
accuracy by incorporating additional biological features, such the accessibility of
miRNA target sites and miRNA expression profiles.
(3) Deciphering transcriptional regulation of miRNAs: To deciphering the
transcriptional regulation of miRNAs, the first step is to determine the location of
transcriptional start sites (TSS) of miRNAs. In this work, we plan to determine
location of miRNA TSS by both experimental evidences and computational tools.
Experimental evidences, such CAGE tags and Solexa tags, derived and sequenced for
genome‐wide identifying TSS of genes provide valuable clues to identify TSS of
miRNAs. Besides, we plan to integrate a variety of tools developed for identifying
TSS of protein coding genes, such as Eponine, EP3, and NNPP. Following the TSS
identification, the cis‐elements located in the upstream of miRNAs will be identified
based on the transcription factor information collected in this work.
(4) Reconstructing miRNA regulatory networks: Following the identification of
miRNA targets and the investigation of transcriptional regulation of miRNAs, we plan
to reconstruct miRNA regulatory networks. Expression profiles of coding genes and
miRNAs will be analyzed to discover co‐expression gene group for cis‐elements
analysis. Tissue‐specific and cancer‐specific miRNA regulatory networks will be
reconstructed and investigated. The Anticipated Results and Scientific Impacts:
During last three years, our group has good research results in miRNA regulations,
such as miRNAMap (Nucl. Acids Res, 2006, 2008), ViTa (Nucl. Acids Res, 2007),
and functions of human miR122a (Hepatology, 2009). We confidently anticipate that
the proposed research goals can be achieved. We also believe that the research topics
of on transcriptional regulation of miRNAs and the regulatory networks involved by
miRNAs will attract more and more attentions. Therefore, the results of this research
plan can provide good biological insights of miRNA regulation and scientific impacts
to this field.
NSC98-2311-B-009-004-MY3 (98N487)

Title:Developing A Fast Docking-based Virtual Screening Method
Principle Investigator:Hui-Ling Hunag
Sponsor:National Science Council
Keywords: Docking, Orthogonal Array, Particle Swarm Optimization, Consensus
         Scoring; Virtual Screening

     This project proposes a fast docking-based virtual screening method to filter
compounds for drug discovery process. Literature reveals that more than 60 docking
programs and over 30 scoring functions have been presented. Many studies indicated
that significant improvements must be achieved in order to develop highly accurate
molecular docking and efficient virtual screening methods. This study investigates
existing docking methods and assesses their parallelization ability of implement. The
docking program SODOCK cooperated with AutoDock is the first method using
particle swarm optimization (PSO). The proposed docking method bases on the
improvement of SODOCK, called PSODOCK, by adding an efficient sampling
method based on orthogonal array for initialization of PSO, which is a highly parallel
program. PSODOCK can be implemented effectively using graphics processing units
(GPU) by refining the parameter settings of PSODOCK.
     The docking-based virtual screening method has been developed successfully
applied to a number of pharmaceutical targets. Generally, an alternative method is to
eliminate unpromising compounds before docking by prescreening drug-like
compounds, by filtering the dataset based on appropriate property and sub-structural
features, by verifying the known interactions with the target receptor, and by
performing diversity analysis. Virtual screening utilizes docking methods and scoring
of each compound candidate. The docking method is based on the prediction of
binding modes and binding affinities of compounds by means of docking to an X-ray
crystallographic structure. The probable binding mode of a ligand to differentiate
correct poses from incorrect ones is based on reliable scoring functions. Scoring
functions are used to direct the docking and predict the binding affinity of the final
pose. It has been demonstrated that consensus scoring is generally more effective than
single scoring for molecular docking. This project would develop a PSODOCK-based
virtual screening method using consensus scoring to filter compounds. The
application of the proposed method is evaluated by filtering compounds from ZINC
database and comparing with published results.
NSC98-2221-E-009-122- (98N298)

Title:Investigation of Microtubule-based Motor Proteins on Neuritogenesis
Principle Investigator:Eric Hwang
Sponsor:National Science Council
Keywords: Microtubule-based

    The establishment of a functional nervous system depends on the intricate
connections between neurons. For neuronal circuits to be correctly wired, various
steps following the generation of a differentiated neuron must be properly regulated,
these steps include the formation, elongation, polarization, branching, and maturation
of neurites. These processes are collectively termed ―neuritogenesis‖. The
microtubule cytoskeleton plays indispensable roles in each of the steps during
neuritogenesis. Microtubule reorganization and microtubule-based transport are
especially critical in these events. Microtubule-based motors have long been shown to
participate in both microtubule reorganization and microtubule-based transport.
Although a handful of microtubule-based motors are known to play important roles
during neuritogenesis, a detailed and comprehensive inhibition analysis on each of the
motors is lacking.
     This investigation intends to carefully examine the role of individual
microtubule-based motors on neuritogenesis by targeted gene silencing using shRNA
introduced by lentivirus. The full motor library investigation, which includes 44
kinesin and 12 dynein/dynactin genes, will be carried out using mouse primary
hippocampal neurons in a high throughput format. The specific design of the shRNA
library allows lentiviral particles containing shRNA clones to be readily produced in
culture, thus allowing high transduction efficiency to be achieved in primary neuronal
cultures. Gene silencing efficiency will be evaluated using a combination of
immunoblotting, immunocytochemistry, and quantitative real-time PCR. The
phenotypic effect of silencing specific microtubule-based motors on neuritogenesis
will be digitally documented following immunocytochemistry. These digitally
documented images will be inspected by automated image analysis software. Specific
microtubule-based motors that caused significant and reproducible phenotypes when
suppressed will be further investigated using a combination of contrast-based and
fluorescent live cell imaging techniques. The specific aims of the two-year project are
as follow:
     1. Establishing high throughput image-based screening procedure (first year).
     2. Improving automated image analysis software (first year)
     3. Conducting microtubule-based motor inhibition investigation and examining
         phenotypic effect using automated image analysis software (second year)
        4. Detailed investigation of selected microtubule-based motors on
           neuritogenesis (second year).
NSC98-2311-B-009-001-MY2 (98N008)

Title:On the Relationship between Structure-Dynamics-Function in Proteins
Principle Investigator:Jenn-Kang Hwang
Sponsor:National Science Council
Keywords: Protein Dynamics, Molecular Dynamics, Atomic Fluctuations, Epidermal
         Growth Factor Receptor, Cancer
      Protein structures are deposited in Protein Data Bank in an amazing speed due to
the progress of structure genomics initiatives. Though knowing the shapes of proteins
is crucial for understanding their biological functions, many important biological
processes cannot be inferred from sheer structures due to large-scale protein dynamics
that frequently occurrs in biological reactions. For example, the ATPase activity and
helicase activity of hepatitis C virus NS3 helicase are dynamically coupled to each
other, while the activation of EGFR, the fist cell-surface receptor to be linked directly
to human cancer, has been shown to be closely related to large scale domain
rearrangements. In other word, protein dynamics is in fact as important as protein
structure in understanding protein function. At present, current methods such as
Molecular Dynamics (MD) are very computationally expensive and, consequently,
are impractical for large biological systems. Here we will develop novel approaches
to compute protein dynamical properties, based on the protein fixed-point (PFP)
model previously proposed by us. This model compute both atomic fluctuations and
motion correlation using the positional vectors of atoms issuing from the fixed point,
which is the point of the least fluctuation in protein. The PFP model is faster than
current methods by about orders of magnitude and its prediction of the average
dynamical properties is much more accurate. Another major advantage of the protein
fixed-point model is that it can be applied to very large proteins. However, the PFP
model cannot be applied to protein complexes or multidomain proteins. Here, we will
propose novel ideas to improve the PFP model. Our preliminary results are very
encouraging. As a practical application of our approach, we will study the protein
dynamics of epidermal growth factor receptor (EGFR), the first cell-surface receptor
to be linked directly to human cancer. Recently. Mutations of the EGFR gene have
been identified in specimens from patients with non-small-cell lung cancer. But the
mechanism of the drug resistance is still unclear despite the availability of the 3D
X-ray structure complex with ATP or the EGFR inhibitors. Our preliminary study
shows that these mutated residues are dynamically coupled. This presents an excellent
chance for us to study the protein dynamics in EGFR. We believe that our results will
help shed light on the mechanism of the drug resistance of EGFR. Finally, we will
build establish a comprehensive Protein Dynamics Database. Though protein
dynamics provides rich information about the relationship between protein structure
and function, the general biologists cannot access such information easily. We will
compute the dynamical properties for all structures in Protein Data Bank and provide
a comprehensive database of protein dynamics for general biologists. We believe that
our results will stimulate interest in investigating protein structure-dynamics-function
NSC98-3112-B-009-002 (98N034)

Title:Genomics Medicine and Biotechnology Bioinformatics Core Facility
Principle Investigator:Jenn-Kang Hwang
Sponsor:National Science Council
Keywords: Biotechnology Bioinformatics Core Facility

    In this project Bioinformatics Core for Genomic Medicine and Biotechnology
Development –Structural Bioinformatics, we will offer bioinformatics service for both
academic and industrial researchers in providing structural-based analysis tools and
databases web service, educational work shop program and research conferences,
which relevant to the users' research topics in genomic medicine and biotechnology.
The current service facility is based on the foundation the current structural
bioinformatics core, which is the only bioinformatics core completely dedicated to
computational structural biology in Taiwan. We will provide structural analysis tools
and database such as protein structure prediction, fast structure alignment, structure
analysis analysis (for example, detection of knots in proteins), protein-protein/ligand
docking tool GemDOCK and other prediction tools. Furthermore, we will also
provide analysis tools and databases in proteomic analysis or genomic analysis, such
as protein subcellular localization predictor, phosphorylaton site prediction, database
of mircroRNA etc. We will also upgrade the current databases such as the
disulfide-pattern protein database and structural entropy database, and continue
providing mirror service of some of the most important structural databases such as
PDB, SCOP and PredictProtein to the local users. In general, our efforts will be
dedicated to three categories: the routine service, technology development and the
collaborative research service. We will provide two types of service to users: the first
type includes the routine web-based service that will automatically return results for
queries submitted by users; the other type of services require special customized
assistance from us in carrying out computational intensive operations. For example,
our structure prediction server will automatically return 3D structures for a limited
number of query sequences submitted by users; however, when the query sequences
are, say, all sequences of certain pathogenic genomes or genes related to certain
cancer that need comprehensive computation and customized/novel tools, we will
provide customized assistance for the users. In general, we will dedicate 65% of our
effort to the routine service for each year; as for the technology development, we will
dedicate 20% of our effort for the 1st year, 15% 2nd year and 10% the third year, and
the rest effort will be dedicated to the collaborative research.
NSC98-3112-B-009-004 (98N038)

Title:Using Controllable Biobricks and Feedback Loop Design to Increase the
       Stability of Genetic Circuits
Principle Investigator:Hsiao-Ching Lee
Sponsor:National Science Council
Keywords: Sing Controllable Biobricks

      Synthetic biology is the design and construction of new biological parts, devices,
and systems, and the re-design of existing, natural biological systems for useful
purposes. Genetic engineering with recombinant DNA is a powerful and widespread
technology that enables researchers to redesign life forms by modifying DNA
fragments. However, most created genetic circuits are unstable and can not function
properly. Therefore, how to design a stable genetic circuit to tolerate intrinsic
parameter fluctuation and to attenuate extrinsic disturbances in order to function
properly is an important topic for synthetic biology. In this project, we will use
controllable biobricks and the feedback loop concept to increase stability of gene
circuits to meet this challenge. The first step in programming and controlling cell
behavior is to establish a library of well-defined components—‗biobricks‘ that serve
as the building blocks of artificial gene networks. Biobricks are the DNA fragments
with specific functions, including promoters, repressors, reporter genes and other
parts required to construct functional plasmids. The main challenge in genetic circuit
design lies in selecting well-matched genetic components that when coupled, reliably
produce the desired behavior. Although the parameter values are calculated by model
equations, it is hard to select the biobricks that reliably implements a desired cellular
function with quantitative values. To overcome this problem, the promoters which can
control the expression of downstream genes are necessary. Two strategies will be
applied. First, degenerated primers designed for PCR are used to generate mutations
in promoter regions. Second, the combinatorial promoter library with multiple
transcription factor binding sites facilitating the integration of multiple signals will be
constructed. The promoter activity can be assay using a bacterial luciferase reporter
cassette on a low copy number plasmid. A library of promoters with different
transcriptional strength will be built to tune the specific parameter values that model
equations indicated. After building genetic circuit, the experimental data can provide
the mathematical property of parameters and refine the circuit design schemes to
increase stability of systems. The stable and noise-resistance circuit design schemes
have potential for applications, such as constructing a synthetic enzymatic pathway
for drug production, and producing metabolic pathway in microbes that churn out
NSC98-2311-B-009-002 (98N029)

Title:Using Directed Evolution to Optimize Metabolic Pathway and Confirm System
       Design Schemes
Principle Investigator:Hsiao-Ching Lee
Sponsor:National Science Council
Keywords: Design Schemes

     The main goal of the nascent field in synthetic biology is to design and construct
biological system with a desired behavior. At present, even the construction of
biological IC circuits has demonstrated the feasibility of synthetic biology, the design
of gene networks is still a difficult problem and most of newly designed gene
networks can not function properly. The major causes for these design failures are
mainly due to both intrinsic perturbations such as gene expression noise, splicing,
mutation, evolution and extrinsic disturbances such as changing extra-cellular
environments, interactions with cellular context. Therefore, how to design a robust
synthetic gene network to tolerate intrinsic parameter fluctuation and to attenuate
extrinsic disturbances in order to function properly will be an important topic for
synthetic biology.
     However, building a genetic circuit in vivo requires tedious measurement of
many unknown parameters of mRNA, protein stabilities, DNA-protein interactions
and so on. To overcome this problem, we will generate a library of genetic devices
with a range of behaviors that can be used to construct genetic circuit and develop
evolutionary strategies for constructing biological IC circuits. A combined rational
and evolutionary design strategy are proposed for constructing biological IC circuits,
an approach that allow the engineer to fine-tune the biochemical parameters of the
gene regulatory networks experimentally in vivo. By applying directed evolution to
genes comprising a genetic circuit, the non-robust genetic circuit containing
improperly matched components can evolve rapidly into a more robust one. The
experimental data can provide the mathematical properties of parameters and our
team can refine the circuit design schemes to increase robustness of bio-systems. In
next step, we will use the robust genetic circuit design to engineer an Escherichia coli
strains to product butanol from glucose via the host s amino acid biosynthetic
pathway. Design of genetic circuits is foreseen to have important applications in
biotechnology, medicine and biofuel, and to revolutionize how we conceptualize and
approach the engineering of biological systems.
NSC98-2221-E-009-182- (98N332)

Title:Study of Helicobacter Pylori Heat Shock Protein 60 on the Activation of
       Transformining Growth Factor-beta Signal Pathway
Principle Investigator:Kuang-Wen Liao
Sponsor:National Science Council
Keywords: Helicobacter Pylori, Heat Shock Protein 60, TGF-â, Immunosuppression

      Helicobacter pylori (H. pylori, Hp) has become an important issue in the field of
gastrointestinal disease and the development of gastric cancer since its discovery in
late 1980‘s. It has been confirmed that Hp can damage human gastric epithelial cells,
cause oncogenesis and suppress host‘s immunity. Our results indicated that H. pyori
heat shock protein 60 (HpHsp60) could bind to TGF-â receptor II and trigger TGF-â
regulated SMAD signal pathway. Thus, at the first stage of this project, we will purify
high quality Hsp60 to treat gastric epithelial cells and observe how the HpHsp60
trigger the TGF-â signaling in the gastric epithelial cells and use the cDNA
micro-array to survey the effect of Hsp60 on gastric epithelial cells. The information
of cDNA micro-array will be focus on the effects to the angiogenesis and metastasis.
At the second stage, we will measure the effect of HpHsp60-induced SMAD signal
pathway on the proliferation for the human regulatory T cells (Treg) to observe how
Hsp60 affects the physiological function, transcription factors, and the whole genomic
expression of the purified Treg. Previous literatures revealed that TGF-â is important
for the function of Treg, therefore Hsp60 should have important influence on Treg. At
this stage, Hsp60 will be investigated its effects on the functions of Treg.
NSC98-2320-B-009-002- (98N333)

Title:Using Mammary Epithelial Cells to Construct A Bioreactor of Artificial

         Mammary Gland Which Can Be Used to Produce Therapeutic
         Recombinant Proteins

Principle Investigator:Chich-Sheng Lin

Sponsor:National Science Council

Keywords:Mammary Epithelial Cells, -casein Gene, Expression Vector,
             Recombinant Protein, Hollow Fiber Bioreactor

      We have established a spontaneously immortalized porcine mammary epithelial
cell line (SI-PMEC) from mammary gland of lactating sow. The SI-PMEC cells can
differentiate into mammary gland-like structures (gland ducts, lateral buds, and
alveoli), and strongly express the transcripts encoding milk proteins and secrete the
proteins in vitro in a matrix- and lactogenic hormone-dependent manner. The cells
appeared to provide an efficient and convenient system for assessing the expression of
transgenes containing mammary gland-specific promoter before transgenic milk
livestock generated. Moreover, the SI-PMEC can reasonably be used as a bioreactor
system for producing exogenous recombinant proteins which have undergone
appropriate post-translational modifications making them structurally and functionally
identical to the native proteins. In this plan, we attempt to use the SI-PMEC cells to
construct a bioreactor of artificial mammary gland which can be used to produce
therapeutic recombinant proteins. We propose three research strategies addressing on
the subject for three years planning, 1) screening a SI-PMEC which can differentiate
into functional structures on collagen substratum, 2) constructing expression vectors
with the regulatory sequence of goat -casein gene, and 3) fabricating a system of
hollow fiber bioreactor.
      In Strategy 1, a SI-PMEC cell line with genetic stability will be screened and
cloned. The cells can duplicate within 24 hours in the medium containing less
hormones and animal serum. Moreover, the cell line should keep the capacity of
differentiation of secretory structures and functions as mammary gland-like when the
cells grow on the collagen substratum.
      In Strategy 2, we attempt to construct the inducible highly expression vectors in
mammary gland and mammary epithelial cells with the regulatory sequences of goat
-casein gene. Sequences between cloning site of the expression vectors are promoter,
exon 1, intron 1, exon 2 of goat -casein gene around 5-6 kb fragment at 5‘ side and a
3-4 kb fragment of the -casein gene from exon 7 to 3‘-flanking sequence at 3‘ side.
The report genes, green fluorescent protein and luciferase, are also applied to
construct the control expression vectors which are used to evaluate the functions of
SI-PMEC and bioreactor. We aim to construct two transgenes which carry the
sequences encoding antibacterial peptides, human cathelicidin LL-37 and porcine
cathelicidin PR-39. Both transgenes will be transfected into SI-PMEC and to screen
the recombinant SI-PMEC (rSI-PMEC) clones.
     In Strategy 3, adhered cultures of rSI-PMEC will be performed in a hollow fiber
bioreactor. The cells are implanted into the extracapillary space (ECS) of cartridge in
a hollow fiber bioreactor and grow to be confluence supplied with growth medium.
Then differentiation medium supplied, the cells will differentiate to be three
dimentional network structures as artificial mammary gland, and then synthesize as
well as secrete recombinant proteins. The produced recombinant protein in the ECS
will be released into the intracapillary space (ICS) through the hollow fibers (small
tube-like filter) and concentrated in the chemical medium in the ICS circulation.
     The aim of this plan is that the rSI-PMEC can be cultivated and maintained in
hollow fiber reactor for at least 42 days to continuously produce cathelicidin LL-37
and PR-39 at an extremely high level. In addition, the system established herein,
SI-PMEC, expression vectors and hollow fiber module, can be applied to produce the
others protein pharmaceuticals.
NSC96-2628-B-009-001-MY3 (96N316)

Title:Studying the Roles of Angiotensin Converting Enzyme II –Angiotensin 1-7
       (ACE2–Ang 1-7) Axis in the Disease Process of Heart Failure
Principle Investigator:CHIH SHENG LIN
Sponsor:National Science Council
Keywords: (ACE2–Ang 1-7) Axis

     The renin-angiotensin system (RAS) has been recognized and strongly
associated with the development of cardiovascular diseases. Although most of the
well-known cardiovascular effects of RAS are attributed to angiotensin converting
enzyme (ACE) – angiotensin II (Ang II) axis (ACE–Ang II axis), much less is known
about the function of angiotensin converting enzyme II (ACE2), an ACE homologue
that efficiently hydrolyses Ang II to form angiotensin 1-7 (Ang 1-7), a peptide that
exerts actions opposite to those of Ang II. Recently, several observations and
experimental evidence suggest a beneficial role for ACE2 and Ang 1-7 in
cardiovascular function. In contrast to the ACE–Ang II axis, however, the roles of the
ACE2–Ang 1-7 axis in cardiac function are largely unclear and remain to be explored.
We are ambitious to study the roles of ACE2–Ang 1-7 axis in cardiovascular function
and test the hypothesis that whether part of the cardioprotective effects of ACE2–Ang
1-7 axis is via the balancing regulation between cardiac matrix metalloproteinase
(MMPs) and tissue inhibitors of MMPs (TIMPs) or not?
     In the present project, we propose three research strategies addressing on the
above subjects. In Strategy 1, the primary human cardiofibroblasts and
cardiomyocytes will be used to study the regulative mechanism of angiotensin
peptides on the ACE2 expression and MMPs/TIMPs balancing regulation through
Ang II–AT1R (Ang II type 1 receptor) and Ang 1-7–Mas (Ang 1-7 receptor) signaling
transduction pathway. In addition, an ACE2 promoter-driving luciferase reporter
vector will be constructed for exploring the modulators and transcriptional factors on
ACE2 gene expression. In Strategy 2, a heart failure (HF) mouse model induced by
doxorubicin administration or coronary artery ligation will be used to explore the
altered expression and/or activity profile of ACE/ACE2, Ang II/Ang 1-7 and
MMPs/TIMPs in the heart tissue during HF development. The fibrotic pathogenesis
and cardiac protective effect of angiotensin peptides will be studied by the operation
of Ang II and Ang 1-7 perfusion in the mice developed HF. In Strategy 3, genetic
manipulation of ACE2 expression in the mouse models, either targeted disruption or
overexpression, will be performed to point to the possible significance of ACE2 and
Ang 1-7 in cardiac function. The cardiospecific overexpressive ACE2 transgenic mice
and ACE2 knockdown by lentivirus-mediated short hairpin RNA (shRNA) will be
applied to study the possible mechanisms of ACE2–Ang 1-7 axis to prevent or retard
pathophysiological processes of developing HF.
     HF is the leading cause of hospitalization among older adults over 65 years of
age. The death rates from HF have tripled in the last 20 years. Difficulty in clinical
treatment of HF represents an enormous clinical challenge in need of effective
therapeutic approaches. Our studies will be valuable in understanding the
cardioprotective effects of ACE2–Ang 1-7 axis and will provide new insight into the
pathophysiological processes of HF. Furthermore, our studies will also illustrate new
potential targets or methods, by the modulation of ACE2–Ang 1-7 axis, for HF
preventions and/or treatments beyond the direct effects of anti-RAS drugs.
NSC98-2313-B-009-002-MY3 (98N194)

Title:Using Orthologous Genes to Develop New Algorithms for Efficient Species
       Identification and Horizontal Gene Transfer Analysis
Principle Investigator:Yeong-Shin Lin
Sponsor:National Science Council
Keywords: Clusters of Orthologous Groups, COG, Identification of Prokaryotes,
         Horizontal Gene Transfer, Database, Algorithm

     Orthologous genes are homologous genes in different species derived from
speciation events. The divergence between orthologous genes has been used to infer
the evolutionary distance between species for a long time. In prokaryotes, the
conserved region of 16S Rrna gene is one of the best examples. Except for inferring
the genetic distance, 16S rRNA gene has also been frequently used for species
identification. However, for recently diverged species, 16S rRNA gene is too
conserved to provide sufficient identification information. In our project, we will
develop algorithms to construct clusters of orthologous groups for species
identification (si-COG) based on protein domain information or some other extra
information. For species with their whole genome projects completed, for example,
Bacillus subtilis and B. anthraci, their genomes would be collected and utilized. We
will also use the constructed si-COG to develop algorithms to identify
species-specific sequence tags (SSST).
    SSST would therefore be used for species identification. The obtained results
would be performed as databases and web servers. Further, based on the generated
si-COGs, we could study the horizontal gene transfer events, and the last universal
common ancestor (LUCA). We may therefore test the hypotheses of the origin of
NSC98-2621-B-009-001-MY3 (98N196)

Title:Prediction of RNA Secondary Structure with Pseudoknots and Its Application
       in Detecting Programmed Ribosomal Frameshifting Genes
Principle Investigator:Chin Lung Lu
Sponsor:National Science Council
Keywords: Algorithms, Bioinformatics, Biological Database, Comparative Genomics,
          Rna Secondary Structure, Pseudoknot

     Programmed ribosomal frameshifting RNA pseudoknots are found in almost all
classes of naturally occurring RNAs and play very important roles in a variety of
biological processes, such as RNA replication, transcription and ranslation. For
example, they can serve as stimulators in the so-called programmed ribosomal
frameshifting (PRF) that is a recoding mechanism commonly observed in many RNA
viruses (such as SARS-CoV). By PRF, the translational ribosome switches from the
initial (zero) reading frame to one of the two alternative reading frame (either -1
mostly or +1) at a specific position and as a result, produces an alternative protein that
are different from that produced by standard translation. It has been reported that for
RNA viruses that use PRF, even small alternations in the efficiencies of PRF can
inhibit their propagation, suggesting that the PRF sites may present a potential target
for antiviral therapeutics. The majority of pseudoknots that have been described to
date are of the so-called H-type pseudoknot in which nucleotides from a hairpin-loop
pair with a single-stranded region outside of the hairpin to form a helical stem that is
adjacent or almost adjacent to the hairpin stem. In fact, more complicated H-type
pseudoknots have also been reported, such as the one, functioning as a stimulator of
PRF in SARS-CoV, has an additional stem formed in long loop. Therefore, rediction
of these H-type pseudoknots, as well as even more complicated pseudoknots, can
improve our understanding of RNA structures and their associated functions. In the
standard thermodynamic model, a pseudoknot-free RNA secondary structure of
minimum free energy (MFE) can be computed in polynomial time. However, when
general pseudoknots are allowed in the RNA secondary tructure, the computation
becomes intractable since it has been shown to be NP-hard. Currently, several
polynomial-time algorithms have been proposed to find an MFE secondary structure
with a restricted class of pseudoknots (containing H-type pseudoknots). However,
they are not yet practical for large-scale sequences due to their high running time
and/or space. In addition, these algorithms may not be effective to detect an
H-pseudoknot that is actually present in the native structure of a long RNA sequence.
In the past, we have proposed a heuristic approach to improve the prediction of
H-type pseudoknots for these algorithms and have also applied it in the detection of
the -1/+1 PRF genes. However, there is still much improvement and enhancement that
can be made regarding their time and space complexities, capability in dealing with
large-scale sequences, prediction accuracy, capability in predicting more complicated
pseudoknots and other types of PRF (such as -2 and +2 PRFs) etc. In this component
project, we aim to develop new algorithms and heuristics for such improvement and
enhancement based on the approaches of comparative genomics, structural and
functional bioinformatics and also to develop databases of RNA secondary structure
with pseudoknots and PRF genes. For its success, therefore, we need to work together
with other team members who will provide expertise from their research areas and
related programs to be developed in their component projects. At the same time, the
programs and databases to be developed in this component project will help our team
members involved in other component projects to study RNA viruses, such as
influenza viruses and enteroviruses, and further explore the relationships between
their sequences, structures, functions and evolution.
NSC97-2221-E-009-081-MY3 (97R317)

Title:Vitamin D Binding Ability of Milk β-Lactoglobulin
Principal Investigator:SIMON J.T. MAO
Sponsor:National Science Council
Keywords:-Lactoglobulin, Vitamin D Binding, Vitamin D Absorption, Quality
               Control of Milk

     β-Lactoglobulin (LG) is a major milk whey protein, constituting about 10% of
the total protein mass.The molecular mass of LG is 18.5 kDa, belonging to the
lipocalin family. In secondary structure, it consists of nine β strands and one α-helix.
The central hydrophobic pocket (calyx) possesses the property to bind vitamin D,
vitamin A, and fatty acids. LG is quite sensitive to thermal denaturation; the
secondary tructure is altered upon heating with a transition temperature at 70-80°C.
In the last five years, we have constructed a detailed thermal denaturation curve for
LG with its time and temperature, which provided the dairy industry with a valuable
reference (Chen et al, 2005, JDS) (1). We have also mapped out a specific amino acid
sequence region that is responsible for the thermal change above 80°C. Such changes
also result in the loss of its ligand binding (retinol and palmitic acid) (Song et al, 2004,
JBC) (2). In addition, we have reported that a monoclonal antibody can only
recognize the dry milk, but not the fresh raw milk (Chen et al, JDS, 2004 and 2006)
(3-4). Reversely, we also developed a monoclonal antibody that only recognizes the
native form of LG, so that the un-denatured LG content in the processed milk can be
determined (Liu et al, JDS, 2007) (5). More recently, we have shown that LG
dramatically stimulates the proliferation of hybridoma B cells, but thermal
denaturation abolishes this ability of LG. It is an important observation that the LG
receptor was identified as membrane Ig M using mass spectrum. For the first time, we
have identified a second vitamin D binding site using synchrotron X-ray (Yang et al,
Proteins, 2007) (6). Patients with vitamin D deficiency are associated with some
diseases such as osteoporosis, and autoimmune disorders. In vivo studies have shown
that LG can be directly absorbed into the circulation through the gastro-intestinal
system because of its acid-resistant property. The immunoreactive LG is recovered
following 2h ingestion of bovine milk. The LG is therefore an effective vehicle in
binding the hydrophobic liagands. In this proposal, we plan to identify the essential
residues of the secondary binding site involved in vitamin D binding using
site-directed mutagenesis. Further, we plan to simulate the binding mode using
computer docking method to investigate whether LG mutant can enhance vitamin D
binding and absorption.
     Therefore, the specific aims in the next 2 years will be to:
1. Use monoclonal antibody as a probe to analyze the thermal sensitive region of LG
   and to relate it in functional role (Year 1).
2. Express LG recombinant protein and its mutant type by site-directed mutagenesis
   in E coli. (Years 1 and 2).
3. Estimate vitamin D binding ability of LG recombinant protein and its mutant, and
   to map essential residues involved in the secondary vitamin D binding site (Years
   1 and 2).
4. Compare binding mode between ligands and LG using Docking program (Years 1
   and 2).
5. Estimate vitamin D binding ability of LG mutants using molecular dynamics
     simulation and docking in order to design LG mutant with high vitamin D binding
     affinity (Years 1 and 2).
6. Determine whether LG can enhance vitamin D absorption using mice as an animal
     model and define the mechanism by which LG enhances absorption of vitamin D.
     (Years 1 and 2).
NSC97-2313-B-009-001-MY2 (97R165)

Title:Development of Nanoscale Visible TiO2/FeO Composite (VIS-NTFC)
       Core-shell Reaction System for Natural Organic Matter and Environmental
       Hormone: Reaction Mechanism and Optimization of the Reaction and
       Regeneration System
Principle Investigator:JILL RU-HSING PAN
Sponsor:National Science Council
Keywords: Titanium Dioxide (TiO2), Zerovalent Iron (ZVI), VIS-NTFC Core-shell,
         Dissolved Organic Matter (DOM), 2,4-dichlorophenol

     Zerovalent iron (ZVI) and titanium dioxide (TiO2) have been widely used in
degradations of organic pollutants. The major obstacle of Fe0 application is the
diminishing reactive efficiency with time due to the formation of a surface oxide layer,
while the electron-hole recombination in TiO2 hinders the photocatalytic reactivity of
TiO2. It is know reduction the reduction potential of the photocatalyst TiO2 is
sufficient to transform ferric ion to ferrous ion. In this study, an innovative method is
developed to synthesize a Visible-Nanoscale TiO2/Fe0 core-shell composite
(VIS-NTFC core-shell) to extend the life span of ZVI by retarding the formation of
surface oxide layer on ZVI. The novel composite will be synthesized with a special
chemical method from TiO2 and Fe0 nanoparticles, prepared in the lab by a neutral
sol-gel process and wet precipitation, respectively. The surface characteristics of the
VIS-NTFC core-shell will be examined by using TEM, UV-VIS, XRD, XPS and
chemical analysis. The photocatalytic effect of the VIS-NTFC core-shell will be
evaluated by the degradation of 2,4-dichlorophenol (2,4-DCP) and dissolved organic
matter (DOM) . The scope of the study includes: (1) synthesis of VIS-NTFC
core-shell, (2) preparation and characterization of VIS-NTFC core-shell, (3) effect of
ZVI and TiO2 ratio on catalytic efficiency of VIS-NTFC core-shell, (4) recovery,
regeneration and reuse of spent VIS-NTFC core-shell, and (5) optimization of a pilot
VIS-NTFC core-shell reactor with automated VIS-NTFC core-shell reuse system. The
ultimate goal is to develop an applicable VIS-NTFC core-shell reactor system of high
selectivity and efficiency to remove persistent organic pollutants (POPs).
NSC98-2221-E-009-021-MY3 (98N115)

Title:Role of the in Vivo Expression Protein Yjcc in the Oxidative Stress Responses
       in Klebsiella Pneumoniae CG43
Principle Investigator:Hwei-Ling Peng
Sponsor:National Science Council
Keywords:Oxidative Stress Responses, Klebsiella Pneumoniae CG43 Yjcc, IVET,
                Soxrs, PDE

    Oxidative stress regulon During infection, defenses against oxidative stress play
an important role indetermining the bacterial virulence. The expression of yjcC,
previously identified by in vivo expression technology (IVET) in Klebsiella
pneumoniae CG43, was found to be inducible by 10
gene organization revealed two oxidative stress regulators SoxR and SoxS encoding
genes are next to yjcC gene. The deletion of yjcC was found to decrease the bacterial
survival under the treatment of paraquat further suggesting the involvement of YjcC
in the oxidative stress defense. We propose in three years to demonstrate the
involvement of YjcC in determining the bacterial virulence and also to assign its role
in the oxidative stress regulon. How the regulation is achieved for an optimal
expression of yjcC or for a functional YjcC will also be investigated. Essentially, the
specific aims are:
(1) To investigate the functional role of YjcC by showing
    - if deletion or overexpression of yjcC affects the LD50 in mouse model, the
       susceptibility to H2O2 and paraquat, and the activity of superoxide dismutase
       and catalase.
     - if YjcC exerts a cyclic di-GMP phosphodiesterase (PDE) activity and if the
        enzyme activity is essential for its function
     - if N-terminal part of YjcC plays regulatory or sensory role for the activity of
(2) To identify the regulatory system(s) for optimal expression of yjcC
     - Using promoter reporter system to determine if the expression is dependent on
        treatment with stress molecules such as H2O2 and paraquat
     - if yjcC expression is under control by SoxRS, OxyR, RpoS, Fur or KvgAS
     - Using Tn-mediated mutagenesis to identify other (if any) regulator(s) for yjcC
(3) To show how and why YjcC is expressed in vivo to encounter the oxidative stress
      - Identification of the targets under control by YjcC using comparative analysis
         of the proteomes of KP CG43 and the yjcC deletion mutant
      - Identification of the YjcC interacting proteins by co-purification or pull-down
      - Investigation of subcellular localization of YjcC using YjcC-GFP fusion under
         various oxidative stress conditions
NSC97-2320-B-009-001-MY3 (97R166)

Title:Identification of the Regulatory Systems Involved in the Counter-Expression of
       Type 1 and 3 Fimbriae in Klebsiella Pneumoniae
Principle Investigator:Hwei-Ling Peng
Sponsor:National Science Council
Keywords: Klebsiella Pneumoniae, Counter-Expression of Type 1 and Type 3
         Fimbriae, Interacting Regulation with CPS Biosynthesis

     Fimbriae (also called pili) play a key role for bacteria to attach to specific host
cell during the establishment of an infection. The expression of type 1 or type 3
fimbriae in the clinical isolates of Klebsiella pneumoniae has been commonly
reported. We have found in the previous study (94IDP11) that the deletion of the type
3 fimbrial major subunit encoding gene mrkA of K. pneumoniae NTUH K2044 or
CG43 significantly increased the expression of type 1 fimbriae. The overexpression of
FimB recombinase was able to switch on the expression of type 1 fimbriae but
reduced the MrkA production. Moreover, deletion of the rcsB gene, which encoding a
major activator for the biosynthesis of capsule polysaccaharides (CPS), was found to
reduce the production of CPS and MrkA, but increased the activity of type 1 fimbriae.
These indicated a counter-expression between type 1 and type 3 fimbirae. The
possibility of an interacting regulation between fimbriae and CPS biosynthesis was
also implicated. It is hence the proposed study is to explore the regulatory
mechanisms involved in the control of counter-expression of type 1 and type 3
fimbriae. The specific aims are:
  1) To investigate if the deletion of the specific regulatory genes affects the
expression: These regulatory genes include fimK, which located at the end of the type
1 fimbrial gene cluster, and phgS and phgM, at the front of the type 3 fimbriae gene
cluster, and KP4551, KP4552, and KP4554 (on the basis of the genome annotation of
NTUH K2044), at the end of the type 3 fimbriae gene cluster. The regulatory gene
with apparent deleting effects on the counter-expression will be cloned and expressed,
and the regulatory mechanism elucidated.
2) To generate transposon-insertion mutant libraries to screen for the regulatory genes:
The mrkA deletion strain or the rcsB deletion strain will be used as the recipient for
the transposon mutagenesis purpose. The mutants reveal a decreased level of yeast
agglutination activity will be isolated. The strain carrying either Pfim-lacZ or
Pmrk-lacZ will be also used as the recipient for the transposon-mediated mutagenesis,
and the mutants showing alteration of LacZ activity will be isolated. After the
insertion sequences determined, the genes of interest will be cloned and the specific
gene deletion mutants generated, and the complementation assay will be carried out to
confirm the regulatory activity. The mechanisms involved in the counter-expression
regulation or interacting regulation between fimbriae and CPS will be further studied.
3) To investigate if envelope stress plays a role in the regulation of the
counter-expression: An envelope stress resulted from the accumulation of MrkB,
MrkC, MrkD, and MrkF at the periplasmic space could be built up in the mrkA
deletion mutant. While in the mutant, expression of the type 1 fimbriae appeared to be
switched on. Moreover, the rcsB deletion which reduced the CPS biosynthesis
affected only the expression of type 1 fimbriae. These implied the changes of the
envelope differentially affect the expression of type 1 or type 3 fimbriae. To
determine if mrkA deletion leads to an envelope stress, expression of the regulatory
genes involved in envelope stress responses will be analyzed by qRCR in the mrkA
deletion mutant, and the mechanisms further characterized.
NSC98-3112-B-009-005 (98N501)

Title:Study the Effect and Regulation of cAMP and CRP on the Stability of OmpA
       mRNA in Escherichia Coli
Principle Investigator:CHING-PING TSENG
Sponsor:National Science Council
Keywords: OmpA, cAMP, CRP, Post-Transcription Regulation

     In Escherichia coli, the OmpA outer membrane protein is a multifaceted protein.
OmpA can function as an adhesin and biofilm formation. It also displays a receptor to
several bacteriophage, which acts as the model for studying pathogenesis. The ompA
mRNA degradation research started from early 1980‘s, Scientists discovered that
some important regulatory factors - RNase E, Hfq and sRNAs involved in ompA
Mrna degradation. Therefore, the research of ompA mRNA degeneration become the
significant model for the mRNA degradation study. Previous studies have reported
that cAMP-CRP manages genes expression by modifying promoter activity, such as
lac and gal. The result of cAMP concentration and ompA mRNA level was
synchronized in cya mutant cultured with glucose supply. As far as we know, the
cAMP-CRP acts as a transcriptional factor in E. coli by binding promoters, and
enhances the interaction with RNA polymerase. Interestingly, wild-type E. coli and
cya mutant strains were cultured in LB and LB+Glc, respectively. We found that the
stability of ompA mRNA was down-regulated when glucose was added or cya was
mutated. These results indicated that the stability of ompA mRNA was elevated by
cAMP. Because the way of cAMP alters ompA RNA stability still unclear, therefore
we propose that post-transcription of mRNA is not directly regulated by cAMP. In
this proposal, We‘ll examine three candidates RNase E, Hfq and sRNAs targeting to
ompA mRNA and discovered which might indirectly participate in the regulation. We
will also examine the interaction between CRP and ompA mRNA inorder to
determine whether the ompA mRNA stability is directly regulated by cAMP-CRP
NSC98-2311-B-009-003-MY2 (98N418)

Title:A study of Curriculum Development and Teaching Experiment on Biomass
       Energy for Senior and Vocational High School
Principle Investigator:C. P. Tseng
Sponsor:National Science Council
Keywords: Biomass Energy, Experiment

     This proposal is the third part of the integrative program—―A Study of
Curriculum Development and Teaching Experiment on Biomass Energy.‖ It aims to
support the ―Biomass Energy‖ courses in high schools and vocational high schools,
and provide them with curriculum evaluation, assistance for curriculum design, and
course-related teachers training programs. The focus of the curriculum evaluation
will be put on the design and practice of the program, materials and equipments, and
students‘ satisfaction, which includes the following categories: The Implementation of
Biomass Energy System on Plants Resources (see proposal one), The Implementation
of Biomass Energy System on Animals and Microbial Resources (see proposal two),
curriculum design (including the outline and purpose of teaching), teaching material
development (including basic knowledge, operation procedures, and application),
teaching experiments (including implementing courses and projects), research
findings (content and publishing), and teaching medium. The courses can be
examined by using the teachers and professional‘s expert knowledge to see if the
expected teaching goal is achieved. Moreover, the research findings will be
beneficial to the development of course design, the editing and selecting of teaching
materials, the improvement of learning efficiency, and the analysis after evaluation.
NSC98-2514-S-009-001-GJ (98N480)

Title:Development of Agricultural Waste Biogas Purification Bioreactor and
      Electricity Generation System
Principle Investigator:C. P. Tseng
Sponsor:National Science Council
Keywords: Marsh Gas, Power Generation, H2S Removal System

     The utilization of renewable energy and development of new energy sources are
government policy. A large amount of biogas derived from wastes is an excellent
resource at present. However, the application of bioconversion energy is restricted by
low purity of biogas and the techniques of power generation. In the former projects,
we isolated an acidophile autotrophic strain with high H2S removal efficiency. The
results of bench-scale biofilter reactor could remove 98% of 2000 ppm H2S in 2
minutes GRT (gas retaintion time). The pilot scale of 200-fold scale-up reactor was
constructed, and the H2S removal efficiency was 96% in 2 minutes GRT (4000 ppm).
In this project, we will focus on sulfide recovery due to the sulfide accumulation,
which caused pH vaule dropping dramatically. Furthermore, the gas flow rate will be
improved and elevated from 100 L/min to 200 L/min after improvement of the reactor
in this project (or shorten half of GRT to 1 minute).
      We have designed a combined chemical-biological reactor in the former project.
The bench scale reactor which eliminated 100% of 2500 ppm H2S in 1 minute GRT.
The 5-fold scale up pilot scale reactor eliminated 95% of 5000 ppm H2S in 1 minute
GRT. In this project, the high efficiency mutant strain will be applied to system for
better H2S elimination performance. Also, the reactor will be 10-fold scaled up to
elevate gas flow rate from 20 L/min to 200 L/min. After that, we will optimize the
operating parameters. The automatic control system will also be constructed in this
system for better security, stability performance and easier to operate. We expect our
system is suitable to use in all piggeries with 3000 pigs and reach electrical
NSC98-2324-B-009-001- (98N489)

Title:Development the Technology of Agricultural Waste Bioconversion to Biogas
        for Electricity Generation and Carbon Dioxide Elimination by Microalgae
Principle Investigator:C. P. Tseng
Sponsor:National Science Council
Keywords: Biogas, Electricity Generation, Microalgae, Desulfurized Bioreactor,
          Biodiesel, Anaerobic Wastewater Treatment

     Biogas is a kind of natural renewable energy source, which consist of 60%
methane and 20 ~ 30% carbon dioxide. The greenhouse effect of methane is 20 times
higher than that of carbon dioxide. The electricity can be generated by combustion of
methane via the generator; whereas carbon dioxide can be removed and transformed
to biodiesel by the algae. Therefore, it contributes to both objectives of energy
generation and the reduction of greenhouse gas effect. This project will set up a model
system to generate electricity in the swine farm at Taiwan Sugar Corporation, and to
develop the swine farm wastewater recycling, the reduction of carbon dioxide
emission and the transesterification technology. The main aim of this project is to
re-construct the aerobic waste treatment pool to the anaerobic waste treatment system.
Biogas collected will be used to remove high concentration of hydrogen sulfide by the
desulfurized bioreactor. Through the scale-up and automatic operation and improve of
the sulfur accumulation problem, the desulphurization system with above 10,000
swines in the swine farm will be used for a long-term treatment. Furthermore,
microalgal strain with high tolerance of biogas and high photosynthesis efficiency
will be screened in the microalgal cultivation project, and the CO2 contained in
desulfurized biogas and the flue gas emitted from the generator will be used to
cultivate the algae. It is expected to reduce the carbon dioxide emission abundantly
and to collect the algal biomass to produce biodiesel by transesterification. The
desulfurized and CO2 reduced biogas will be used in the long-term operation of the
existing 30 kW generator and other monitoring parameters, to construct 100 ~ 300
kW of large scale generator for the electricity generation with the 10,000 swines. Due
to the biogas production is low under the low temperatures during winter in Taiwan,
to increase biogas production in the sub-project, the model and pilot system of
anaerobic wastewater treatment will be addressed to optimize the biogas production
parameters in order to obtain the maximum biogas production in the swine farm
anaerobic wastewater treatment for the whole year. It is expected that the generator
can produce 70 kwh from biogas while carbon dioxide can be reduced up to 3600
NSC98-3114-B-009-002 (98N542)
Title:Study the Removal of Water Soluble Organic Waste Gases from

        Semiconductors and Photonics Industry by Biological Systems

Principal Investigator:C. P. Tseng

Sponsor:National Science Council

Keywords:PGMEA, Oluble Organic Waste Gases, Photonics Industry

   The aim of this project focus on removal of soluble organic waste gases in
semiconductors and photonics industry.

    In the first year, we choose the target pollutants:acetone, IPA(isopropanol) and

PGMEA(Propylene Glycol mono-methylether acetate) Then we will try to eliminate

these organic waste gases by three type reactors below, biofilters we developed before,
fludized bioscrubbers and modified bio-plate towers. There will be some strains we
screened in laboratory fixed in three type reactors, to do related analysis during
operation. In the second year, we will try to analyze the relationship between
mass-transfer velocity, biochemical-reaction rate and removal of organic waste gases
in different operation conditions, in further, we will set up a theoretical model and
make a proof with some given known parameters, to make sure this theoretical model

could work for scale-up. In the last year, we will set up a pilot-scale(scale-up 50

folds) modified bio-plate towers to eliminate organic waste gases. To do comparison

with the theoretical model, then modify (if necessarily) related coefficients of
operation parameters. Finally, to do a economic analysis for first cost and operation
cost, this could be a good reference for scale-up in the future.
NSC98-2324-B-002-CC1 (98N963)

Title:Rapid Analysis of Bio-Macromolecules Interaction Using Bio-electrical Signal
       and Nano-scale Image Study Simultaneously
Principle Investigator:Shin-Hua Tseng
Sponsor:National Science Council
Keywords: Nano-scale Image Study Simultaneously
     In this study we will find a general model correlating the electrical behavior of
bio-molecule-modified electrode with bio-macromolecules interaction occurred on
electrode surface. Based on this model, a bioelectrical signal analysis is applied to
detect bio-macromolecule interaction for the construction of intra- and extra-cellular
signal transduction pathway. We will also develop a special bioelectrical signal
measurement instrument and install it on the AFM platform to be capable of studying
biomacromolecules interaction on electrode surface by bioelectric signal analysis,
AFM image and single molecule force spectroscopy simultaneously. About
bioelectrical signal analysis, it is intuitive that both the capacitance of electric double
layer on electrode surface and the conductivity of electrode surface will decrease
when other molecules in solution bind or absorb to electrode-surface-bonding
macromolecules. Moreover, the binding strength between these bio-macromolecules
can further be studied in this project by single molecule force spectroscopy of AFM or
adjusting static or alternative electrical field of the analyzing electrode. In summary,
the goals of this proposal include: (a) Find a general model for bio-electrical signal
measurement to correlate bio-macromolecules interaction. (b) Develop a bio-electrical
signal measurement instrument which can integrate with AFM system and become
fast screen and analyzing system of bio-molecule interactions. (c) Develop
bio-sensors for interaction analysis of biomacromolecules. Particularly, these
inventions have high potential to be transferred to biomedical application and
biological research. Furthermore, the network of protein interaction with p53 can be
expanded using the bio-electrical signal measurement instrument combining with
NSC98-2112-M-009-021-MY2 (98N502)

Title:MRI Contrast Agents of Gadolinium(III) Complexes and Surface Modified Iron

          Oxide for Molecular Imaging

Principal Investigator:Yun-Ming Wang

Sponsor:National Science Council

Keywords:Magnetic Resonance Imaging Contrast Agent, Thermodynamic, Kinetic,

               Relaxivity, Gadolinium      Complex,                 Water       Exchange        Rate,
               Superparamagnetic Iron Oxide
     Recently, MRI has been developed to examine living organisms down to the
cellular and molecule level. To exploit the advancement of MRI technique for cellular,
molecule and functional imaging, there are increasing needs for developing new MRI
contrast agents and techniques for cell and molecule labeling to report the localization,
movement, mass, and functions of cells in vivo. Therefore, two major classes of
contrast agents are available for MRI such as small molecular weight Gd chelates
and iron oxide nanoparticles. The purpose of research is tuning the lipophilicity of
Gd complexes and conjugating with peptide substrates of MMP-7, Legumain
protease or cRGD. MMP-7, Legumain protease and αvβ3 receptor are greatly related
to tumor invasion and metastasis, and are also highly expressed in majority of human
tumors, which make them as the very representative cancer proteases and membrance
receptor. To find the optimum lipophilicity of Gd complexes that can be stably
incorporated into cell membranes may serve as a useful tool for tumor cell labeling
and tracking. Ideally, these Gd chelates should label intact cell membranes
noninvasively at low concentrations and with fast kinetics, and should remain on
labeled cells over a period of time to allow repetitive imaging. The chemical and
physical properties of these Gd3+ complexes will be characterized, including
thermodynamic stability constant, relaxivity (r1), the number of inner-sphere water
and kinetic parameter (water exchange rate and rotational correlation time). Finally,
the MR imaging will be conducted as well. On the other hand, in the case of targeting
to tumor cell, the surface of iron oxide nanoparticles modified with dextrin or PEG
(polyethylene glycol) and then conjugated with herceptin or targeting peptide will be
synthesized. The nano-sized superparamagnetic iron oxide(SPIO) particulates
selectively shorten the transverse relaxation time (T2) of nearby water protons and
generally produce negative enhancement by decreasing signal intensity. The geometry,
structural features, and physical properties of magnetite nanoparticles will be
characterized. The longitudinal relaxivity (r1) and transverse relaxivity (r2) will be
measured by 20 MHz relaxometer at 37.0 ± 0.1℃. To prove effectiveness of this MR
probe, the flow-cytometric analysis and MR imaging will be used.
NSC97-2113-M-009-016-MY3 (97R309)

Title:The Research on Bio-Mimetic Solar Cell Device Design and Photovolatic
       Energy Conversion Mechanism
Principle Investigator:WU TUNG-KUNG
Sponsor:National Science Council
Keywords: Bio-Mimetic System, Porphyrin, Genetic Engineering, Directed Evolution,
             Site-saturated Mutagenesis, Femtosecond Spectroscopy

     The needs of the planet for the energy will double and triple by mid-century and
the end of century, respectively, with the fossil fuels that can no longer furnish. Solar
radiation is a plentiful, clean, and ultimate source of power. However, how to develop
new materials with the ability to tune the electronic bandgap for maximal solar energy
conversion efficiency becomes one of society‘s important challenges. One route to the
solution is the development of bio-mimetic photosynthetic system with diverse
bandgap properties and high energy conversion efficiency at the molecular level. The
ultimate goal of this proposal aims at developing an integrated bio-mimetic
photosynthetic system, single artificial protein complex or protein complex array with
integrated photoinduced charge separation and electron transfer properties, which
enables thermal stability and efficient photovoltaic conversion of solar energy into
electricity. In parallel, understanding of the distributions in energy levels and electron
transfer dynamics of the bio-mimetic system is of fundamental importance and
prerequisite for efficient energy absorption and photovoltaic conversion. Therefore,
we propose herein with three inter-connected subprojects to develop the bio-mimetic
photosynthetic systems and to study the reaction mechanisms as well as
electron/energy transfer dynamics, with the integration of multidisciplinary research
fields. In subproject 1, we propose to design and synthesize various
metallo-porphyrin-based donor-acceptor systems as prosthetic group, which will be
reconstituted into engineered artificial protein to form complexes for photovoltaic
applications and for fundamental studies described in subproject 2 and 3. In
subproject 2, artificial proteins with different oxidation states or redox potential for
bandgap adjustment will be designed and constructed, using apomyoglobin as a model
template, and subjected to prosthetic group reconstitution and bio-mimetic
photovoltaic applications and for interfacial electron transfer dynamics and reaction
mechanism studies. In subproject 3, the bio-mimetic solar cell device will be
fabricated and characterized; the fundamental process controlling the important
photovoltaic energy conversion functions will be probed with the state-of-the-art
spectroscopic technology -femtosecond time-resolved spectroscopy.
NSC98-2627-M-009-007 (98N497)

Title:The Research on Bio-Mimetic Solar Cell Device Design and Photovolatic
       Energy Conversion Mechanism
Principle Investigator:WU TUNG-KUNG
Sponsor:National Science Council
Keywords: Bio-mimetic System, Porphyrin, Genetic Engineering, Directed Evolution,
           Site-saturated Mutagenesis, Femtosecond Spectroscopy

     The needs of the planet for the energy will double and triple by mid-century and
the end of century, respectively, with the fossil fuels that can no longer furnish. Solar
radiation is a plentiful, clean, and ultimate source of power. However, how to develop
new materials with the ability to tune the electronic bandgap for maximal solar energy
conversion efficiency becomes one of society‘s important challenges. One route to the
solution is the development of bio-mimetic photosynthetic system with diverse
bandgap properties and high energy conversion efficiency at the molecular level. The
ultimate goal of this proposal aims at developing an integrated bio-mimetic
photosynthetic system, single artificial protein complex or protein complex array with
integrated photoinduced charge separation and electron transfer properties, which
enables thermal stability and efficient photovoltaic conversion of solar energy into
electricity. In parallel, understanding of the distributions in energy levels and electron
transfer dynamics of the bio-mimetic system is of fundamental importance and
prerequisite for efficient energy absorption and photovoltaic conversion. Therefore,
we propose herein with three inter-connected subprojects to develop the bio-mimetic
photosynthetic systems and to study the reaction mechanisms as well as
electron/energy transfer dynamics, with the integration of multidisciplinary research
fields. In subproject 1, we propose to design and synthesize various
metallo-porphyrin-based donor-acceptor systems as prosthetic group, which will be
reconstituted into engineered artificial protein to form complexes for photovoltaic
applications and for fundamental studies described in subproject 2 and 3. In
subproject 2, artificial proteins with different oxidation states or redox potential for
bandgap adjustment will be designed and constructed, using apomyoglobin as a model
template, and subjected to prosthetic group reconstitution and bio-mimetic
photovoltaic applications and for interfacial electron transfer dynamics and reaction
mechanism studies. In subproject 3, the bio-mimetic solar cell device will be
fabricated and characterized; the fundamental process controlling the important
photovoltaic energy conversion functions will be probed with the state-of-the-art
spectroscopic technology -femtosecond time-resolved spectroscopy.
NSC98-2627-M-009-008 (98N498)

Title:Molecular Interaction Networks and 3D-Domain Interologs in Medical
Principle Investigator:Yang,Jinn-Moon
Sponsor:National Science Council
Keywords: 3D-Domain Interologs, Conserved Pathway, Consensus Interacting
         Pattern, Orthologous

     proteins, protein-protein interaction, protein-ligand interaction, protein-DNA
interaction A major challenge of postgenomic biology is to understand the networks
of interacting genes, proteins and small molecules that produce biological functions.
The large number of protein interactions, generated by large-scale experimental
methods, provides opportunities and challenges in annotating protein functions,
protein-protein interactions (PPI) and domain-domain interactions (DDI), and in
modeling the cellular signaling and regulatory networks. An approach based on
evolutionary cross-species comparisons is valuable for addressing these issues. We
have utilized "3D-domain interologs" to discover potential protein-protein and
protein-DNA interactions which are predicted as orthologous interactions among
different species, in particular about inferred interacting domain pairs and binding
models (e.g. hydrogen-bond interactions and conserved residues). The 3D-domain
interologs is defined as "Domain a (in chain A) interacts with domain b (in chain B)
in a known 3D complex, meaning that their inferring protein pair A' (containing
domain a) and B' (containing domain b) in the same species would be likely to
interact with each other if both protein pairs are homologous". We have demonstrated
that 3D-domain interologs is useful to understand the interacting evolution
(co-evolution) of PPIs and DDIs across multiple species. In this project, we will
enhance and modify 3D-domain interologs to understand the mechanisms of
biological networks across multiple species and its applications on biomedical
applications. We will achieve the following specific aims: (i) Developing
knowledge-based scoring functions for calculating the binding affinity of
protein-protein and protein-DNA interactions; (ii) Determining interacting domains,
binding models (e.g. hydrogen-bond interactions and conserved residues), and
consensus patterns of protein-protein/protein-DNA interactions across multiple
species;    (iii)   Identifying     the   conserved     networks     (pathways)   of
protein-protein/protein-DNA networks across multiple species; (iv) Using consensus
patterns of protein-protein/protein-DNA interactions to understand the mechanisms of
protein-ligand binding models and to realize the probable mechanism of pathogenicity;
and (v) Applying these methods and scoring functions on new disease targets,
cancer-related pathways and drug discovery. Currently, we have achieved some
preliminary results, such as a 3D-partner server for protein-protein interaction
prediction, a 3D-interologs database for examining orthologous interactions across
multiple species, and two knowledge-based scoring systems for measuring the
binding affinity of protein-protein and protein-DNA interactions. Our methods and
scoring functions have successfully identified the protein-protein interactions (i.e.
CDK2/CKS1 complexes across 56 species) and protein-DNA interactions (Myc/Max
across 72 species) of cancer-related pathways of lung cancer. Our long-term objective
is to combine the known cellular processes and our predicted protein-protein,
protein-ligand, and protein-DNA/RNA interactions to construct molecular interaction
networks, which are essential for many medical applications, therapeutic strategies
and drug development.
NSC98-3112-B-009-003 (98N036)

Title:Comparative Analysis of Molecular Interactions and Pathways in Biological
Principle Investigator:Jinn-Moon Yang
Sponsor:National Science Council
Keywords: Comparative Analysis, Molecular Interactions, Pathways

      During the first-term ―Program for Interdisciplinary Research Projectin
Bioinformatics (2005-2008)‖,we have achieved specific goals and published ~40
journal papers (see appendix A). Based on these achievements (correlations between
sequence, structure, function, and biochemical pathways), the primary theme of our
integrated project is to investigate molecular interactions and evolution relationships,
and build various pathways (i.e. signaling pathways and metabolic pathways) and
networks (e.g. gene regulatory networks). Disease, drug and biomedical researches
correlated closely with the complications among gene sequence, protein and
biological functions. The fruitful outcomes of our project will be in accurately
analyzing and predicting molecular interactions and biochemical network components,
moreover, drug developments. Furthermore, our predictions will be immediately
confirmed by cellular and viral experiments. This project consists of four subprojects:
network evolution for studying molecular interactions and pathways (subproject 1);
intelligent optimization methods for reconstruction and analysis of gene networks
(subproject 2); data management and exploration of molecular interactions and
pathways (subproject 3), and protein subcelluar localization prediction and
protein-protein interactions: from genomic sequences to protein functions (subproject
4). This integrated project studies molecular interactions and networks on genes,
proteins, signaling pathwayys, metabolic pathways, and gene regulatory networks.
The specific aims of our project are listed as follows:
1. To identify the interactions and binding models of protein functional domains, as
well as the protein-protein, protein-DNA and protein-RNA consensus patterns across
multiple species. We will develop evolutionary and multiple species-based scoring
functions to predict molecular binding affinity and interacting models by using
evolutionary relationships across species. Based on these predicting models and
scoring functions, we can infer potential interactions from known interaction
networks. In addition, we are able to compare interaction networks across different
species to discover the conserved interaction pathways (Subprojects 1, 2 and 4).2. To
establish an integrated database system, including sequences, structures, structural
visualized interactions, molecular interactions, biochemical pathways, and gene
regulatory networks. The database, which will be updated and integrated
automatically, is designed as a comprehensive, broad species coverage and
non-redundant molecular interaction repository (Subprojects 1, 2, 3 and 4). 3. We will
develop methods for predicting molecular interacting sites (e.g. protein-ligand,
protein-protein, and protein-DNA) and molecular recognitions in integrated ways
(Subprojects 1, 2 and 4). These methods will be used to practical applications, such as
immunogenicity of MHC class I and II binding peptides, structure-based drug
screening (e.g. dengue virus and cancer targets) and protein-protein interactions
(Subprojects 1 and 2). 4. To reconstruct the gene regulatory networks and to revise
deficiency of experimental data (noise and nsufficient data) by optimization methods,
such as genetic algorithms, support vector machines, and neural networks. We will fit
the reconstructed gene regulatory networks to the natural biological systems based on
the molecular interactions models (Subprojects 1, 2 and 3) and the biological
databases. 5. To identify protein locations and interactions of bacterial and viral
proteomes by the well-developed tools, (e.g. CELLO and 3D-partner). Based on the
localizations and interactions of host and viral proteins within the host cells, we will
be able to predict and investigate the extensive interactions involved in disease
mechanisms. These predictions can be verified by experiments (Subprojects 1 and 4).
6. To cooperate with biological experiments to evaluate our computational results on
some important biochemical interactions and networks, such as the mechanisms in
diseases, cancer therapy and production of biomass energy. We will use multiple
strategies to explore protein-protein, protein-DNA and protein-RNA interactions, and
find out the roles of these interactions in these networks (Subprojects 1, 2, 3 and 4).
NSC98-2627-B-009-003- (98N481)

Title:Development of An Ultrasensitive and Real-time Sensing Nanowire Field
       Effect Transistor Biosensor for Influenza Virus
Principle Investigator:Yuh-Shyong Yang
Sponsor:National Science Council
Keywords: Development of An Ultrasensitive, Real-time, Influenza Virus

     We propose the use of semiconductor device as transducer for molecular
diagnosis to develop an ultrahigh sensitivity, label-free, real time detection and easy
to use biosensing system using silicon nanowire field effect transistor (SNW FET).
One of the unique features of this project is to use poly silicon nanowire field
transistor (poly-SNW FET) for its fabrication is compatible with current commercial
semiconductor process. It is important for the future products will rely on the
availability of the SNW FET. We will immobilize DNA or antibody, which is
complementary to influenza virus RNA or against influenza virus‘ surface protein,
respectively, on the nanowire surface of SNW FET. The high sensitive and high
specificity diagnosis will be achieved when the complementary RNA or antigen
protein interacts on the nanowire surface of SNW FET. The electronic response of
SNW FET is very sensitive to the variation of charges on its surface. Avian influenza
viruses will be our primary targets especially for H5 and H7 subtypes. In vitro test in
the laboratory will be established by following the clinical test in collaboration with
medical center. We will concurrently improve the sensing sensitivity through the
fabrication of semiconductor chip and through the biomolecular modification of the
nanowire surface. In the future, a portable diagnostic instrument with micro array
SNW FET chip will be explored as the basis for commercial product development.

Title:Organic Thin Film Transistor as Non-Invasive Biomedical Sensor
Principle Investigator:Yuh-Shyong Yang
Sponsor:National Science Council
Keywords: Non-invasive, Organic, Biomedical Sensor

     Organic thin-film transistors (OTFTs) possess great potentials in realizing large
size, low-cost, one-time-use, and yet non-invasive sensors for medical and
environmental applications. Development of such highly selective, sensitive and
flexible gas sensor array will direct the core course for this project, in which it
comprises the development of various self-assembled monolayer (SAM) materials
and immobilized biomolecules techniques. In addition, the effects of gas sensing
mechanism and selectivity due to the OTFTs active layers will be particularly
addressed in our efforts by various active layermaterials, deposition process
conditions, surface morphology, and functional group modification of polymeric
conjugated materials. Furthermore, this study will also undertake the vacuum
deposition equipment integration which enables the continuous process for thin film
deposition. For the biomedical application of the gas sensor, this research will be
targeted on cirrhosis clinical diagnosis. The ammonia presence in patient‘s breath is
one of cirrhosis clinical symptoms. This is due to blood ammonia diffused during
respiration. The exhaled ammonia concentration can be detected as the measurement
of blood ammonia concentration. As a result, the medical personnel can monitor the
progress of cirrhosis according the breath ammonia reading. During the course of
study, the patients‘ breath samples will be provided by collaboration with China
Medical University Hospital. The database will be established and compared with the
results of gas chromatography. Finally, we will apply machine learning and the
current methods of bioinformatics in order to extent the applications on complex
gases detection and precise reading. The future goal is to fabricate the home care
ammonia gas detector for cirrhosis patients.
NSC98-2627-B-009-006- (98N477)

Title:Elucidating the Functions of Pathogenesis Factors by Functional Genomic
      Approaches in Candida Albicans
Principle Investigator:Yun-Liang Yang
Sponsor:National Science Council
Keywords:       Pathogenesis,       Regulatory       Network,      DNAmicroarray,          Functional

     Yeast infections in human have increased significantly in recent years. Among
the pathogens, Candida albicans is the most dominant one. It has emerged as the
fourth most common cause of nosocomial infections in the United States. The
estimated cost for treating the Candida nosocomial infections approaches 1 billion US
Dollars per year in the United States. Currently available antifungal drugs have
undesirable issues such as side effects, ineffective against new or reemerging fungi,
and the emerging of resistance. Therefore, development of a new concept and strategy
for anti-fungal effort is in demand. Although several factors associated with C.
albicans pathogenesis, such as environmental cues, nutrition availability, drug
resistance, and virulence factors (for examples, morphogenesis, extracellular
hydrolytic activities, and phenotype switch) have been identified, the overall picture
of the global networking and the coordination of those regulatory and signaling
pathways contributing to the pathogenesis is still missing. Therefore, the long-term
goal of this research is to elucidate the regulatory network and mechanisms of
pathogenesis in C. albicans, which will allow us the identification of potential targets
for anti-fungal purposes. Here, we propose to employ molecular genetics and
functional genomics tools, particularly homologous replacement and DNAmicroarray,
to analyze and explore (1): the function of signaling/regulatory pathways contributing
to the C. albicans pathogenesis induced by the environment cues, iron availability,
host anti-microbial responses, and morphogenesis/virulence factors and (2): their roles
in the whole gene network of pathogenesis. There are three objectives in this project.
The first objective of this study is to focus on the functional study of Efg1 pathway
known to be the key regulatory factor of several pathogenesis pathways, starting from
the CaEno1, a known glycolytic proteins specifically regulated by Efg1 and involved
in the pathogenesis process as well as being an important cell wall component and the
major antigen of Candida infection. What is its function and role in the Efg1 pathway
and Candida pathogenesis? Due to the lack of known sexual cycle and plasmids,
mutagenesis to study the function of this gene will rely on the homologous
replacement technique. The second one isto construct and employ a microarray
platform to study the whole-genome profiling induced by selected pathogenesis
factors and the regulatory pathways thereof and their global effects on C. albicans.
The third is to unveil the relationship of those regulatory/signaling pathways,
especially in connection with the Efg1, a major controlling point of pathogenesis, and
to map the global gene network of pathogenesis. In addition to satisfy the need for
understanding the basic mechanism of fungal pathogenesis, this research may allow
us to define particular gene products or signal transduction pathways that can be used
as targets to block the transition from commensal to pathogen or to kill the fungal
cells specifically. And in the next stage, they may assist in the design and
development of new antifungal drugs and/or new antifungal strategies on the
molecular level. This study is highly relevant to the aim of innovative research of the
―National Research Program for Genomic Medicine Program‖ .
NSC98-3112-B-009-001 (98N033)

Title:Theoretical Investigation on the Spectroscopy of Polyiodide-Amylose Complex

Principal Investigator:Jen-Shiang Yu

Sponsor:National Science Council

Keywords:Polyiodide-Amylose Complex
      The double helices of amylose can form blue complex with polyiodide ions. The
maximum absorption peak in UV/visible spectrum locates at the range of 588 to 620
nm, depending on the source of the starch. It is aimed in this proposal to study in
silico the structural periodicity of the helical formation, the spectral properties of the
blue complex, as well as the thermodynamic stability by quantum mechanical theories.
Methodologies that will be utilized include molecular mechanics and semi-empirical
theories for the geometrical optimization for the most stable structures and
conformations of the amylose double-helices, and density functional theories for the
complex involving binding with the polyiodide ions. Thermal stability of the complex
will be computed, and theoretical spectra of the absorption of the complex will be
modeled with time-dependent density functional theories using random phase
transition theory, as well as symmetry adapted cluster configuration interaction
computation. Direct algorithm of the SAC-CI theory will be applied to study these
large-size molecules in the target system.
NSC97-2113-M-009-001-MY2 (97R020)

Title:Theoretical Study and Design on the Metal Coordinated Macromolecules
Principle Investigator:Jen-Shiang K. Yu
Sponsor:National Science Council
Keywords: Theoretical Study

     Ab initio methods and density functional theories (DFT) are utilized to study a
series of metal coordination complexes and their host proteins. Target molecules
include MIII⋅Salophen (M=Cr, Mn, and Fe) and Cr⋅cyclam compounds. DFT will be
employed with proper basis sets to optimize the theoretical geometries of these
compounds, followed by spectral calculations using high-level ab initio methods and
compare with experimental crystal structures and spectroscopy. The interaction
between coordination compounds and host proteins will be investigated by QM/MM
and ONIOM methods. The study will help to understand the physical properties of
these metal coordination compounds and the binding mechanisms. Results would be
beneficial to design proper scoring functions for the molecular docking software and
design of anticancer metal drugs, as well as the synthesis of artificial proteins.
NSC98-2113-M-009-010-MY2 (98N183)

Title:Research on the Role and Mechanism of Human Mammalian Sterile 20-Like
       Protein Kinase 3 (Mst3) in Apoptosis
Principle Investigator:Chiun-Jye Yuan
Sponsor:National Science Council
Keywords: Mst3, Apoptosis, Apoptosis-Activation Factor, Placenta, Preeclampsia

     The main objective of this project is to understand the physiological function of
mammalian Ste20-like protein kinases 3 (Mst3) in placenta during pregnancy disorder
and its molecular mechanism in the stress-induced apoptosis. In previous studies, we
have demonstrated that overexpression of Mst3 could induce apoptosis in several cell
lines. Recently, we found that endogenous Mst3 may be present in both mitochondria
and cytoplasm. In mitochondria, Mst3 resides in the intermembrane space and
associates with pro-apoptotic proteins, including apoptosis-inducing factor (AIF) and
endonuclease G (EndoG), forming a pro-apoptotic complex. Therefore, in this
three-year proposal, we want to extend this study to further investigate the role of
Mst3 in the formation of Mst3/AIF/EndoG complex and in regulating the activity of
AIF and EndoG in response to apoptotic signals. Furthermore, the mapping of the
binding domain in Mst3 for AIF and/or EndoG will be performed. The effect of Mst3
without this domain will be studied. The results of these studies may provide
evidences explaining how Mst3 mediates apoptosis via AIF and EndoG. Recently,
Mst3 was found to mediate oxidative stress-induced placental apoptosis through both
the caspase-dependent and -independent pathways. Interestingly, the expression of
Mst3 was also found in the placenta from woman complicated with preeclampsia.
Preeclampsia, a disorder of pregnancy, is the leading cause of maternal and fetal
morbidity and mortality. However, the role of Mst3 in preeclampsia is unclear so far.
Hence, immunohistochemical study and explant culture will be performed first to try
to elucidate the role of Mst3 in placenta with preeclampsia. Subsequently, a human
trophoblast cell line will be used as a model to further investigate the molecular
mechanism Mst3 in the hypoxia-induce trophoblast apoptosis. The results from these
studies may reveal, at least partially, the role and molecular mechanism of Mst3 in
NSC97-2311-B-009-001-MY3 (97R164)

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