Project Number :

							Project Number :              1
Supervisor :                  Prof. Ian Marison
Office :                      Room X209
Literature survey :           Bioethanol production
Project :                     The application of liquid-core microcapsules for the in-situ
                              product recovery of ethanol from yeast fermentations.


Due to the continued reduction in oil supplies and the increasing costs of fossil fuels, there
is an increasing interest in white biotechnology, which is aimed at producing chemicals or
fuel replacements using durable renewable resources. Of the many different fossil fuel
alternatives ethanol has become increasingly explored, since it can be used in combustion
engines to levels of up to 20% without any modification to the engine. Furthermore it is
considered as CO2 neutral, thereby having no net effect on greenhouse gas emissions. In
order to produce so-called bio-ethanol, a cheap source of carbon and other nutrients is
required and a high productivity essential in order to minimize production costs.

All bioprocesses require the presence of high cell densities in order to achieve high
productivities. Furthermore continuous processes, particularly those with cell retention or
cell recycle provide the highest productivities. One way to achieve this is to immobilize the
yeast, or incorporate a cell recycle system, such that the majority of cells are retained during
continuous operation. However, ethanol is toxic to the yeast above a critical concentration.
Therefore in order to obtain high concentrations of ethanol it is necessary to use in-situ
product recovery techniques (ISPR). This can involve evaporation, liquid-liquid extraction,
adsorption or membrane-based techniques such as transmembrane distillation, pervaporation
etc.
In this project a series of alcohol tolerant Saccharomyces cerevisiae yeast strains will be
grown on a defined medium (to facilitate the work) under batch conditions and the growth
and alcohol production kinetics determined. This should also yield information about the
inhibition constants with respect to alcohol. Continuous suspension cultures will then be
undertaken and the kinetics re-determined. Finally, the continuous cultures will be repeated
at a specific dilution rate and liquid-core microcapsules added to the bioreactor in order to
continuously remove the ethanol, thereby overcoming the toxicity. The amount of
microcapsules necessary to remove the ethanol at a rate at least equal to the formation rate
will be determined. The microcapsule size, shape, composition and mechanical resistance
will be optimized. This is a highly novel application of microcapsules for in-situ product
recovery.


Project Number :              2
Supervisor :                  Prof. Ian Marison
Office :                      Room X209
Literature survey :           Production of recombinant proteins using methylotrophic
                              yeasts
Project :                     Production of recombinant avidine using fed-batch
                              cultures of Pichia pastoris


Many systems exist for the production of recombinant proteins using bacterial, yeast and
animal cell systems. Bacterial systems are frequently preferred due to the high levels of
expression and high productivities. However, the downstream processing to recover the
active, intact protein at high purity is often limited by the accumulation of the protein as
inclusion bodies, resulting in the need for cell breakage and protein denaturation/
renaturation which usually results in significant reduction in protein recovery. Animal cell
systems are clearly only useful for proteins which have specific properties or require post-
translational modifications such as glycosylation.
Yeasts are becoming increasingly used for therapeutic and analytical protein production
since they combine the high growth rates, high productivities, ability to secrete and perform
a wide range of post-translational events and, in particular to result in protein concentrations
of >1 g/L. Of all the yeasts employed P. pastoris, a methylotrophic yeast, is becoming
increasingly popular due to the ability to induce protein formation using the highly active
AOX1 promoter (alcohol oxidase) and the addition of methanol. Under standard protocols,
P.pastoris is grown on a multi-stage fed-batch culture, first on glycerol to achieve high
biomass densities, followed by fed- batch growth on methanol to achieve induction and
protein expression at reduced growth rates.
The project will involve setting-up a bioprocess to optimize the growth conditions on both
glycerol and methanol in stirred tank bioreactors. Initially this will involve batch cultures on
each substrate, followed by mixed substrates in order to determine whether glycerol is a
catabolite repressor, the optimum methanol concentration for induction and the toxicity
level of methanol. These will be then be followed by fed- batch cultures on glycerol and
methanol, in which the feed rates will be varied and the effect on growth and protein
production determined. The aim will then be to measure as many culture parameters as
possible in order to determine growth and production kinetics as well as to enable mass and
energy balances to be formulated.


Project Number         3
Supervisor             Dr. Greg Foley
Office                 X-203
Literature Review      Processing of cheese whey
Project                Design of an integrated milk processing plant using Superpro
                       Designer


Process design is the conceptual work done prior to building, expanding or retrofitting a
process plant. Nowadays, process design is usually done with the aid of process simulators.
These are sophisticated software tools that enable the user to readily represent and analyze
integrated processes and have been in use in the petrochemical industries since the early
1960s.
SuperPro Designer is a process
simulation package that has
been developed specifically for
batch bioprocesses. SuperPro
handles material and energy
balances, equipment sizing and
costing, economic evaluation,
environmental             impact
assessment,              process
scheduling,        and       de-
bottlenecking. It is widely used
in industry, especially in
companies like Wyeth where
efficient scheduling of the
production process can have               Figure 1 A simple two-unit process in Superpro
huge economic benefits.

The aim of this project will be to design, analyse, schedule and cost a pilot scale plant to
process skim milk with the aim of producing a range of novel milk products including low
carbohydrate milk, milk protein concentration (MPC) and glucose. The possibility of using
the whey to produce ethanol will also be examined. In your design, you will consider all
aspects of the process including scheduling, equipment operation and cleaning, and waste
minimisation. You will assume that the equipment available is similar to what is available in
DCU. The ultimate aim of the project will be to produce a sample design that can be used in
future years for teaching bioprocess design.


Project Number         4
Supervisor             Greg Foley
Office                 X-203
Literature Survey      Industrial diafiltration processes
Project                Minimisation of ultrafiltration-diafiltration process cost using
                       mathematical modelling

The classic approach to concentrating a macrosolute (protein) and diafiltering a microsolute
(salt) is to use batch ultrafiltration (UF) with constant volume diafiltration (CVD), where the
latter is performed at the optimum concentration, Cg/e. Diafiltering at this concentration
minimises the total process time for the case where the rejection coefficient, , of the
protein is equal to 1.

In many real situations, the protein may have <1. This means that some of the protein will
pass through the membrane and be lost in the permeate. We have shown that in this case, the
calculation of the optimum concentration is a bit more complicated and requires some
numerical integration [1]. The aim of this project will be to extend our analysis of the  < 1
case to find the dialfiltration concentration that will minimise the total process cost – a
problem that has not been addressed before. The process cost will be given by an expression
of the form:

 Cost  k1  total process time  k2   water used in diafiltration step   k3   solute lost 
where the constants k1 would be determined by pumping costs, k2 by the unit water cost and
k3 by the value of the lost product. This problem is thus more involved, and more
interesting, than simply minimising the process time

The aim of this project will be to set up the relevant equations on the computer and
investigate (i) how the optimum concentration differs from the classic Cg/e value and (ii)
how varying the rejection coefficient affects the optimum. The problem can be solved using
Excel and a web-based applet for doing numerical integrations. Part of the project will
involve doing some research to find realistic cost data. The project will suit someone with
an interest in engineering analysis and the use of computers.

1. G. Foley (1999) Minimisation of process time in ultrafiltration and continuous
diafiltration: the effect of incomplete macrosolute rejection. J. Memb. Sci. 163 (2), 349-355.



Project Number    5
Title             Modelling of product transmission in filtration processes
Literature Review The method of lines and its use in solving chemical engineering
                  problems
Supervisor        Greg Foley
Office            X-203

A very interesting problem that arises in membrane filtration processes is the transmission
of soluble products through the membrane. From a practical point of view, this problem is
very important because good product transmission is essentially for efficient product
recovery. When recovering a soluble product from, say, a cell suspension, the product can
adsorb to the membrane and/or the filter cake. As well as being of practical importance, this
is a an interesting theoretical problem because, in any filtration process, the cake thickness
increases with time. This is unlike classic adsorption process such as chromatography where
the bed length is fixed.

As a first step in trying to understanding the mechanism of product transmission in filtration
processes, this project will examine a simplified problem, namely, diffusion of a solute
through a combination of a membrane and a filter cake that is increasing over time in a
dead-end process. The project will involve formulation of the problem mathematically and
solution of the equations using finite difference and method of lines techniques. The
solution will be implemented using the software package Berkeley Madonna. This is an
easy-to-use package for the solution of ordinary differential equations and is used
extensively in bioprocess modelling.

The literature survey section of this project will give you time to familiarise yourself with
some of the mathematical aspects of the work. Nonetheless the project would suit someone
with an interest in engineering analysis. It should be noted that this project has excellent
potential to be extended at a postgraduate level and would ideal for someone wishing to
purse and MSc/PhD in bioprocess engineering and wishing to get some initial experience in
numerical methods.
Project Number:                6
Supervisor:                    Dr. Thecla Ryan
Office:                        Room X207
Literature Survey:             Genome Evolution in Mammals: Alternative gene splicing
                               and its consequences
Project:                       Unravelling the complexities of tissue specific gene
                               transcripts and splicing patterns


Spring of 2000 found molecular biologists placing bets trying to predict the number of
genes that would be found in the human genome when the sequencing of its nucleotides was
completed. Estimates at the time ranged as high as 153,000. After all, since humans are
known to make in the range of 90,000 different types of proteins, so we would have
expected to have at least as many genes to encode them. And given our complexity, we
ought to have a bigger genetic assortment than the 1,000-cell roundworm, Caenorhabditis
elegans, which has a 19,500-gene complement, or corn, with its 40,000 genes.
With the publication of first draft of the human sequence, the sequencing team announced
their calculated estimate of 30,000 to 35,000 protein-coding genes for humans. This
estimate has been revised downward still further in the intervening years to fewer that
25,000.
How then do we produce such a variety of different proteins, often in a tissue specific
manner, from so few genes? It now appears that the old axiom “one gene, one protein” no
longer holds true. Rather, the more complex an organism, the more likely it became that
way by extracting multiple protein meanings from individual genes. These
different/alternative expression products (of a single gene) are called isoforms.
It is now becoming clear that, in addition to differential promoter usage, differential RNA
processing events can result in a large number of different isoforms. Recent estimates
suggest that as many as three quarters of all human genes are likely to be subject to
alternative splicing. Furthermore the prevalence of alternative splicing appears to increase
with an organism’s complexity. In addition, scientists are also beginning to understand how
faulty gene splicing can result in cancer and congenital diseases as well as how splicing
mechanisms can be used therapeutically.
Taking a genomics approach, this project will attempt to document some of these events at a
molecular level, for a few selected genes. Starting with a particular gene, (i) the gene
structure (with repect to exons, introns, control regions, etc) will have to be mapped, and (ii)
the gene family data will need to be documented. Following this, both natural (tissue
specific and developmental) and aberrant (if filed) transcripts will be analysed and mapped.
Using this approach it should be possible to identify the mechanism(s) by which each
individual isoform was created.




Project Number:                7
Supervisor:                    Dr. Michael Parkinson/ Dr Greg Foley
Office:                        Room X224/ X203
Literature Survey:             Dynamic methods for measuring settling.
Project:                       Characterisation of settling using a multi-sensor array
Settling is an important industrial process, and modelling of the rate of settling has been
developed using greatly simplified models based on single point determinations of the
interface. However, until recently, there have been few methods to determine settling
dynamically at a number of points. We have constructed a multi-sensor array based on a
light sensor/ detector array that simultaneously measures cell density at 16 levels in a
column of settling solids, and shown that the settling is a rather more complicated
phenomenon than the simplified model would suggest. This project will examine the settling
of a range of solids under a variety of conditions to explore the capability of the settling
apparatus, and help to refine models of settling.
The project builds on previously unpublished work in our laboratory, which has developed
the settling apparatus, and carried out preliminary work into the kinetics of settling. It is
expected that the results of this work will provide the basis for a scientific publication.

Project Number:               8
Supervisor:                   Dr. Michael Parkinson
Office:                       Room X224
Literature Survey:            Methods for measuring growth of plant cell cultures.
Project:                      Characterisation of a multi-sensor micro-plate array for
                              measuring the growth of plant cell cultures.

Plant cell cultures are a potentially valuable source of a number of important
pharmaceuticals, and may be used for phenomenally efficient micropropagation of a number
of crop species through embryo culture. For all these activities, it is necessary to measure
growth of the cultures. A wide variety of methods have been employed, however, the most
effective and efficient methods would be simple, cheap, rapid, automatic, non-destructive
and non-invasive, and few if any existing methods meet these criteria. In my laboratory we
have constructed a micro-sensor array that is capable of simultaneously monitoring the
growth of up to 24 cultures to a high degree of accuracy and sensitivity. In this project, you
will use the array for measuring the growth of a range of cultures and define the sensitivity,
specificity and range of the method.
The project builds on previously unpublished work in our laboratory, which has developed
the multi-sensor array, and carried out preliminary work into monitoring of plant cell
cultures. It is expected that the results of this work will provide the basis for a scientific
publication.


Project Number:               9
Supervisor:                   Dr. John Tobin
Office:                       Room X216
Literature Survey:            Fate of antibiotics released into the environment
Project:                      Characterisation of the interactions of microbial biomass
                              with antibiotics in solution.

Antibiotics have emerged as a cause of growing concern in recent years because of their
potential as environmental pollutants. A range of antibiotics has been detected in both
sewage treatment plant (STP) effluents and surface water in the US and Europe. This
suggests that current STP treatments are insufficient in the removal of these products and
that there is a necessity for alternative methods to reduce their environmental loading.
Furthermore, it has been suggested that significant fractions of the antibiotic loading may
end up associated with the activated sludge solids rather than being biodegraded. This in
turn may lead to problems where land-spreading of sludge is practised (such as in Ireland).
In some European countries, Switzerland for example, this practice is currently banned for
environmental reasons.
Adsorption is a well-established technique for the treatment of domestic and industrial
wastewaters with activated carbon being the most effectively used adsorbent for dye
removal. However, the surface properties of many bacteria, yeast, fungi and algae enable
them to adsorb organic and inorganic compounds from solution. In previous work in our
laboratory*, Rhizopus arrhizus biomass has been shown to have the high sorption capacities
for both metal ions and textile dyes in aqueous solutions.
The aim of this project is to characterise the adsorption potential of Rhizopus arrhizus and
activated sludge biomasses for the removal of antibiotics from aqueous solutions. Process
parameters including pH, temperature and initial concentration are to be optimised.
Experimental work will include setting up batch antibiotic/biomass contacting systems and
antibiotic analysis by HPLC. The results will elucidate both the potential for microbial
adsorption systems for antibiotic removal and also the risks posed by agricultural use of
sludges.

      T. O’Mahony, E.Guibal and J.M. Tobin (2002) ‘Reactive dye biosorption by Rhizopus arrhizus
       biomass’, Enzyme Microb. Technol. 31(4), 456-463




Project Number:           10
Supervisor:               Dr. John Tobin
Office:                   Room X216
Literature Survey:        Yeast fermentations for citric acid production.
Project:                  Optimisation and scale up of citric acid production by
                          fermentation

         Citric acid is one of the most commonly used acids in the food and pharmaceutical
industries on account of its high solubility, palatability and low toxicity. Although it can be
extracted from citrus fruits, it is principally produced by submerged fungal fermentation
processes - notably using Aspergillus niger. However, in cultures of filamentous fungi the
growth of interlocking hyphae may result in high viscosity fermentation broths and
consequent mass transfer and operational problems. Hence, for efficient citric acid
production, growth of the Aspergillus in pellet form is desirable and this can be achieved by
process optimisation.
Recently, a number of alternative organisms have been identified as potential high yield
citric acid producers. These include the yeasts Yarrowia lipolytica and Y. guilliermondii.
Various carbon sources may be used for citric acid fermentation. For commercial reasons
the use of molasses, sucrose or glucose syrups is favoured. The use of molasses in particular
is desirable because of its availability and low cost.
Considerable research effort has been expended in developing the citric acid production
protocol. However, because it is a high volume, low value product, commercial profitability
is dependent on keeping technical and scientific advances proprietary. Consequently,
industrial developments in the citric acid production techniques are closely guarded by
confidentiality agreements.
Factors which affect biomass growth and citric acid yields are many and include: substrate
and nitrogen concentrations, pH, dissolved oxygen and cation (especially Fe2+, Mn2+ and
Cu2+) levels of the medium. Recently we have developed protocols for citric acid production
in the School of Biotechnology pilot plant and are producing appreciable citric acid
concentrations (g/L) in both shake flasks and 10L fermenters.
The aims of this project are to optimise protocols for the production of citric acid by Y
lipolytica fermentation and:
     to scale up production to the Schools 10L computer-controlled Biostat fermenter
        systems. Citric acid will be assayed using standard kit assays. Cell and product
        concentrations profiles will be determined and optimised for various media
        (including molasses) and growth conditions
     to develop a protocol for the production of citric acid by fermentation as a future
        BT4 Bioprocessing laboratory process.




Project Number:               11
Supervisor:                   Dr. John Tobin
Office:                       Room X216
Literature Survey:            Modelling metal removal by biosorption in packed bed
                              reactors.
Project:                      Metal removal by peat biomass in continuous flow
                              columns: studies and modelling

  Adsorption is well-known and widely used industrial separation in which varying species
are selectively bound on the surface of solid. Previous batch studies in our laboratory have
shown that peat moss is a potentially useful biosorbent for treating metal-contaminated
wastewaters. In process applications however, a fixed-bed column is preferable as an
effective process for cyclic sorption/desorption. Beads of peat are easily prepared from
commercially available raw materials, are stable in strong acid solutions and can be utilised
in conventional hydrometallurgical processing equipment. The sorption mechanism is
reversible and metal ions can be eluted from the beads with dilute acids.
The purpose of this project is to continue and complete a recent study in our laboratory.
Laboratory scale packed beds containing peat biomass will be set up and used to determine
the metal removal characteristics when single metal and multi-metal solutions are pumped
through the beds. Metal analysis will be by atomic absorption spectroscopy. A key focus
will be on the mathematical modelling of the metal biosorption process. We have recently
adapted existing mathematical models to provide a simplified method to predict the
performance of such peat beds**. This project will continue and develop that investigation.
Consequently an interest in modelling and computational methods will be an advantage in
this project. It is expected that the experimental results combined with the modelling
component will provide the basis for a scientific publication.

** M. Wei and J.M. Tobin (2004) ‘Determination and modelling of effects of pH on peat
biosorption of chromium, copper and cadmium’ Biochemical Engineering Journal, 18(1), 33-40.

M. Wei and J.M. Tobin (2003) ‘Development of multi-metal binding model and application to
binary metal biosorption to peat biomass’ Water Research 37(16), 3967-3977.
Project Number:       12
Supervisor:           Dr. John Tobin & Dr. Anne Morrissey
Literature Review:    Measuring antibiotics in the environment
Title :               Optimisation of polarograph for antibiotics analysis
Office Number         X216 & X 210

Trace analysis or the measurement of low concentration levels of key elements or
compounds is of fundamental importance in all aspects of biotechnology and biological
sciences. In industry, and particularly biotechnology industries, routine and rapid
measurement of concentration levels down to parts per billion (ppb) is frequently required.
Analysis based on electrochemical techniques is the method of choice. The School has an
EG&G pc-driven electrochemical analytical system. The item is a versatile and powerful
analytical system that allows measurement of both elements (including most metal ions) and
organic compounds at trace levels in matrices ranging from aqueous media, foodstuffs, plant
and animal tissue to soils and sediments. The core of the system is a polarographic analyser
that is controlled by a personal computer using Windows-based software. The software
allows both analytical methods and data processing routines to be developed and stored.
This project involves optimisation and preparation of new standard operating procedures for
the unit for use in determination of trace levels of antibiotics.
Many of the most common drug types (for example antibiotics) are used in quantities
similar to those of many agrochemicals but they are not required to undergo the same level
of testing for possible environmental effects. Consequently, they and their metabolites have
been subject to many years of unrestricted emissions, primarily into water bodies, and until
very recently little has been known about their environmental levels and the resulting
impacts of living systems. In the last five years new research in the EU and US has
quantified the extent of the problem in selected river/water systems and shown that
significant environmental impacts are occurring. A current research project in our laboratory
is investigating both pharmaceutical residue levels and their consequences in Irish water
systems. This project will feed into that EPA-funded study.

The aims of this work are to

       to optimise the system for analysis of selected common antibiotics (e.g. clofibric
        acid, and selected basic compounds). This is to be done in pure solutions and also in
        multicomponent solutions
       to prepare standard operating protocols for polarograph use for antibiotic
        determination

The work will be undertaken in close consultation with technical and academic staff. The
project represents an exciting opportunity to become familiar with and validate advanced
analytical systems, and to design and implement operating procedures. An interest in
environmental biotechnology and instrumentation/analytical systems would be of benefit in
this work.
Project No.        13
Title :            Evaluation of the government’s strategy on biodegradable waste
                   management
Literature Review: The environmental impact of biodegradable waste in landfills
Supervisor:        Dr. Anne Morrissey
Office Number      X 210

Project Description:

Government policy on waste management has changed dramatically over the last 10 years.
Prior to the publication of the Waste Management Act in 1996, Ireland relied almost totally
on landfill for the disposal of municipal waste. The latest statistics from the Environmental
Protection Agency show that while Ireland’s dependence on landfill is decreasing, 87% of
the household and commercial waste collected in Ireland still ends up in landfill compared
to 32% in Sweden, 12% in Denmark and 13% in the Netherlands. In addition, the number of
landfills in Ireland is decreasing, with 87 active local authority landfills in 1995, compared
to less than 50 local authority landfills in operation today. Ireland is coming under
increasing pressure from the European Union to meet recycling and recovery targets (e.g.
the packaging waste, waste electronic equipment and landfill directives). In particular, the
EU Landfill Directive requires reductions in the rate of biodegradable waste going to
landfill to 75% of 1995 levels by 2016. The Department of the Environment, Heritage and
Local Government published a draft strategy on the management of biodegradable waste in
2004, but so far, the strategy has not been finalised. The objective of this project is to
evaluate the effectiveness of the proposed strategy and to compare it to strategies in other
EU countries. This work will involve questionnaires, surveys and a lot of legwork.
Project No.        14
Title :            An environmental audit of the activities of the school of
                   Biotechnology
Literature Review: A review of Strategic Environmental Assessment
Supervisor:        Dr. Anne Morrissey
Office Number      X 210

Project Description:

Environmental Impact Assessment (EIA) is the term applied to the systematic examination
of the likely impacts of development proposals on the environment prior to the beginning of
any activity, whereas an environmental audit is carried out on a set of activities or
programmes once they are up and running to determine its environmental performance.
There are currently a number of international standards, (ISO 14010 – 14012), which
provide guidance on environmental auditing. This audit normally features a comprehensive
identification and evaluation of all the significant environmental impacts and issues relevant
to the organisation’s activities at the site. Audits are normally conducted via site surveys,
discussions with relevant site personnel and other third parties as relevant. Depending on the
site requirements, environmental monitoring of, for example, surface water, groundwater,
soil or air may be conducted. Nowadays, computer tools are available to assist with the EIA
and auditing process. One of these is EnviroPro Designer, a subset of SuperPro designer.
EnviroPro Designer is an environmental process simulator designed to enhance the
productivity of engineers and scientists engaged in the design, development, and assessment
of integrated water purification, wastewater treatment, and waste disposal processes. It
enables the user to efficiently develop, assess, and optimise environmentally beneficial
technologies. Further details of this software can be found at http://www.intelligen.com/

In this project you will use EnviroPro Designer software to carry out an environmental audit
of the activities of the school of Biotechnology. The output will be a set of
recommendations for preventing waste being produced in the first place and for reducing the
amount of waste that is produced. This project is related to Project XXX (Dr. Greg Foley).
The two students who select these projects are encouraged to work together.
Project No.           15
Title :               The management of biological waste at Dublin City University
Literature Review:    Biological waste treatment options
Supervisor:           Dr. Anne Morrissey
Office Number         X 210

Project Description
Dublin City University, like other organisations must comply with the legislative
requirements governing the disposal of waste. The responsibility for the waste management
programme lies with the Estates Office. At present, the university generates in the region of
24 tonnes of waste per month, of which approximately 80% can be recycled. Procedures are
currently in place to recycle waste streams such as paper, glass bottles, litter and aluminium
cans. The recycling of biological waste (e.g. garden waste, restaurant waste) is next on the
list for investigation. Preliminary figures show that DCU generates about 2.5 tonnes of
biological waste per month.

The objective of this project is in two stages.
 The first stage, or literature review stage, is to examine what methods are available for
   managing and treating biological waste in general. These could include composting,
   vermiculture, biological treatment etc. The literature review will be carried out in
   semester one.

   The second stage of the project, will be carried out in semester 2 and will involve
    examining what method of biological waste treatment would be suitable for DCU.

Both stages of the project will involve working very closely with the Estates office.
Project No.        16
Title :            The development of a standard operating procedure for the detection
                   of pharmaceuticals in water using HPLC
Literature Review: High Performance Liquid Chromatography
Supervisor:        Dr. Anne Morrissey and Dr. John Tobin
Office Number      X 210


In recent years the occurrence and effects of pharmaceuticals and related chemicals in the
aquatic environment have emerged as a new and growing environmental concern. A
multitude of persistent organic compounds, including active ingredients in pharmaceuticals
and personal care products, which are used in large quantities throughout the world enter
wastewaters daily. Of these, medicines form the largest sub group, of which there are in
excess of three thousand substances. While these compounds are partly degraded by the
body, thousands of tons still enter the environment each year by excretion in an uncontrolled
way. It is only in the last five years new research in the EU and US has quantified the extent
of the problem in selected river/water systems and shown that significant environmental
impacts are occurring.

The aim of this project will be to develop and optimise a standard operating procedure for
the detection and quantification of both acid and basic pharmaceutical compounds using
High Performance Liquid Chromatography (HPLC). HPLC is a method of analysis used
extensively in both biotechnology and chemical industries and due to its versatility and
ability to separate and quantify simultaneously, it is a suitable method for the detection of
pharmaceuticals in environmental matrices.



Project Number:                17
Supervisor:                    Dr. Patricia Johnson
Office:                        Room X204
Literature Survey:             Influenza Virus Predisposing for Bacterial Disease
Project:                       Influenza Modulation of Dendritic Cell Response to
                               Bacterial Lippopolysaccharide (LPS)

Influenza virus causes yearly epidemics of respiratory illness worldwide and is one of the
most prevalent causes of hospitalisation for acute respiratory disease.          Bacterial
superinfections are a major cause of morbidity and mortality in influenza infections and
while the host appears to be immunosuppressed during influenza infection, the mechanisms
underlying this predisposition to bacterial infection remain poorly defined.

Dendritic cells are potent antigen presenting cells, which drive T cell responses to infection.
How these cells mature in response to infection can often dictate the clinical outcome.

In order to examine further the role of these cells in the increased susceptibility to bacterial
superinfection during influenza viral infection this project will investigate whether influenza
virus can modulate dendritic cell maturation and cytokine production in response to
bacterial LPS.
Project Number:               18
Supervisor:                   Dr. Michael O’Connell
Office:                       Room X212
Literature Survey:            Interactions between regulatory proteins and promoters
Project:                      Investigation of the rhtX promoter region of Sinorhizobium
                              meliloti by site directed mutagenesis
Recently the RhtX permease was identified by my research group as the primary
representative of a novel transport mechanism for iron in bacteria (O Cuiv et al, Journal of
Bacteriology, April 2004, available on line). It is of interest to understand the system by
which the gene, rhtX, encoding this protein is regulated, in particular by iron. Using a
random mutagenesis method we have identified a number of bases that influence the
regulation of the promoter. However, the random mutagenesis technique that we used
always resulted in multiple mutations. We now plan to remake the mutations individually by
site directed mutagenesis and in this way to ascertain which of the individual base changes
is causing the mutant phenotype. The project will involve the design of PCR primers to
introduce the appropriate base changes, use of PCR to introduce the base changes
individually into a plasmid carrying the rhtX promoter linked to a reporter gene, the transfer
of the individually mutated plasmids to the S.meliloti host by bacterial conjugation and an
assessment of the phenotype resulting from the individual mutations by monitoring the
reporter gene.



Project No.                   19

Supervisor :                  Brendan O’Connor
Office :                      Block X , Room X2-22
Literature Survey :           Biopharma – new generation of drugs

Project :                     A study of a prokaryotic ‘glycoprotein-binding’ lectin.

The fundamental challenge facing the global biopharmaceutical industry to-day is the
development of effective and efficient new bioanalytical tools to be able to cope with the
dramatic change from ‘traditional’ chemical drugs to biochemical drug molecules. Over
80% of new Pharaceutical products in final phase 3 testing are now known to be
Glycoproteins.
Consequently, the analytical tools required to effectively monitor and understand the
dynamics of molecular changes occurring in these systems must be faster , more powerful
and sophisticated than the conventional methods used in the chemical pharmaceutical
industry.
In this project (being run in conjunction with Britol-Myers-Squibb and the newly
established Center for Bioanalytical Sciences CBAS), we will specifically focus on the
preliminary investigation of the sheer diversity of lectin biorecognition of glycoproteins.
Lectins are cell ‘agglutinating’ and ‘sugar-specific’ proteins. They have been shown to
occur widely in plants , animals and microorganisms. They have been found to be extremely
useful tools for the investigation of carbohydrates on cell surfaces, in particular of the
changes that occur in malignancy, as well as for the isolation and characterization of
glycoproteins.
Our group has already identified a number of novel lectins with very important specific
‘glycoprotein’ binding patterns The project will involve the selection of certain procaryotic
lectins followed by their cloning, expression & production. The use of expression vectors in
a bacterial background will enable us to produce large amounts of recombinant tagged lectin
in a manner that facilitates highly efficient and easy chromatographic purification. It is also
hoped to manipulate/mutate these new recombinant lectins. The genetic basis for the
production of many of these lectins is well known and they are amenable to large scale
production in bacterial backgrounds. In the short term the project will be directed towards
investigating lectins of P. aeruginosa and Ralstonia solanacearum.



Project Number:    20
Supervisor:        Ciarán Fagan
Office:            Room X2.21
Literature Survey: Trypsin: a model serine proteinase with notable applications.
Project:            Cloning and expression of recombinant pig trypsin.

Trypsin is a well-described serine proteinase that cleaves peptide bonds at Arg and Lys
residues and has uses in protein chemistry, cell culture, insulin production and peptide
synthesis. Recombinant trypsins have been cloned into E. coli, Aspergillus sp. and
transgenic maize Zea mays and the enzyme has been mutated. Unfortunately, trypsin is
prone to autolysis following activation from the zymogen and is not very thermostable. To
counter these drawbacks, it has been subjected to immobilization and chemical modification
procedures. Your review should describe the structure, function and applications of trypsin.
Comparisons of different trypsin sequences and structures in the various databases should
be included, using bioinformatic tools.
        Our laboratory has published papers (e.g. Biotech Bioeng, 1998, 58, 365-373), and
produced 2 PhDs (1996, 2003), on peptide synthesis by chemically-stabilized trypsin. We
wish to extend this work into protein engineering of recombinant trypsin. Recently we took
delivery of a synthetic pig trypsin gene in an E. coli cloning vector. The project will involve
PCR-based cloning of the trypsin gene into a convenient expression vector derived from
pQE60. The proposed vector contains a sequence directing the inserted recombinant gene to
the bacterial periplasm, avoiding the problem of inclusion body formation. The vector also
attaches a hexa-His tag to the protein’s C-terminus, allowing convenient, one-step
purification by nickel affinity chromatography. Screening of clones for trypsin activity is
conveniently done on milk agar plates, where a clear zone (due to digestion of milk protein
by secreted trypsin) indicates positive colonies. Optimization of host cell growth and trypsin
production will take place once re-cloning has been accomplished. This project, which will
be of real long-term value to the host laboratory, should be very satisfying and rewarding
for someone seeking to gain skills in cloning/ expression, enzyme assay and protein
purification.
Project Number:                21
Supervisors:                   Ciarán Fagan & Michael Parkinson
Office:                        Room X2.21 & X2.24
Literature Survey:             Enzyme stability and inactivation under bioprocessing
                               conditions.
Project:                       Use of a ramped temperature method to measure enzyme
                               stability.

The stability of proteins in use or during storage is often a critical attribute in biotechnology.
Indices of protein stability include folding (conformational) stability (Tm and G) and
kinetic (long-term) stability (half-life). These parameters may not correlate well with actual
biocatalyst performance under process conditions. Your literature survey should describe
and contrast the main parameters for protein stability measurement. Pay special attention to
models of biocatalyst performance/ inactivation in industrial situations and to material
published in the past 5 years.
      A paper by M. Boy et al. (Process Biochem 1999, 34: 535-547) outlines an
experimental strategy that allows the measurement of relevant biocatalyst stability
parameters in a single experiment. The method involves an initial constant-temperature
incubation followed by programmed steady heating to a high inactivating temperature. This
interesting method has the potential to save time and materials. The project will apply this
method to trypsin by using a pH-stat to follow the degradation of albumin. Experimental
data fitting to models outlined in the paper will be a key element of the project, in order to
derive the appropriate descriptive equation and values for particular data sets. Data
processing using Sigma Plot will be as important as the experiments themselves. Someone
with an interest in process description and modelling will find this project very satisfying.


Project Number:              22
Supervisor: Dr Donal O'Shea
Room: X220
Literature Review: In-situ product recovery (ISPR) from fermentation broths.
Project: – In-Situ Peristraction of Geldanamycin from Fermentation Broths


The antibiotic geldanamycin is a benzoquinone ansamycin produced as a secondary
metabolite by the Actinomycete Streptomyces hygroscopicus var. geldanus in submerged
culture. Geldanamycin is currently under investigation for its anticancer properties through
its interactions with Heat Shock Protein 90 (Hsp90), and it also possesses a broad spectrum
antibiotic activity. Hsp90 is a ubiquitous protein, present in the cytosol of both Eukaryotic
and Prokaryotic cells. It plays the role of “molecular chaperone”, binding and stabilising
proteins, aiding their assembly and transport across membranes. Geldanamycin binds with a
high affinity to the ATP binding pocket of Hsp90 resulting in cancer-causing proteins being
left malformed, and thus leaving them readily degradable by the cells own mechanisms.

Geldanamycin is a low molecular weight hydrophobic compound which is both
thermolabile and photolabile.     It readily degrades in fermentation and as such,
methodologies of recovering the compound from the fermentation broth, while production is
ongoing, are desirable. The use of ionic and hydrophobic resins in conjunction with low
molecular weight cutoff membranes is currently being investigated as a means of effectively
conducting ISPR in this process. This project will further develop this work undertaken in
the laboratory and will require the student to apply fermentation and analytical skills
learned throughout the course.


Project Number:                23
Supervisor: Dr Donal O'Shea
Room: X220
Literature Review: Optimisation of fermentation processes.
Project – Optimisation of Fermentation Conditions for Geldanamycin Production
The antibiotic geldanamycin is a benzoquinone ansamycin produced as a secondary
metabolite by the Actinomycete Streptomyces hygroscopicus var. geldanus in submerged
culture. Geldanamycin is currently under investigation for its anticancer properties through
its interactions with Heat Shock Protein 90 (Hsp90), and it also possesses a broad spectrum
antibiotic activity. Hsp90 is a ubiquitous protein, present in the cytosol of both Eukaryotic
and Prokaryotic cells. It plays the role of “molecular chaperone”, binding and stabilising
proteins, aiding their assembly and transport across membranes. Geldanamycin binds with a
high affinity to the ATP binding pocket of Hsp90 resulting in cancer-causing proteins being
left malformed, and thus leaving them readily degradable by the cells own mechanisms.

Our laboratory has been involved in a multi-iteration optimisation process that has resulted
in a 1000 fold increase in fermentation yield, through altering growth media formulation. It
is proposed that further optimisation is feasible through stringent in-process environmental
control. Instrumented bioreactors can control environmental conditions such as shear and
dissolved oxygen tension. They are a significant step forward from the use of agitated shake
flasks, which is current practice in the laboratory. This project will call on students
experience in microbiology, bioreactor engineering and analytical techniques learned during
the course.


Project Number:                24
Supervisors:                   Prof. Richard O’Kennedy and Dr. Stephen Hearty
Office: X 206.                 Lab: X273

Literature Survey :             Current trends in antibody production and engineering.

Project:                       Production of recombinant antibodies against         Listeria
                               monocytogenes

Overview: Listeriosis is a severe disease caused by ingestion of contaminated food. In
2000, the Centres for Disease Control (CDC) reported that of all the foodborne pathogens
tracked by the CDC, L. monocytogenes had the second highest case fatality rate (21-30 %)
and the highest hospitalisation rate (90.5 %). It is also the fourth most common cause of
adult meningitis. Thus, early detection of L. monocytogenes contamination is of paramount
importance. Unfortunately current detection methods are neither specific enough nor ‘rapid’
enough for reliable analysis.
We have produced a monoclonal antibody that is specific for a L. monocytogenes
pathogenicity-associated marker. The key objective of this project will be to clone the gene
that encodes this antibody’s specificity and construct a recombinant form of the antibody
(scFv and Fab). This will allow ‘tailoring’ of the antibody’s binding affinity and also the
incorporation of various affinity tags and/or labels. The research will incorporate exposure
to a diverse range of classical and contemporary techniques that will include the following:
cell culture, RNA extraction, cDNA synthesis, PCR, cloning, phage display, affinity
chromatography, SDS PAGE, Western blotting, immunoassay development and SPR-based
biosensor analysis.


References:
Krebber, A., Bornhauser, S. Burmester, J. Honegger, A. Willuda, J. Bosshard, H.R.
Plückthun, A. (1997). Reliable cloning of functional antibody variable domains from
hybridomas and spleen cell repertoires employing a re-engineered phage display system. J.
Immunol. Methods, 201:35-55.
Leonard, P., Hearty, S., Wyatt, G., Quinn, J. and O’Kennedy, R. (2005). “Development of
a surface plasmon resonance-based immunoassay for Listeria monocytogenes.” J. Food.
Protect., 68(4), 728-735.


Project No.           25
Supervisor:           Dr Rosaleen Devery
Office:               X211

Literature Survey: Sphingolipids- Metabolism and function, cellular responses and
                   potential targets for cancer therapy
Project:           Quantitative determination of ceramide in
                   MDA-MB435-S-F breast cancer cells.

Sphingolipids play a major role in cell signalling pathways including cell differentiation,
proliferation and apoptosis. Of particular interest are sphingolipid mediators known as
sphingosine and ceramide. Ceramide is a well-established marker of apoptosis and can be
seen at elevated levels in response to numerous apoptotic inducers including non steroidal
anti-inflammatory drugs, cytokines and physical stress. The objective of this project will be
to establish a reproducible assay for ceramide quantification in cancer cells exposed to
cytotoxic fatty acids, including conjugated linoleic acids (CLA) and to determine if
sphingolipid signalling is involved in CLA-induced growth arrest. The techniques involved
in this project will include tissue culture and ceramide analysis by HPLC.
Project Number:       26
Supervisor:           Dr Christine Loscher
Office:               Room X218
Literature survey:    The role of co-stimulatory molecules in the cross talk between
                      dendritic cells and T cells
Project:              Effects of dietary fatty acids on expression of cell surface molecules
                      on dendritic cells


Dietary polyunsaturated fatty acids (PUFA) can modulate immune cell function and
therefore represent novel anti-inflammatory strategies to prevent or treat inflammatory
diseases such as inflammatory bowel disease and rheumatoid arthritis. These diseases are
associated with a dysregulated T helper cell type 1 (Th1) response. Inhibition of this Th1
response by anti-inflammatory molecules results in a decrease in inflammation and relief of
diseases symptoms. Th1 cells are generated from naïve T cells by activated immune cells
called dendritic cells (DC). The expression of cell surface molecules on these cells, are of
critical importance for the activation of Th1 cells.
Assessing the effects of PUFA on these surface molecules will provide us with important
information about how fatty acids may affect the interaction between DC and T cells and
whether this can lead to inhibition of Th1 responses.
This project will firstly determine the effects of PUFA on expression of cell surface
molecules in both resting and activated DC using immunofluorescence analysis/flow
cytometry techniques. It will then examine how PUFA may mediate this effect by assessing
the involvement of a number of cell signalling molecules. This will involve the use of
specific inhibitors to these signalling molecules aswell as SDS gel electrophoresis and
western blotting.