RACHEL TAM

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					                                       RACHEL TAM
PERSONAL DETAILS________________________________________

Email: rachel.tam@student.unsw.edu.au
Nationality: Australian and New Zealander

EDUCATION_____________________________________________

Doctor of Philosophy (2002 - 2007)
Department of Physiology and Pharmacology, School of Medical Sciences, University of New
South Wales, Sydney
Thesis Title: ‘Using Yeast as a Model System to Characterize Human Topoisomerase IIα and
Topoisomerase II Poisons.’

B. Science (Hons) in Pharmacology (2001)
The University of New South Wales, Sydney, Australia
Thesis Title: ‘In Vivo Detection of Trapped Cleavable Complexes by DNA Binding Topoisomerase II
Poisons.’
Achievement: Class II Division I Honours based on distinction average results

B. Science in Physiology and Pharmacology (2000)
The University of Auckland, New Zealand
Achievement: Credit average results

Rangitoto College, New Zealand (1997)
Subjects: Maths with Calculus, Maths with Statistics, Biology, Chemistry and Economics
Achievement: ‘A’ Grade in New Zealand Bursaries

RESEARCH EXPERIENCE____________________________________

Visiting PhD Candidate (Jan 2005 - Apr 2005)
Department of Biochemistry, Vanderbilt University, Nashville, TN, USA
Supervisor: Prof. Neil Osheroff
Techniques employed:
          Recombinant protein purification
          In vitro protein characterization

Doctor of Philosophy (2002 - 2007)
Department of Physiology and Pharmacology, School of Medical Sciences, University of New
South Wales, Sydney
Supervisors: A/Prof LPG Wakelin and Prof. Ian W Dawes
Thesis Title: ‘Using Yeast as a Model System to Characterize Human Topoisomerase IIα and
Topoisomerase II Poisons.’

Techniques employed:
          Bacterial cell culture
          Yeast Biology, yeast transformation techniques
          Yeast culture techniques
          Medicinal chemistry
          Clonogenic assay, drug-treated yeast transformants
          Yeast total protein isolation - glass beads method
          PCR techniques
          Recombinant DNA technology
          DNA sequencing techniques
          SDS protein gel, phast gel system and DNA agarose gel
          Western blotting
          Random and site-directed mutagenesis
          Recombinant protein purification
          In vitro protein characterization
          Computational protein modeling

Full details in attached appendix

Summer Research Scholar (2001 – 2002)
High Blood Pressure Research Unit, John Curtin School of Medical Research, Australian
National University, Canberra
Supervisor: Prof JA Whitworth and Dr. Christopher Schyvens
Project Title: ‘The Effect of Vitamin E on Glucocorticoid-Induced Hypertension.’

Techniques employed:
          Whole animal experimentation, animal handling, acclimatization and drug administration
          Blood pressure measurement by tail cuff method
          Harvesting of organs and tissues from rats
          Biochemical analysis, plasma and serum separation from whole blood
          Electrolyte determination of urine samples by flame photometry
          Determination of nitrate/nitrite levels by spectrophotometry
          Preparation of drug aliquots and data analysis, statistics analysis and presentation

Honours Student (2001)
Department of Physiology and Pharmacology, School of Medical Sciences, University of New
South Wales, Sydney
Supervisor: A/Prof LPG Wakelin
Thesis Title: ‘In Vivo Detection of Trapped Cleavable Complexes by DNA-Binding Topoisomerase II
Poisons.’

Techniques employed:
          Growth inhibition assay, drug-treated CCRF-CEM human leukaemia cell culture
          Preparation of CCRF-CEM cells lysis with Sarkosyl
          Ultracentrifugation, isolation of trapped cleavable complexes through CsCl step gradient
          Dot-blotting, applying the complexes onto nitrocellulose membrane
          Immunoassay, utilize monoclonal primary antibody, secondary antibody and horseradish
          peroxidase-mediated chemiluminescence detection
          Densitometry, quantify the trapping signals from western blots

Biomedical Research Summer Studentship (2000 - 2001)
Department of Surgery, Faculty of Medicine and Health Sciences, University of Auckland,
New Zealand
Supervisor: A/Prof Susan Stott
Project Title: ‘Gene Expression in Chrondrogenic Cell Culture.’


Techniques employed:
          PCR primer design
          RNA extraction from micromass culture and embryo chick limb buds
          Reverse transcription from RNA to cDNA
          RT-PCR and agarose gel electrophoresis, defining gene expression as the cells underwent
          differentiation
          Completed data collection on the expression of 10 different genes over the time course of
          the experiment (using four time points for each gene)

EMPLOYMENT HISTORY______________________________________

Freelance Web Site Designer (Aug 2005 – present)
Amok Production, Sydney, Australia

Freelance Wedding Photographer and Planner (Sept 2005 – present)
Amok Production, Sydney, Australia

Co-founder of Amok Production, Sydney, Australia (June 2005 – present)

Dissecting room technician (Apr 2002 - Aug 2002)
Department of Anatomy, University of New South Wales, Sydney

Customer Service Representative (Dec 1999 - Feb 2000)
Net Depot Ltd., Tsuen Wan, Hong Kong

Voter Registration Assistant (Dec 1998 - Feb 1999)
Hong Kong Voter Registration Section of the Registration and Electoral Office

Assistant Clerical Officer (Dec 1997 - Feb 1998)
Hong Kong Voter Registration Section of the Registration and Electoral Office

ADMINISTRATION EXPERIENCE_________________________________

Lab Manager (June 2003 - Jan 2005)
Duties:
        Managing supervisor and students
        Accounting
        Research reagents and lab consumables ordering
        Training users of tissue culture facilities, gel-doc systems, coulter counter technology and
        western blotting techniques
        Assisting students with technical difficulties with their research projects
        Arrange meetings with other lab members and sales representatives
TEACHING EXPERIENCE______________________________________

Co-supervisor (June 2004 - March 2005)
Conducting research supervision of a master of biopharmaceutical student, Miss Ansy Joseph

Demonstrator (Aug 2004 – present)
Fundamentals of Molecular Biology
Department of Biochemistry, University of New South Wales, Sydney


Co-supervisor (Mar 2003 - Nov 2003)
Conducting research supervision of an Honours student, Mr. Andre Serobian

Demonstrator (Aug 2003 - Nov 2003)
Genetics
Department of Biochemistry, University of New South Wales, Sydney

Demonstrator (Mar 2002 – June 2002)
Physiology
Department of Physiology and Pharmacology, University of New South Wales, Sydney

KEY SKILLS_____________________________________________

Computing
I am a proficient user of all Microsoft Office programs, Adobe Photoshop and Dreamweaver programs. I
am experienced in the use of advanced Internet search engines. I have extensive knowledge of a range
of bioinformatics software packages including Graphpad Prism, SPSS, Swissplot, Pymol, BioEdit,
Chemdraw and Accelrys Insight II.

Language
Fluent in English, Cantonese and Mandarin (both spoken and written)

SCHOLARSHIPS RECEIVED____________________________________

Postgraduate Travel Grant (2005)
School of Medical Sciences, University of New South Wales
Attended as the first and presenting author of a poster at the American Association for Cancer
Research’s 96th Annual Meeting, Anaheim, California, USA

Postgraduate Travel Grant (2004)
School of Medical Sciences, University of New South Wales
Attended as the first and presenting author of a poster at the 25th Annual Conference on the
Organisation and Expression of the Genome, Lorne, VIC, Australia

University of New South Wales Medicine Deans Scholarship (2002 - 2005)
School of Medical Sciences, University of New South Wales
Selection is based on academic achievement and potential research ability
The Australian National University Summer Research Scholarship (2001 - 2002)
Selection is based on academic achievement and potential research ability
Biomedical Research Summer Studentship (2000 - 2001)
The University of Auckland
Selection is based on academic achievement and potential research ability

CONFERENCE PRESENTATIONS_________________________________

          Tam, R., Suzuki, M., Denny, W.A., Dawes, I.W., Wakelin, L.P.G. (2005) Mutations Within
          the α4 Helix of Human DNA Topoisomerase IIα Confer Differential Sensitivities to
          Topoisomerase Poisons. Oral presentation at the Auckland Cancer Society Research Centre
          Symposium, Auckland, New Zealand, Nov. 2006

          Tam, R., Suzuki, M., Denny, W.A., Dawes, I.W., Wakelin, L.P.G. (2005) Mutations Within
          the α4 Helix of Human DNA Topoisomerase IIα Confer Differential Sensitivities to
          Topoisomerase Poisons. Poster presented at the American Association for Cancer
          Research’s 96th Annual Meeting, Anaheim, California, USA, Apr. 2005

          Tam, R., Denny, W.A., Dawes, I.W. and Wakelin, L.P.G. (2004). Saccharomyces
          Cerevisiae as an Eukaryotic Model System to Study the Actions of Human DNA
          Topoisomerase IIα/β-Targeted Drugs. Poster presented at the 25th Annual Conference on
          the Organization and Expression of the Genome, Lorne, VIC, Australia, Feb. 2004

PUBLICATIONS___________________________________________

   Tam, R., Denny, W.A. and Wakelin, L.P.G. (2007) Activity of Acridine-4-carboxamide
     Topoisomerase Poisons and Bis(9-aminoacridine-4-carboxamide) Threading Agents in Yeast
     Bearing Human Topo IIα and Topo IIβ. Submitted.

   Christopher G. Schyvens, Miles C. Andrews, Rachel Tam, Trevor A. Mori, Kevin D. Croft, Katja
      U.S. McKenzie, Judith A. Whitworth, Yi Zhang. (2007) Antioxidant Vitamins and
      Adrenocorticotrophic Hormone-Induced Hypertension in Rats. Accepted by Clinical and
      Experimental Hypertension.

   Tam, R., Byl, J.A.W., Suzuki, S., Dawes, I.W., Wakelin, L.P.G. and Osheroff, N. (2007) Mutations
     Within the α4 Helix of Human DNA Topoisomerase IIα Alters Topoisomerase II-Mediated DNA
     Cleavage. In process.

   Rachel Tam, Motoshi Suzuki, Ian W. Dawes, Neil Osheroff and Laurence P. G. Wakelin. (2007) A
      Mutation in Human Topoisomerase IIα That Confers Resistance to Multiple Classes of
      Topoisomerase II Poisons. In process.

   Rachel Tam and Laurence P. G. Wakelin. (2007) Mutations in DNA Topoisomerases II and
      Resistance to Anti-Cancer Drugs. Review. In process.

   Tam, R. (2007). Using Yeast as a Model System to Characterize Human Topoisomerase IIα and
     Topoisomerase II Poisons. PhD Thesis, UNSW, Sydney.

   Rachel N. Tam, Motoshi Suzuki, William A. Denny, Ian W. Dawes, Laurence P. G. Wakelin
      (2005). Mutations Within the α4 Helix of Human DNA Topoisomerase IIa Confer Differential
       Sensitivities to Topoisomerase Poisons. Abstract presented at the American Association for
       Cancer Research’s 96th Annual Meeting, Anaheim, California, USA.

   Tam, R., Denny, W.A., Dawes, I.W. and Wakelin, L.P.G. (2004). Saccharomyces Cerevisiae as an
     Eukaryotic Model System to Study the Actions of Human DNA Topoisomerase IIα/β-Targeted
     Drugs. Abstract presented at the 25th Annual Conference on the Organization and Expression
     of the Genome, Lorne, VIC, Australia.

   Tam, R. (2001). In Vivo Detection of Trapped Cleavable Complexes by DNA-Binding
     Topoisomerase II Poisons. Honours Thesis, UNSW, Sydney.

REFEREES______________________________________________

A/Prof. LPG Wakelin
School of Medical Sciences
University of New South Wales
Sydney 2052, Australia
Tel: (+612) 9385 2563
Fax: (+612) 9385 1059
Email: l.wakelin@unsw.edu.au

Prof. Ian W Dawes
University of New South Wales
Sydney 2052, Australia
Tel: (+612) 9385 2089
Fax: (+612) 9385 1059
Email: i.dawes@unsw.edu.au

Prof. Neil Osheroff
Neil Osheroff, Ph.D.
Professor of Biochemistry and Medicine
John G. Coniglio Chair in Biochemistry
Department of Biochemistry
654 Robinson Research Building
Vanderbilt University, TN, USA
Tel: 1-615-3322-4338
Fax: 1-615-343-1166
Email:neil.osheroff@vanderbilt.edu

RESEARCH ABSTRACT______________________________________

The DNA binding cytotoxins amsacrine, mitoxantrone and the acridine-4-carboxamides related to
DACA, function by poisoning DNA topoisomerase IIα. In this work, I explored the topoisomerase II
selectivity of acridine-4-carboxamides and investigated whether topoisomerase II activity is involved
in the mechanism of action of threading bis(9-aminoacridine-4-carboxamides), using the drug-
sensitive JN394 (MATa, ura3-52, leu2, trp1, his7, ade1-2, ISE2, rad52::LEU2) strain of
Saccharomyces cerevisiae. Its temperature-sensitive mutant JN394top2-4, wherein the ISE2
permeability mutation facilitates drug uptake and the rad52 mutation renders the yeast defective in
double-stranded DNA break repair. Whilst these compounds are known to intercalate into DNA and
trap the enzyme in its ternary complex, it is not clear whether they do so by interacting with the
same amino acid residues within the cleavage domain. I attempt to resolve this issue, since if the
compounds interact with different residues, this has implications for new drug discovery, as well as
for their clinical use, since it suggests that natural mutations conferring resistance on one drug may
leave tumours sensitive to another within these classes. In human topoisomerase IIα, amino acids
760 to 772 correspond to the α4 helix as seen in the yeast enzyme. This region is the
breakage/rejoining domain that interacts with DNA. We have examined a series of proline
substitutions, within, and near, the α4 helix in human topoisomerase IIα. We have discovered that
the T767P mutation confers 5-fold resistance upon the yeast to both amsacrine and AS-DACA at the
non-permissive temperature. In contrast, the mutation N774P shows an 8-to-10-fold hypersensitivity
to amsacrine and AS-DACA. Both mutants retain wild-type sensitivity to mitoxantrone. A third
mutant, N770P, exhibits 30-fold resistance to mitoxantrone and 5-fold resistance to AS-DACA but
retained wild-type sensitivity to amsacrine. These results demonstrated that mutations in positions
767, 770 and 774 in human topoisomerase IIα impart marked differences in sensitivity to a range of
drug type (an anilinoacridine, a 9-aminoacridine-4-carboxamide and an anthracenedione). I then
purified and characterized these mutants in vivo and found that T767P displays 3- to 5-fold lower
levels of DNA cleavage than did the wild-type enzyme while N774P shows 2- to 3-fold higher DNA
cleavage activity. T767P also confers 4- to 6- fold higher relative DNA cleavage activity in the
presence of etoposide which suggests that position 767 in human topoisomerase IIα may interact
with the drug in the DNA-topoisomerase cleavage complex.

				
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