Cell Culture Specialist, Biochemistry, Molecular and Cell Biology

RESUME

RUSUDAN KOTARIA, Ph.D.

Address: *** **** ********** ***. **** Bluff, IL 60044

Cell phone: 847-***-****

E-mail: adj4ys@r.postjobfree.com

Visa Status: USA Citizen

OBJECTIVE

Seeking position as a Cell Culture Specialist, Biochemistry, Molecular and Cell Biology, Protein Chemistry, Cystic Fibrosis Physiology and Biophysics, Cancer Cell Biology

PROFESSIONAL RECORDS

11.25.19-1/31/21 Research Associate, Center for Genetic Diseases, Rosalind

Franklin University of Medicine and Sciences/Chicago Medical

School, North Chicago, IL, 60064

2014-6/28/19 Research Associate, Department of Physiology and Biophysics,

Rosalind Franklin University of Medicine and

Science/Chicago Medical School, North Chicago, IL, 60064

2000-2014 Research Associate, Department of Biochemistry and

Molecular Biology, Rosalind Franklin University of Medicine and

Science/Chicago Medical School, North Chicago, IL, 60064

1998-2000Postdoctoral Fellow, Department of Biochemistry and molecular Biology, FUHS/Chicago Med. School, North Chicago, IL, 60064

1990-1998 Senior Research Associate, Department of Biophysics,

Tbilisi State University, Tbilisi, Georgia

Supervised Ph.D. and M.S. program students,

1996-1998Assistant Professor, Institute of Medicine, Tbilisi, Georgia

Taught General Physiology for first year Medical Students

EDUCATION

Ph.D. – Physiology/Radiobiology, Institute of Biophysics,

Moscow, Russia

M.S. - Biology and Chemistry, Physiology of Human Being and

Animal, University of Tbilisi, Tbilisi, Georgia

PROFESSIONAL EXPERIENCE

I‘ve been working with Antisense oligonucleotide (ASO) and small compounds for the treatment of cystic fibrosis, with primary HBE cell cultures derived from the bronchi of CF patients that are used for the routine lab testing of compound potency and efficiency; Using electrophysiological methods to study the regulation, pharmacology and biophysics of ion channels including CFTR; I developed successfully CFF-16HBEge W1282X mutant Single Cell Clones with High Resistance for Functional TECC Assay; I’ve also developed the method of culture primary Nasal cells derived by brushed from the CF patients. Nasal brushing of turbinate offers an attractive source of human airway cells that are used for developing new way for lab testing of compound potency and efficiency. For testing Cells Transfection with ASO, or Lipofectamine and analyzing CFTR cDNA Splicing and activity, RNA extraction, isolation, Reverse transcription and radiolabeled PCR were used.

I’ve worked also with 3T3 cells to produce Conditioned Media, which produces Growth Factor and helps Primary and transfected cells lines to grow well. I also improved: A. Protein Extraction protocol for different cell lines, like FRT, HEK 293, CFBE, T-84, primary HBE and nasal CF Cells; These cells were expressing CFTR protein with WT, or CF with single and double amino acid mutations; B. Western Blot protocol for CFTR Protein, extracted from cells mentioned above; images were developed with Fluorescence Way by Odyssey Infrared Image System (LI-COR Scanner). All data were quantified with Image Studio Software. I have studied the method of detection of CFTR expression at Plasma Membrane with Biotinylation and Up regulation to monitor CFTR cell surface expression, I used CF patient Primary HBE cells and cultured cell Lines like FRT, HEK 293, CFBE; Also studied Electrophysiological Methods of Measuring CFTR Chloride Channel. Using Conductance Assay to study ion transport; Accelerate goal of increasing throughput (8-fold) to screen compounds for CFTR modulators; I also worked with Simple Western Size Assays (WES), automated, capillary-based immunoassays; The digital image of CFTR from primary HBE, primary Nasal cells, FRT CFTR HCAI Mutants and 16HBEge Mutants lysates were analyzed and quantitative results were presented in Compass software.

I worked on the project dealing with study of the structure, function, and regulation of mitochondrial anion transport proteins at the molecular and atomic levels. A major focus of our lab has been the Mitochondrial Citrate Transport Protein (CTP) at high resolution to define the function of specific amino acid residues in the translocation process and identify those residues within the Yeast CTP that are mechanistically essential and otherwise important for function. Elucidation of amino acid residues comprising the substrate translocation pathway within the east CTP; Identification of conditions enabling the growth of large X-ray quality crystals of yeast CTP. Also studied Plasma Membrane Citrate Transporter (PMCT), Yeast, P. Pastoris were grown in Fermenter, PMCT protein overexpression, purification, and crystallization were performed; identified two selective inhibitors and developed a homology model based on the crystal structure of the Leucine transporter.

Was undergoing intensive training in Molecular Biology in the Department of Structural and Cellular Biology, University of South Alabama. Worked on the project dealing with study y-Globin gene role in Sickle Cell Anemia. The techniques which I learned during the period include: small and large-scale preparation of plasmid DNA and mammalian genomic DNA; electrophoresis (both PAGE and Agarose gel) for protein and DNA segregation; RNA extraction and RNAase protection assay, as well as other molecular biology techniques. I have also learned techniques for DNA-DNA binding (triple helix formation) and DNA-protein binding and used gel-mobility shift assay to analyze the bindings.

Worked on the Project investigating X-ray influence on the Prostaglandin’s functions on the Cancer cells. The study covered Cancer both, chemically induced by Benzo(a)pyrene and transplanted tumors (Erlich Ascite Carcinoma and Zaidela ascite hepatome) in Microsomal Membranes of Rats and Mice;

Successfully presented Dissertation for Candidate of Sciences Degree (Ph.D.) at the Institute of Biophysics in Moscow, Russia. This research examined the effect of Ionizing Radiation on the Transport of the Sodium, Potassium Ions and Water in frog skin.

RESEARCH SKILLS

Cell biology: Very experienced working with Primary Human Bronchial Epithelial cell (HBE) and Nasal (HNE) cells; also with CFF-16HBEge Single Cells Cloning, 3T3, FRT, HEK 293, CFBE, T-84, Lymphoblasts transfected culture cell Lines with ASO or Lipofectamine. RNA extraction, isolation, Reverse transcription and radiolabeled PCR used to analyze CFTR cDNA Splicing and activity during development Antisense oligonucleotide technology. Erlich Ascites Carcinoma and Zaidela Ascite Hepatoma Cells used for cancer research.

Physiology: Electrophysiological Methods of Measuring CFTR Chloride Channel. Using TECC Assay to study Chloride ion transport; Accelerate goal of increasing throughput (8-fold) to screen ASO and small compounds for CFTR modulators;

Biochemistry: Column Chromatography, including FPLC; Electrophoreses of proteins and DNA; Colorimetric quantification of proteins and DNA; Yeast, P. Pastoris were grown in Fermenter; Overexpression, isolation and purification of Single-Cys CTP mutant and PMCT; incorporation into phospholipid vesicles and measurement of transport kinetics using ion-exchange chromatography; determination of the molecular weight of CTP by size- exclusion chromatography and charge- shift native Gel Electrophoresis..

Molecular biology: To test Antisense oligonucleotides and analyze CFTR cDNA Splicing and activity, RNA extraction, isolation, Reverse transcription and radiolabeled PCR were used; Performed Transfection of Primary HBE and Immortal Cell lines with ASO or Lipofectamine; In vitro Site- directed mutagenesis to switch amino acids; Gene amplification by PCR; cloning and expression of recombinant protein in E. coli and Pichia pastoris system; Small and large scale preparation and purification of plasmid DNA; Restriction Enzyme Digestion; RNAase Protection Assay; DNA-DNA binding (triple helix formation) and DNA-protein binding and gel-mobility shift assay to analyze the bindings

Immunochemistry: Monoclonal antibodies (IgG) purification; Fab and Fc-fragments isolation; ELISA; Western, WES, Northern and Southern blot analysis.

Crystalography: Formation of CTP microcrystals using the Hanging Drop Vapor- diffusion method; Determination of the quality of the resulting crystals by microscopic examination and SDS- PAGE analysis of washed crystals for X-ray diffraction analysis.

Animal husbandry: More than 5 years working experience with frogs, also with chemically induced and tumor transplanted Rats and Mice.

Computer Skills: Microsoft Word, Excel software, Power Point, Graph pad Prism,

Image Quant, Compass software, Image Studio Software, Datatel Colleague System to create Purchase Order

Language Skills: English, Russian, Georgian.

PROFESSIONAL AFFILATIONS

American Society for Biochemistry and Molecular Biology; Georgian Cancer Research Society;

PUBLICATIONS

32 papers and abstracts published in leading biological journals and were presented on different Conferences; eleven full-length publications in top U.S. journals; twelve posters presentation at American Biophysics Society Meetings.

RECENT PUBLICATIONS

1. Kotaria R, Mayor JA, Walters DE, Kaplan RS, Oligomeric State of Wild-tape and Cystein -Less Yeast Mitochondrial Citrate Transport Proteins. J. Bioen. Biomemb. 31: 543- 549 (1999).

2. Kaplan R S, Mayor JA, Brauer D, Kotaria R, Walters DE. The Yeast Mitochondrial Transport Protein: Probing the Secondary Structure of Transmembrane Domain IV and Identification of Residues that Likely Comprise a Portion of the Translocation Pathway. J. Biol. Chem. 275: 120**-***** (2000).

3. Kaplan R S, Mayor J A, Kotaria R, Walters DE, Mchaourab HS The Yeast Mitochondrial Transport Protein: Determination of Secondary Structure and Solvent Accessibility of transmembrane Domain IV Using Site- Directed Spin Labeling. Biochemistry, 39: 9157-9163 (2000).

4. Ma C. Kotaria R. Mayor JA, Eriks LR, Dean AM, Walters DE, Kaplan RS. The Mitochondrial Citrate Transport Protein: Probing The Secondary Structure of Transmembrane Domain III, Identification of Residues that Likely Comprise a portion of Citrate Transport Pathway, and Development of a model for the Putative TMDIII-TMDIII’ Interface. J. Biol. Chem. 279:1533-1540 (2004).

5. Ma C, Kotaria R, Mayor JA, Remani S, Walters DE, Kaplan RS. The Yeast

Mitochondrial Citrate Transport Protein: Characterization of Transmembrane Domain III Residue Involvement in Substrate Translocation. J. Biol. Chem. 280: 2331-2340 (2005).

6. Ma C, Remani S, Kotaria R, Mayor JA, Walters DE, Kaplan RS.The mitochondrial transport protein: Evidence for a Steric interaction Between Glutamine 182 and Leucine 120 and its Relationship to the Substrate Translocation Pathway and Identification of Other Mechanistically Essential Residues. Biophysica at Biochemica Acta (Bioenerg.), 1757: 1271-1276 (2006).

7. Ma C, Remani S, Sun J, Kotaria R, Mayor JA, Walters DE and Kaplan RS. Identification of Substrate Binding Sites within the Yeast Mithochondrial Citrate Transport Protein. J. Biol. Chem. 282: 172**-***** (2007).

8. Remani S, Sun J, Kotaria R, Mayor JA, Breownlee HT, Walters DE, and Kaplan RS.

Tha Yeast Mitochondrial Citrate Transport Protein: Identification of the Lysine Residues Responsible for Inhibition Mediated by Pyridoxal 5'-Phosphate. J. Bioenerg. Biomemb. 40: 577-585 (2008).

9. Mayor JA, Sun J, Kotaria R, Walters DE, Oh KJ, and Kaplan RS. Probing the Effect of Transport Inhibitors on the Conformation of the Mitochondrial Citrate Transport Protein via a Site-Directed Spin labeling Approach. J. Bioen. Biomemb.42: 99-109 (2010).

10. Aluvila S, Kotaria R, Sun J, mayor JA, Walters DE, Harrison DH and Kaplan RS.

The Yeast Mitochondrial Citrate Protein. Molecular Determinants of its Substrate Specificity. J. Biol. Chem. 285: 273**-***** (2010).

11. Sun J, Aluvila S, Kotaria R, Mayor JA, Walters DE and Kaplan RS. Mitochondrial and Plasma Membrane Citrate Transporters: Discovery of Selective Inhibitors and Application to Structure/Function Analysis. Mol. Cell Pharm. 2(3) 101-110 (2010).

LIST OF REFERENCES

1.Michelle L. Hastings, PhD

Director, Center for Genetic Diseases, Professor, Chicago Medical School

Vice-Chair, Cell Biology and Anatomy Rosalind Franklin University of Medicine and Science. 3333 Green Bay Rd. North Chicago, IL 60064; Phone: 847-***-****

E-mail: adj4ys@r.postjobfree.com

2. Robert J. Bridges, Ph.D.

Center for Genetic Diseases, Professor, Dept. of Physiology and Biophysics, Rosalind Franklin University of medicine and Science/The Chicago Medical School 3333 Green Bay Road, North Chicago, Illinois 60064 Phone: 847-***-****; E-mail: adj4ys@r.postjobfree.com

3.Ronald S. Kaplan, Ph.D.

Vice President for Research, RFUMS Vice Dean for Research, CMS; Center for Proteomics and Molecular Therapeutics, Professor, Dept. of Biochemistry and Molecular Biology Chicago Medical School Phone: 847-***-****

E-mail: adj4ys@r.postjobfree.com

4.Barbara M. Vertel, Ph.D

Professor, Department of Cell Biology and Anatomy; Rosalind Franklin University of medicine and Science/The Chicago Medical School 3333 Green Bay Road, North Chicago, Illinois 60064 Phone: 847- 578-3443 E-mail:adj4ys@r.postjobfree.com

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