Cell Biology Ph D

RESUME

Rusudan Kotaria, Ph.D.

Lombard, IL *****

SUMMARY

Seeking a position as a Protein Purification Specialist, including FPLC. ACTA AVANT, HPLC UHPLC Chromatography Cell Culture Specialist, Biochemistry, Molecular and Cell Biology, Protein Chemistry, Cystic Fibrosis Physiology and Biophysics, Cancer Cell Biology.

Worked on the project dealing with the purification of RNA-binding protein, Native and Recombinant, a component of a ribonucleoprotein, RNP complex, clinically important target of autoantibodies in patients with rheumatoid diseases. A major focus of our lab has been RNP as a Antigen, Polyclonal and Monoclonal antibodies (IgG) Protein Purification by Column Chromatography, including FPLC. ACTA AVANT 150, using Affinity, Ion-Exchange, Size-Exclusion Columns, AminoLink, Fab, F(ab1)2 Kits. Highly used Immunochemistry experiments like: ELISA; SDS-PAGE, Western blot analysis.

Worked with Antisense oligonucleotide (ASO), a small compound 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 CRISP/Cas9 edited 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 ways 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.

Worked also with 3T3 cells to produce Conditioned Media, which produces Growth Factor and helps Primary and transfected cells lines to grow well. I improved Protein Extraction protocol for different cell lines, like FRT, PCP-R, HEK 293, ESC, iPSC, 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; 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 Biotinylating and Up regulation to monitor CFTR cell surface expression, I used CF patient Primary HBE cells and cultured cell Lines; 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; Also highly used Patch Clamp (PC) Method to study CFTR/CL channel. I also worked with Simple Western Size Assays (WES), automated, capillary-based immunoassays; The digital image of CFTR from cells lysates were analyzed and quantitative results were presented in Compass software.

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 Yeast CTP that are mechanistically essential and otherwise important for function.

Identification of conditions enabling the growth of large X-ray quality crystals of yeast CTP.

I also studied Plasma Membrane Citrate Transporter (PMCT in Human, Rat, Mouse) and Drosophila INDY Transporters. These will permit to design of specific inhibitory drags of Human obesity and its consequent risks. Yeast, P. Pastoris were grown in Fermenter, PMCT and INDY proteins overexpression, purification, and crystallization were performed.

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 Ascites Carcinoma and Zaidela ascites hepatoma) in Microsomal Membranes of Rats and Mice.

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

M.S. - Biology and Chemistry, Physiology of Human Being and Animal, Tbilisi State University, Tbilisi, Georgia

PROFESSIONAL EXPEREINCE

Thera test Laboratories, Advance Immunoassay, INC 1120 North DuPage Ave, Lombard, IL 60148 Director of Research and Development Department 07/2021-01/2025

Rosalind Franklin University of Medicine and Sciences/Chicago Medical School, Center for Genetic Diseases 3333 Green Bay Rd. North Chicago, IL, 60064.

Research Associate 11/2019-1/2021

Rosalind Franklin University of Medicine and Science/Chicago Medical School, Department of Physiology and Biophysics North Chicago, IL, 60064.

Research Associate 06/2014-6/2019

Rosalind Franklin University of Medicine and Science/Chicago Medical School, Department of Biochemistry and Molecular Biology North Chicago, IL, 60064

Research Associate 05/1998-2014

Tbilisi State University, Tbilisi, Georgia

Department of Biophysics, Senior Research Associate 1990-1998

Institute of Medicine Tbilisi, Taught Physiology for first year medical students 1996-1998

RESEARCH SKILLS

Biochemistry: Very experienced working with Protein Purification by Column Chromatography, including FPLC. ACTA AVANT 150, HPLC, UHPLC Chromatography, Using Affinity, Ion-Exchange, Size-Exclusion Columns. Protein Purification with AminoLink, Fab, F(ab1)2 Kits. 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.

Immunochemistry: RNP Antigen, Polyclonal and Monoclonal antibodies (IgG) purification; Fab, F(ab1)2 and Fc-fragments isolation; ELISA; Western, WES, Northern and Southern blot analysis.

Cell biology: Very experienced working with HEK293T, Hep-2 cells; Primary Human Bronchial Epithelial cell (HBE) and Nasal (HNE) cells; with CRISPR/Cas9 edited CFF-16HBE Single Cells Clones, 3T3, FRT, CHO, CFBE, T-84, PCP-R, ESC, iPSC, Lymphoblasts transfected culture cell Lines with ASO or Lipofectamine, E-coli and Yeast Cell Cultures. 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 Ascites Hepatoma Cells are 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. Also highly used Patch Clamp (PC) Method to study CFTR/CL channel.

Molecular biology: To test Antisense oligonucleotides and analyze CFTR cDNA Splicing and activity, DNA and 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;

Crystallography: Formation of CTP microcrystals using the Hanging Drop Vapor- diffusion method; Determination of quality of 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, Unicorn 7; Python, Image Quant, Compass software, Image Studio Software, Datatel Colleague System to create Purchase Order

Language Skills: English, Russian, Georgian.

PUBLICATIONS

33 papers and abstracts published in leading biological journals and were presented on different Conferences; twelve full-length publications in top U.S. journals; thirteen posters

presentation at American Biophysics Society Meetings.

PROFESSIONAL AFFILATIONS

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

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