- Ph.D.,Department of Biotechnology Panjab University, Chandigarh, India. 2004.
- Thesis title:Studies on the molecular alterations in the pathogenesis of small cell and non-small cell lung carcinomas.
- M.Sc.,Life sciences, Pondicherry University, with first class (7.7 GPA) in 1998).
- B.Sc.,Botany (hons) Madras University, with first class, 1996.
RESEARCH EXPERIENCE PROFESSIONAL TRAINING:
- Working as Assistant Professor in the department of Biotechnology, Thapar Institute of Engineering & Technology, Patiala (July 2011-present). Here I am teaching Postgraduate students and B.Tech students the subjects of Virology, Animal biotechnology, Stem Cell and Tissue engineering, Animal Cell Culture Technology, Diagnostic and Clinical Microbiology.
- Worked (August 2009-June 2011) as an Application Scientist in Flow Cytometry in Beckman Coulter, here my job profile included Instrument and Applications training on Beckman Coulter Flow Cytometry to ensure a high level of end-user satisfaction. I also organized training programs for channel partners and Application Specialists to raise and strengthen the technical bar and capability on product knowledge and applications of Beckman Coulter products particularly those related to Flow Cytometry, immunology and cell biology. Synergize and coordinate within business segments and with customers for timely and operationally efficient delivery of various training programs. Work in coordination with sales, service and other application associates towards achieving the business objectives. Support a culture of constructive feedback and continuous improvement within the organization. Understanding requirements of customer’s experiment and provide appropriate technical and product solutions through discussions, presentations and/or product/application demonstrations to positively impact sales and increase productivity.
- Jan 2007 to June 2009: Post Doctoral Fellow, Department of Pathology, University of Miami Miller School of Medicine, USA. Here I have used a flow analyzer which can simultaneously measure Electronic Cell Volume and stem cell marker expression to study stem cells in HPC-A samples. In approximately 35% of the samples analyzed, a significant and distinct population of CD34positive cells with CD45very dim and small electronic volume was present. These cells had the smallest size, positive expression of stem cell markers (such as CD90, CD117, and CD133) and were not platelets. In addition to HPC-A, I have also used umbilical cord blood (cord blood) to study expression of CD34, CXCR4, ALDH and SP cells. The above study was to develop a rapid and innovative method for characterization of early stem/progenitor cell populations based on electronic cell volume in combination with marker expression. I have also studied the expression of Aldehyde dehydrogenase (ALDH) in stem cells.In a recent study, I have used the Cell Lab Quanta analyzer to determine CD34, CD90, CD117 CD133 expression and electronic cell volume in ALDHbright and ALDHdim cells. Approximately 70-77% of the ALDHbright cells had positive stem cell marker expression. The above work resulted in three publications. [Cytometry Part A 2008; 73 (2):16 0-7., Cytometry. Part B, Clinical Cytometry 2008; 74 (3):182-8 and Cytometry. Part B, Clinical Cytometry 2010]. I also worked on the expression of many stem cell markers (ALDH1, CD44, CC24, CD34 and CD45) in female adenocarcinoma patients. This resulted in a recent publication in Cytometry B, 2010. I also studied the side population (SP cells) in HPC-A samples and correlated their presence with expression of other stem cell markers like CD34, CD90, CD117 and CD133
- January 2005-November 2006; Post-doctoral fellow in the Centre for Proteomics and Genomic Research in the Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, South Africa. My collaborative project with Procognia (a London based proteomics company) was focused on cloning and expression of genes involved in drug metabolism (CYP3A4, NADPH, Cytochrome b5) in E.coli and Baculovirus for the creation of functional protein array. My main focus of work was to clone CYP3A4 (and its different polymorphic variants), NADPH and Cytochrome b5 into the C-terminal and N-terminal BCCP- His Tag vector as different truncated constructs and to express them E.coli. The different constructs were purified and expressed successfully in E.coli system. Similarly I had also cloned and expressed the above said genes as fusion proteins in Baculovirus expression system in Insect cells.
- June 2004- November 2004: Research associate Ranbaxy laboratories Gurgaon, India. I was involved in the cloning of ion channel genes and also in Muscaranic receptor genes. Here I was involved in the cloning of cDNAs encoding for five Muscaranic subtypes (M1-M5) in different promoters in various eukaryotic vectors and each subtype was expressed in different mammalian cell lines. CHO-K1 cell line was the best for generating stable cell lines expressing muscarinic receptors.
- September 2002 to January 2004:Research associate (Scientist Grade II) In the Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER, Mohali). I worked on the role of oxidative stress genes and risk towards oral carcinogenesis in North Indian populations. Initial work was on the caloric restriction of yeasts and its influence on aging, this resulted in a publication in [Free Radical Research 39:55-62], apart from this I also was involved in the cloning of phytase gene from AspergiI!us niger var. teigham in Pichia pastoris.
- January 1999-Sept 2002: Phd Student, Worked on a research project for Doctoral Thesis titled “Studies on the molecular alterations in the pathogenesis of small cell and non-small cell lung carcinomas”. This work involved detecting novel markers if any for early risk assessment and diagnosis of lung cancers. Studying the role of detoxifying genes like CYP1A1, CYP2EI, CYP2D6, GSTM1, GSTT1 and also tumour suppressor genes like p53 and the role of Telomerase in small cell and non-small cell lung cancer pathogenesis using molecular biology techniques like PCR, RFLP,TRAP assay, SSCP, etc. The above work resulted in following publications [Molecular Cellular Biochemistry 2004, Vol266 :(1-2):1-9, BIOMARKERS (2003). Vol 8 No.5: 41 5-428. and Cancer Genetics and Cytogenetics 156 (2005) 68–73.]
- July-September 1999: Worked in the Regional Research Laboratories of the Council of Scientific and Industrial Research, Jammu, Genetic Engineering Unit on a project “EMS induced Mutagenesis in an enantio-specific lipase producing yeast
- April-August 1997: I spent two months learning tools for molecular biology at the Chittranjan National Cancer Institute in Calcutta.
TECHNIQUES LEARNT DURING ACADEMIC AND RESEARCH CAREER:MolecularBiologytools
- DNA isolation (from clinical samples i.e. blood, biopsies, body fluids), fungal, Bacterial, Viral DNA, Insects etc purification and quantification.
- RNA isolation, purification and quantification
- Plasmid preparation (Mini & Maxi prep) Bacmid large construct
- Gene cloning, Sub-cloning, Site directed mutagenesis, Restriction Digestion, DNA ligation, Competent cell preparation, DNA Transformation, Expression of clone, Screening of Transformants,
- Generation of Fusion-proteins with different affinity tags like HIS, BCCP and Myc
- INVERSE-PCR (IPCR), RFLP-PCR, RAPD, Multiplex-PCR, Allelic specific-PCR, Reverse transcriptase PCR.
- Use of IPCR for creation of truncation mutation and addition of sequences.
- Mutation detection: SSCP, RFLP.
- Protein expression in E.coli, Baculovirus and Pichia Pastoris.
- SDS-PAGE, In-Gel assays for enzymes like catalase, super oxide dismutase and Glutathione peroxides.
- Protein purification using various affinity tags (His-tag, Myc-tag and BCCP).
- TRAP assay (Telomere rapid amplification protocol) for detection of telomerase activity
- Comet Assay, Chromosomal culture (from blood for metaphase slide preparations).
Flow Cytometry and Stem Cell analysis
- In hand experience in the Cell Lab QuantaTMSC, EPICS-XL-MCL, FC-500 MoFlow Sorter, Gallios, Navios and CyanADP flow machines.
- Flow Cytometry for measurement of DNA content and aneuploidy levels.
- Flow Cytometry analysis for stem cell markers like CD34 (ISHAGE protocol), CD90, CD117, CXCR4, CD133 etc. Also surface and nuclear markers like Ber-Ep4, TTF-1, ER/PgR, p53, p63 etc.
- Side Population (SP) Cell Analysis and ALDH (Aldefluor) assay using flow cytometry
- Three color, Four and 6-color Multiparametric flow cytometric analysis.
- Flow Cytometry measurement of Apoptosis, S-phase cell cycle,PNH.
Insect & Animal Cell Culture
- Handling and maintenance & passaging of insect cell lines like Sf-21 and Sf-9. Viral plaque assays for finding out viral titre values.
- Transfection of genes into different insect cell lines.
- Handling & maintenance & passaging of human cancer cell lines A 549 (lung cancer), MCF-7 & OvCar (breast cancer), H441, H293, CEMs, H9s (lymphoids).
Enzymatic Assays, (Glutathione peroxidase, Phytase, NADPH) Fluorescent enzyme assay for CYP3A4. Protein Assays (Bradford etc).
Bioinformatics & Statistical software’s
- Statistical software like SPSS,Statistica,Prism-Graphpad,MedCal,Chem-draw.
- Data mining software’s like Text analysis and Path way assist,Basic Knowledge in proteomic software’s like PD-Quest.
- Software used in Flow Cytometry like WinMidi,Winlist,Modfit,Quanta,Summit(Cyan)
Basic microbiological techniques like maintenance of culture stocks, Streaking and Spread plating, Dilution plating, Spot assays, Mutation treatments etc.
- 1. Sharma Siddharth, Shariatmadar Sherry; Krishan Awtar. Electronic Volume, Aldehyde dehydrogenase and Stem Cell Marker Expression in Cells from Human Peripheral Blood Apheresis Samples. Cytometry B (Clinical Cytometry), 2010; 78B: 123-129.
- 2. Krishan Awtar, Deepti Sharma, Sharma Siddharth, Ron Hamelik, Parvin Ganjei-Azar, Mehrdad Nadji. ALDH+/CD44+/CD24- Expression in Cells from Body Cavity Fluids. Clinical Cytometry (Cytometry B), 2010 78B, 176-182.
- 3. Sharma Siddharth; Cabana Raquel; Shariatmadar Sherry; Krishan Awtar Cellular volume and marker expression in human peripheral blood apheresis stem cells. Cytometry Part A 2008; 73 (2):160-7.
- 4. Shariatmadar Sherry; Sharma Siddharth; Cabana Raquel; Powell Scott; Ruiz Phillip; Krishan Awtar. Electronic volume of CD34 positive cells from peripheral blood apheresis samples. Cytometry. Part B, Clinical Cytometry 2008; 74 (3):182-8.
- 5. S. Agarwal*, S. Sharma*, V. Agarwal and N. Roy (2005) Caloric Restriction Augments ROS Defense in S. cerevisiae, by a Sir2p Independent Mechanism. Free Radical Research 39:55- 62. *both authors contributed equally
- 6. Genetic polymorphisms of CYP2D6, GSTM1, and GSTT1 genes and bladder cancer risk in North India R.C. Sobti,*, A.I. Al-Badran, S. Sharma, S.K. Sharma, A. Krishan, H. Mohan. Cancer Genetics and Cytogenetics 156 (2005) 68–73.
- 7. Genetic polymorphism of the CYP1A1, CYP2ET, GSTMI and GSTTI genes and lung cancer susceptibility in North Indian population. Sobti, R.C, Sharma, S, Joshi, A., Jindal; 5K., and Janmeja; A. (2004) Molecular & Cellular Biochemistry Vol266:(1-2):1-9
- 8. CYPIAI and CYP2D6 polymorphism and risk towards lung cancer in a North Indian population. Sobti, R.C, Sharma, S, Joshi, A., .Jindal, S.K., Janmeja, A. BIOMARKERS (2003). Vol 8 No.5: 41 5-428.
- 9. RAPD-PCR analysis in two species of Catopsilia. Gill, T., Sharma, V., Mamtesh, Sharma, S., Sobti, R.C. CAROLOGIA (2003). Vol: 56: 100-1 05
- 10. Molecular markers in lung cancer. Nucleus, 2000 vol 43(1,2) 71-82.
- 11. Molecular genetics of cancer with special reference to cancers of GI tract, Lung, Head and Neck and Cervix. JPAS. Vol 1(2001): 63-67
- 1. Siddharth Sharma and Awtar Krishan. Electronic Volume of Hematopoietic Stem Cells. In:Flow Cytometry Applications in Stem Cell Research, Eds. Awtar Krishan, H Krishnamurthy and Satish Totey, John Wiley-Blackwell Publications; 2010.
- 2. Molecular genetics of Gastrointestinal Tract Cancers. (2002). In book:Some aspects of Chromosome Structure and Function. Pp 177-1 92 (Eds) Sobti, R.C. eta!. Publishers (Kluwer & Narosa).
- 3. Telomere Telomerase and Cancer. (2002), In book: Some aspects of Chromosome Structure and Function. pp 168-191 (Eds) Sobti, R.C. et at Publishers (Kluwer & Narosa).
- 4. Molecular pathogenesis of lung cancer (2002). In book: Some aspects of Chromosome Structure and Function. pp 193-205 (Eds) Sobti, R.C. et at Publishers (Kluwer & Narosa).
Awarded independent project grant from DST young scientist scheme on project ”Role of detoxifying genes in oral cancer pathogenesis in North Indian population”.
Was awarded the Young Scientist award by the Panjab Academy of Sciences (PAS) held at Thapar College of engineering, Patiala, Feburary-9-11, 2002.
- 1. Stem cell marker expression in cells from body cavity fluids. Awtar Krishan, Deepati Sharma, Siddharth Sharma, Ronald Hamelik, Parvin Ganjei-Azar, and Mehrdad Nadji. Presented at 24th Annual Meeting of the Clinical Cytometry Society October 16–18, 2009, Jacksonville, Florida, USA.
- 2. Electronic Volume and ALDH expression in Stem cells from human Apheresis samples by Siddharth Sharma, Sherry Shariatmadar, Raquel Cabana, and Awtar Krishan. Presented at The 23rd Annual Meeting of the Clinical Cytometry Society October 12–14, 2008 Portland, Oregon, USA
- 3. Electronic Volume of Stem Cells in Human Apheresis Samples by Siddharth Sharma, Raquel Cabana, Sherry Shariatmadar, Awtar Krishan. Presented at the ISAC XXIV International Congress, May 17-214. Electronic Cell Volume and Marker Expression in Stem Cells from Human Apheresis Samples by Siddharth Sharma, Raquel Cabana, Sherry Shariatmadar, Awtar Krishan. Presented at the AACR at San Diego, CA, USA on April12-16 2008.
- 5. Electronic Volume of Hematopoietic Stem Cells by Siddharth Sharma, Raquel Cabana, Sherry Shariatmadar, Awtar Krishan. Presented at the 22nd Annual Clinical Cytometry society at Washington D.C. on October 16-19 2007.
- 6.“P450-BIOCHIPS by Siddharth Sharma Natasha Beeton and JM Blackburn. Presented at the international conference on “A Molecular Meander in the Midlands” organized by South African Society for Biochemistry and Molecular Biology at PeiterMaritzburg, South Africa, on July 3-56h, 2006.
- 1. Attended workshop on Microarray data analysis from june19-23, 2006 at IIDM, UCT.
- 2. Attended workshop on statistical analysis from march10-15, 2005 at UWC
- 3. Attended INDO-US workshop on Flow Cytometry (18-24 Feb, 2002 at Biotechnology Dept Panjab University)
- 4. Attended workshop on Microgel (Comet assay) Electrophoresis at Dept. of Biotechnology, Panjab University, Chandigarh
- Prof. Awtar Krishan, Department of Pathology, University of Miami Miller School of Medicine, Miami, USA. 33136. Email: firstname.lastname@example.org.
- Prof. R.C.Sobti, Vice-Chancellor, Panjab University, Sector-14. 160014. Email: email@example.com
- rof. Jonathan M Blackburn, Director, Centre for Proteomics and Genomic Research, Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Observatory 7925, Capetown, South Africa. E-mail: firstname.lastname@example.org
- Raquel Cabana, Director Laboratory, NPE systems, Pembroke Pines, Florida, USA. Email: email@example.com, firstname.lastname@example.org