First post-doctoral training (July 2010- September 2011)
To broaden my research expertise in the role of epigenetic changes involved in diseases, and aging in itself is a cause of multitude of diseases, I wanted to familiarize myself with aging-related in vivo models. I applied and was accepted as a post-doctoral candidate in Dr. Eugenia Wang’s laboratory at University of Louisville (Gheens Aging Center). Dr. Wang is well-recognized in the field of aging and role of small RNAs like microRNAs in aging-related diseases. First, I described the change the gain of survival signaling by altered expression of miRNAs in the brain of long-lived mutant and diet restricted mice. We could identify multiple miRNAs targeting the same pathway and how their expression was dependent on ILS pathway. I could describe the role of miR-34a in apoptosis and antagonistic pleiotropy. It gives me immense satisfaction the role of miR-34a in apoptosis is now exploited to design an anti-cancer miRNA-based therapy by Austin-Based Mirna Therapeutics. Latest results show promising results in phase 2 of clinical trials. My work in Dr. Wang laboratory resulted in 2 first author publications and one co-author expression in Aging (Albany NY) and Aging cell. My work also described the altered expression of miRNAs in Ames dwarf mice.
Second Post-Doctoral training (October 2011- 2016)
The desire to undertake more mechanistic work on understanding aging and aging-related diseases like cardiovascular diseases (CVD). I decided to pursue a second post-doctoral position using invertebrate models like C.elegans that have played a pivotal role in developmental biology and aging. Consequently, I searched for a mentor for my second post-doctoral training that had expertise in studying the role of dietary and genetic factors in aging using invertebrate models. Thus, I joined Dr. Pankaj Kapahi’s lab at Buck Institute, who has provided excellent mentorship to pursue my goals. The experience in his laboratory has acquainted me with developmental biology and aging-related techniques and taught me much about the effect of diet on health span. My project involves the characterization of gut flora specific metabolites in development, health span and aging. This study has helped me shape a couple of first author papers. Subsequently, I have developed a project that investigates the role of trimethylamine a gut-specific metabolite and its effects on aging and health span. We have developed a novel technique to check the impact of metabolites and different bacterial mutants on gut permeability and have developed C. elegans as a model system for the same.
In July 2007, I got selected into Dr. Stefan Stamm lab at University of Erlangen-Nurnberg. The focus of their laboratory was to describe the role of small nuclear RNAs (HBII-52 and HBII-85) in Prader-Willi syndrome. My doctoral studies focused on the role of these snoRNAs on alternative splicing. I used invitro cell system and Prader-Willi mouse to understand the role of snoRNAs in Prader-Willi syndrome. The main conclusions were: 1) identification of a novel target genes that were alternatively spliced in presence of these snoRNAs and critical in progression of pathologies related to Prader-Willi syndrome, 2) Using combination of snoRNA-specific RNA probe pull down assay and mass-spectrometry, we identified novel proteins associated with these snoRNAs that were also involved in alternative splicing, 3) identified set of novel small RNAs that are cleaved out from the precursor snoRNA and potentially work as RNA switch in modulating alternate splicing . These studies resulted in 2 first author publications in Human Molecular Genetics and RNA biology journals. Also, my work in graduate school contributed to and two co-authored publications in Gene and Nucleic Acid Research journals. I presented my work as oral and poster presentations at several national and international conferences and won the best speaker award. I was chosen to instruct a group of international Ph.D. and medicine students about latest techniques and assays that we developed in our lab to study alternate splicing at Montpelier France. Throughout my doctoral studies, I interacted and worked with undergraduate students and assisted them with their projects. My contribution in their projects provided a co-authorship paper. I also had an opportunity to work with my mentor in the preparation of grants that were eventually funded by the NIH. All these experiences in my doctoral program provided me a firm grounding in cardiovascular sciences, and I thought to pursue my career as an academic research scientist.
In July 2002, I qualified a highly competitive national entrance test for enrollment into master at National Center Human Genome Studies and Research, India. I was fortunate to do research for my master’s degree under the excellent mentorship of Drs. Tapas Mukhopadhyay and Rajnikant Mishra. While pursuing my masters, I learned various aspects of genetics and molecular biology. My master’s thesis described the development of a novel strategy for in vivo ligation and gene transfer using bacterial cells as a model system. This cloning method involved a high throughput gene transfer without the utilization of any restriction enzymes and was based on homologous recombination technology. I had an opportunity to present these findings at an annual Cell Biology meeting in India. During my masters, I visited various national research institutes and gathered experience and on research different technologies. This program laid the foundation and motivated me to pursue a career in biomedical research and to enter a graduate program.
During my M.S program, I heard a seminar by Dr. Deepak Kaul and was completely captivated by his work, which describes the role of small molecules and epigenetic changes that regulate gene transcription in diseases. I approached him and joined as a research assistant in his laboratory at Post Graduate Institute of Medical Education and Research (PGIMER), India. Initially, I determined the role transcriptional regulation of PPAR-γ in human immunomodulatory cells using siRNA technology. This study proposed that PPAR-γ gene has the inherent capacity to inﬂuence the lipid-mediated inﬂammation process. Another project I was involved identified novel microRNA (hiv1-mir-H1) coded by HIV genome and its role in modulating expression of apoptosis antagonizing transcription factor (AATF) in HIV-positive patients. Together, these projects culminated into two publications in Molecular and Cellular Biochemistry and Proceedings-Indian National Science Academy. After completing these projects in the role of epigenetic factors in gene regulation, I continued to focus on this field.
All the experiences I have gained so far have prepared me well to lead an aging group in academia.
- Primary neuronal culture
- Mammalian cell culture. Cell line handles: N2a, HEK 293, NIT1 and HELA cells
- Macrophage cell culture
Molecular Biology: RNA/DNA based techniques
- Cloning of double-stranded RNAs (small and long RNAs)
- RNase protection assay (RPA)
- Next Generation Sequencing (NGS)
- RNA Pull down assay
- Northern Blotting
- Southern Blotting
- Colony Hybridization
- Total RNA Isolation: Animal tissues and cells
- Transient transfection.
- PCR, Reverse Transcription PCR and Real Time PCR,
- Minigene cloning
- Restriction enzyme and bacteriophage lambda based cloning
- Primer-Designing and Gene Arrays
- In-situ hybridization and immunostaining
- Primary Neuronal Culture
- SDS-PAGE of Proteins,
- Western Blot, Silver Staining of Proteins
- Two-Dimensional Gel Electrophoresis
- Preparation and Staining of Metaphysics chromosomes & Barr Body.
- Immunohistochemistry: Macrophage staining
Radiation Safety Certification from Radiation Safety University of Kentucky Animal Handling Mouse dissection: Mouse tissues and macrophage culture
Bioinformatics: BLAST Alignment of Pair sequences, Multiple Sequence Alignment, Prediction of RNA Secondary Structure Gene and SWISS-PROT, Database Search for Similar Sequences. Developed an online resource: BioInoformatics Support and Analysis (www.bisa.co.in)
Animal Handling Mouse dissection: Mouse tissues extraction, primary neuronal and macrophage culture
- Multifactor Dimensionality Reduction (MDR) software for Gene-Gene and Gene-Environment interactions.
- MASCOT software for analyzing the MALDI-TOF results
- PD quest for analyzing the 2D dimensional results
- ImageJ and Quantity One 3.1 software for studying the DNA/PCR results.
- Haploview version 3.2 for studying the Linkage disequilibrium for the association between SNPs.
- PHASE version 2.1 for generating Haplotypes of genes
- Primer 3 software for designing the primers/oligos.
- End Note 9 software