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  1. 1. Aneesa Fasim #23/24, L-15, Palace Garden Apts, Phone: (91) 98454-00955 Vasanthnagar, Bangalore, India -560020 Email: PROFILE SUMMARY: Postdoctoral Fellow with hands on experience in Molecular Biology, Protein BioChemistry, Cell Biology, Cell Culture and BioPhysical techniques. EDUCATION:  Assistant Professor – Biochemistry, DSU, Bangalore, KA, India 05/16 - Current  Project Manager, Indian institute of Sciences, Bangalore, KA, India 08/15 – 04/16  Post-Doctoral fellow, Indian Institute of Sciences, Bangalore, KA, India 09/09 – 07/15  Ph.D, Biochemistry, Osmania University, Hyderabad, AP, India 10/04 – 04/09  M.Sc, Biotechnology, Andhra University, Visakhapatnam, AP, India 07/00 – 07/02  B.Sc, Life Sciences, Andhra University, Visakhapatnam, India 07/98 – 07/00 TECHNICAL EXPERTISE:  Molecular Biology: Gene cloning, Subcloning, Site directed mutagenesis, Quantitative real time PCR, Overlap extension PCR, routine DNA, RNA , Genomic DNA extraction from E.coli, S.aureus and cell lines, PCR and RT-PCR primer designing.  Protein Chemistry: Recombinant protein expression and purification from E.coli, Affinity chromatography, Gel filtration chromatography, Ion exchange chromatography, HPLC, Preparation of affinity matrices (coupling of Sepharose to antibodies).  Biochemical Techniques: Electrophoresis, Spectroscopy (UV/Visible), Enzyme assays, Flowcytometry, AKTA Purification, Raising antibodies, Immunoprecipitation, Co- immunoprecipitation, ELISA, Western blotting, AEC and ECL staining, Peptide mass finger printing, Kinase assays.  Cell Culture: Maintenance, storage, revival of animal cell lines (Adherent cell lines: SK- NMC, NEURO2A, MCF10A, MCF7, MDA-MB231; Suspension cell lines: U937, Transfection of GFP-profilin, DSRed-actin, Profilin-1 SiRNA. Isolation of platelets, lympocytes etc.  Biophysical Techniques: Thermal melt analysis, Circular dichroism, Dynamic light scattering, Surface plasmon resonance, Crystallographic techniques.  Microscopy: Normal, Fluorescence and Confocal, Immunoflourscence studies by staining cells with FITC, Rhodamine- Labelled antibodies, DAPI, Propidium iodide staining etc  Cancer Biology: Invasion assays, migration assays, gelatin zymography  Computational Biology: Proficient in various sequence and structure analysis web servers like EXPASY, BLAST, CLUSTALW, Pymol, ESPRIPT, Well versed with scientific graphing softwares like Sigmaplot and Graphpad prism. RECOGNITIONS:  Designated as Project Manager on the Biocatalysis and Enzyme Bioengineering Project, IISc, Bangalore from August 2015- Current.  Awarded Research Associate fellowship at IISc, Bangalore from 02/2015- 07/2015.  Awarded DBT-Postdoctoral fellowship by Department of Biotechnology(DBT), Delhi from 01/2010- 12/2014.
  2. 2.  Awarded Senior and Junior Research Fellowship by Department of Science and Technology (DST), Delhi from 09/2004-2008.  Awarded Research Assistant Fellowship by Defense Research and Development Organization (DRDO): 2003-2004.  Qualified for Government of India’s Central Scientific Industrial Research (CSIR) - Lecturership via National eligibility test. TEACHING/ RESEARCHERS and STUDENTS MENTORED  Teaching Ist semester B.Sc Biochemistry and Biotechnology  Teaching IVth semester M.Sc Biochemistry (Molecular physiology)  Project interns mentored - 5  PhD students mentored - 1  Short Laboratory Course – RT-PCR for new personnel GRANTS WRITTEN  Drafted and submitted a research grant project titled “Functional role of Profilin2 in Brain cancers” to DST-Early career research on September 30th, 2016. The grant is under review. ROLE AND PROJECTS:  Assistant Professor- Biochemistry: Dayananda Sagar University, Bangalore. I am presently teaching theory and conducting practicals for the Cell Biology course to Ist semester B.Sc Biotechnology and Inorganic chemistry theory and practicals to 1st semester B.Sc Biochemistry. I am also taking theory classes on molecular physiology course for IVth semester M.Sc Biochemstry.  Project Manager: Molecular Biophysics unit, Indian Institute of Sciences, Bangalore, Karnataka in collaboration with IIT-Bombay and HTBS Pvt. Ltd, Pune. Project title: Biocatalysis and Enzyme Bioengineering of alcohol dehydrogenases, ω- Transaminase and Nitrilases. Research Objective: Aim of this project is to bioengineer an (R)- Alcohol dehydrogenase ω- Transaminase and nitrilase, with high activity and enantioselectivity for biocatalysis of cost effective active pharma ingredients. Project Investigator: Prof. B. Gopal, MBU, IISc, Bangalore In collaboration with Hitech Biotech Pvt. Ltd Pune.  Research Associate-I: Molecular Biophysics Unit, Indian Institute of Sciences, Bangalore, Karnataka. Project title: Structural and functional studies of proteins involved in the Agr quorum sensing machinery of Staphylococcus aureus. Research Objective: To understand the role of Agr two component system in S.aureus, which not only helps in virulence but also involved in quorum sensing mechanism. . Project Investigator: Dr. B.Gopal, MBU, IISc, Bangalore.  Doctor of Philosophy: Dept. of Biochemistry, Osmania University, Hyderabad, India Thesis title: Studies on the role of profilin and phosphoprofilin on cell invasion. PhD Guide: Dr. Surya Satyanarayana Singh, Osmania University, Hyderabad. Research Objective: Understanding profilin (an actin binding protein) and phosphorylated profilin’s role in cell migration and invasion of breast cancer cell lines.  Research Assistantship: Dept. of Biochemistry, Osmania University, Hyderabad, India Project title: Protein-protein interaction mediated by profilin in bovine platelets. Project Investigator: Dr. Surya Satyanarayana Singh, Osmania University, Hyderabad. Project Duration: Nov 2003-Oct 2004
  3. 3. Research Objective: Evaluate phosphorylated profilin’s interaction with other proteins. PUBLICATIONS  Kalagiri Rajasree, Aneesa Fasim and Balasubramanian Gopal. Conformational basis for the conditional differences in AgrA-mediated gene expression. Biochemistry Biophysics Reports. Vol 6, 124-134, 2016.  Sandeep Srivastava┴, Rajasree Kalagiri┴, Aneesa Fasim┴, Gayathri Arakere and B.Gopal. Influence of the AgrC-AgrA complex in the response time of staphylococcus aureus quorum sensing. Journal of Bacteriology. 1;196(15):2876-2888. 2014. ┴- Equal contribution  Wasia Rizwani, Aneesa Fasim, K. Sathish and Surya S. Singh. S137 phosphorylation of profilin 1 is an important signaling event in breast cancer progression. Plos one. DOI: 10.1371/journal.pone.0103868, 2014  Sandeepta Burgula, Aneesa Fasim, Padma B. and Surya S. Singh. Profilin activates Bacillus thuringiensis phosphoinositide specific PLC. International Journal of applied biology and pharmaceutical technology, 3(3) 360-366, 2012.  K.V.N. Radhika┴, M.S. Achary┴, Aneesa Fasim, K. Sathish, Wasia Rizwani., M. Sairam, H.A. Nagarajaram and Surya S. Singh. Regulation of profilin binding to poly (L- proline) by oligomerization. International Journal of Biological Macromolecules 45(3):265-73, 2009. ┴- Equal contribution  S. Kasina, R. Wasia , Aneesa Fasim, K.V.N. Radhika, Surya S. Singh. Phorbol ester mediated activation of inducible nitiric oxide synthase results in platelet profilin nitration. Nitric Oxide, 14 (65-71), 2006. INVITED REVIEWS  Surya S. Singh, K.V.N. Radhika, Wasia Rizwani, K. Sathish and Aneesa Fasim. Profilin: From allergen to tumor suppressor. Cellular Signalling and Apoptosis Research. 2007 (159-184). Nova Science publishers, New York.  Surya S. Singh, Wasia Rizwani, Sandeepta B., Aneesa F. and K.V.N.Radhika. Microarrays: A boon in disguise. Proc. of AP Akademi of Sciences. 9 (5), 201 – 208, 2005. CONFERENCES, ORAL//POSTER PRESENTATIONS:  Participated in International Conference on Biomolecular Forms and Functions organized by Indian Institute of Science, India from 8th-11th January 2013.  Participated and gave an oral presentation titled “Structural and functional studies of the Agr quorum sensing mechanism in Staphylococcus aureus” in the MBU-In House Symposium organized by Molecular Biophysics Unit, Indian Institute of Sciences, Bangalore, India 2012.  Participated in International conference on Recent Developments in Staphylococcus aureus Biology and Infections organized by Sir Dorabji Tata centre for research in tropical diseases, IISc, Bangalore, 2011.  Participated and presented a poster titled “Regulation of profilin by oligomerization in breast cancer” in the conference International Conference on Recent Advances in Biomedical and Therapeutic Sciences, Bundelkhand University, Jhansi, Uttar Pradesh. 2004.
  4. 4. REFERENCES 1. Prof. B. Gopal, Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India. Ph: 91-80-22933219, Fax: 91-80-23600535; E-mail: 2. Prof. Surya Satyanarayana Singh Department of Biochemistry, Osmania University, Hyderabad 500007, India. Phone #: 91-40-27097044, 91-40-27682245 (Office)/ 91-9848450411 (cell). E-mail: 3. Gayathri Arakere, Ph.D Sir Dorabji Tata Centre for Research in Tropical Diseases Indian Institute of Science Campus Bangalore 560012 Ph: 91-9739820737 E-mail: Detailed description of Post- Doctoral and PhD work are as annexures. Annexure 1 Current Project: Biocatalysis and Enzyme Bioengineering. Introduction: Biocatalysis is an emerging field where native enzymes are bioengineered to harbor important features like, high activity, stability under harsh conditions (temperature and pH), highr stereo selectivity, specificity towards many substrates etc. Bioengineered enzymes catalyze chemical reactions in cost effective and environmentally friendly manner to produce pharmaceutical products. Objective: Aim of this project is to bioengineer an (R) Alcohol dehydrogenase and w- Transaminase, with desired features for biocatalysis. Results: -Alcohol Dehydrogenases:We started working on alcohol dehydrogenases (ADH) from 2 different organisms. ADH from Leucomnostoc carnosum( showed high activity but is not stable beyond 30°C where as ADH from Burkholderia terrae (B.ter) is low on activity but very stable at higher temperatures. As a first step to bioengineer an ADH we tried crystallizing these proteins. Using Anion exchange (Q sepharose) chromatography,we purified the proteins and set up crystallization trays. We obtained crystals which diffracted upto 2Å. We are currently processing the data. W-Transaminases: For a start we plan to clone, express, purify and crystallize w-transaminase ATA-117 from arthrobacter sps, to gain insights into its activity, stability and other key features. Product inhibition being one of the drawbacks of this engineered w-transaminase, we focus on finding out the residues involved in feedback inhibition by the product. Further optimization of the enzyme will be carried out by directed mutagenesis of these residues for unobstructed function of the enzyme. To circumvent the problem of low and stable enzyme yield and the use of exorbitant inducers we plan to integrate the gene into E.coli genome with 5 core tac promoter clusters to achieve constitutive and high level gene expression.
  5. 5. Annexure 2 Current Project - We are currently working on a project to gain insights into the role of phosphorylation on Agr two component system using S.epidermidis host system. Introduction: Staphyloccocus aureus is a gram positive coccus. The expression of many virulence factors in this pathogen is regulated by peptide quorum sensing system encoded by the accessory gene regulator (Agr) operon. The Agr locus consists of two divergent transcription units driven by the promoters P2 and P3. The P2 operon encodes a two- component signaling pathway consisting of four proteins, of which AgrC is the receptor and AgrA is the response regulator where as AgrB is the processing protein and AgrD is the propeptide which is processed by AgrB and secreted out as an autoinducing peptide (AIP), which in turn binds AgrC and triggers the expression of virulent genes with the help of P3 promoter. AgrA, is the master transcriptional activator of Agr operon. The P3 transcript, RNAIII, modulates the expression of virulence genes in a RNAII (P2 transcript) dependent manner and thus strictly relies upon AgrA. AgrA is also know to activate certain leukcocidal peptides (phenol soluble modulins) in an RNAIII independent manner which contributes to a highly virulent phenotype of community associated S.aureus (M.otto 2009). It is well known that any mutations in AgrA directly affect its binding to DNA and apparent avirulent phenotype of staphylococci. Phosphorylation of AgrA was considered to be an important event in downstream signaling. It is well documented that phosphorylated AgrA shows high affinity towards both promoters and its approximately 10 times more for P2 promoter than P3(Koenig et al 2004). Recent studies by Wigneshwaraj et al with recombinant AgrA demonstrated that unphosphorylated AgrA can activate transcription from P3 promoter but not from P2 promoter. There is growing body of evidence that phosphorylated and unphosphorylated AgrA exists in dynamic equilibrium and phosphorylation might be an additional mechanism to regulate the transcriptional expression from P2 or P3 promoter. Objective: The focus of this study is to correlate the transcriptional response to the phosphorylation of histidine kinase (AgrC) and response regulator (AgrA). These studies would allow us to evaluate if signal recognition is influenced by differential phosphorylation as well as binding affinity of response regulator to P2 or P3 promoters which in turn, mediate the transcriptional regulation of virulent gene expression. To begin with, S.epidermidis AgrA and its phosphorylation mutant AgrAD59A are cloned in a shuttle vector and these constructs will be overexpressed in S.epidermidis to study how phosphorylation effects the transcriptional response by real time PCR. A GFP driven reporter plasmid will also be constructed by inserting P2/P3 promoter fused GFP. The florescence of GFP will be directly proportional to the binding affinity of AgrA/ AgrAD59A to the promoters. Annexure 3 Synopsis of Post-Doctoral Project - Structural and functional studies of proteins involved in the Agr quorum sensing machinery of Staphylococcus aureus. Introduction: Staphyloccocus aureus is a gram positive coccus. The expression of many virulence factors in this pathogen is regulated by peptide quorum sensing system encoded by the accessory gene regulator (Agr) operon. The Agr locus consists of two divergent transcription units driven by the promoters P2 and P3. The P2 operon encodes a two- component signaling pathway consisting of four proteins, of which AgrC is the receptor and AgrA is the response regulator where as AgrB is the processing protein and AgrD is the propeptide which is processed by AgrB and secreted out as an autoinducing peptide (AIP), which in turn binds AgrC and triggers the expression of virulent genes with the help of P3 promoter. Based on the primary amino acid sequence of AIP’s, S.aureus can be subdivided into 4 different Agr groups (I, II, III, IV). Remarkably, the Agr locus has diverged such that the AIP’s of the four different S.aureus groups self activate but cross-inhibit Agr expression. The
  6. 6. Agr locus is conserved throughout the Staphylococci but the autoinduction circuit consisting of signal receptor AgrC, the AIP, processed from AgrD and AgrB show significant sequence variations. Although the physiological relevance of the quorum sensing phenomenon in this coccus is still under investigation, it appears likely that the Agr system could have therapeutic potential. Objective: The focus of this project was to study receptor peptide interactions where in, we wanted to correlate the transcriptional response to an AIP stimulus with the expression and phosphorylation levels of AgrA and AgrC, which in turn, mediates transcriptional changes in virulent gene expression. To achieve these objectives, we raised polyclonal antibodies against AgrA which were used to analyse the levels of AgrA in S.aureus cell lysates at different time points under agonistic and antagonistic stimuli. RNA was isolated from different strains of S.aureus grown in activating and inhibiting conditions and Real time PCR was performed on genes which were regulated during virulence and quorum sensing Results: To analyse the response of the agonistic stimuli (activating AIP) and antagonistic stimuli (cross inhibiting peptide), RT-PCR was carried out to evaluate expression of Agr locus genes and some toxin genes at different time points. It was observed that the levels of exotoxins increased with the increase in Agr locus genes under agonistic stimuli and it decreased with addition of antagonistic stimuli. This phenomenon was also observed at protein levels measured by immunoprecipitation of AgrA. Overall, this data showed surprising consistency in terms of the strength of extracellular stimulus as evident from growth profiles to gene expression levels as well as variation in signal sequences (different Agr types). These studies thus reveal an exquisite response that is sensitive to signal type and signal intensity. Annexure 4 Synopsis of Doctoral Thesis work - Studies on the role of profilin and phosphoprofilin in cell invasion Introduction: Profilin is an actin-binding protein which either inhibits actin polymerization by sequestering actin monomers or promotes polymerization by rapid ATP exchange. Profilin interacts with PIP2 (phosphoinositide) linking signaling pathway with actin cytoskeleton. Profilin also interacts with poly (L) proline containing proteins assisting in their association with actin cytoskeleton. Recent work has showed that profilin undergoes post-translational modifications such as phosphorylation which allows it to interact with greater affinity to actin and proline rich proteins. It has been increasingly evident that profilin is associated with important signaling proteins known to be involved in transformation, apoptosis (PI3K) and tumorigenecity (PKC). Objective: Differential expression of profilin was reported in different cancers suggesting the complexity in the role of profilin in cancer and also studies supporting its role in cellular proliferation, migration and invasion differed. Thus, necessitating further studies in this area and also, as phosphorylation of profilin preferentially by PKC-ζ alters its affinity towards actin and poly-L-proline. The present study aims at understanding profilin and phosphorylated profilin's role in cell migration and invasion. Results: For an in-depth analysis on the role of profilin, profilin-1WT, R74E (actin mutant) and H133S (PLP mutant) were cloned into eGFP-C1 mammalian expression vector. Polyclonal antibodies raised against recombinant profilin-1 were used for various experiments like immunoprecipitation, western blots and immunoflourescence assays.
  7. 7. Levels of profilin, phosphoprofilin and PKCζ were analyzed in three breast cancer cell lines (MCF7, MDA-MB231 and T47D) and one normal breast cell line (MCF10A). Profilin and phosphoprofilin levels were highly elevated in cancer cell lines. mRNA levels of both profilin- 1 and profilin-2 were analyzed by RT-PCR and Realtime-PCR which were again found to be elevated in cancer cell lines compared to normal. To test the effect of overexpression of profilin 1 and its mutants (as control and for ligand interactions) on cell invasion, transfections of GFP-profilin1, R74E (actin mutant) and H133S (PLP mutant) were carried out in a weakly invasive breast cancer cell line MCF7. It was observed that GFP-wild type overexpressing cells invaded much more when compared to controls. Earlier results on platelets indicating profilin phosphorylation in the presence of PDBu and its inhibition in the presence of LY294002, confirmed that profilin phosphorylation is a downstream event with respect to PI 3-kinase activation and is mediated by PKCζ. This supports present data on increased profilin phosphorylation in breast cancer cell lines and its inhibition in the presence of LY294002 and wortmanin – PI3-kinase inhibitors. PDBu treatment of profilin overexpressing cells enhanced the invasive ability, while PI3K inhibitor – LY294002, decreased the invasion levels indicating that phosphoprofilin may be involved in cell invasion. To further confirm the results, profilin-1 SiRNA was used to silence profilin-1 in a highly invasive breast cancer cell line MDA-MB-231 and its effect was seen on invasion. Abrogation of profilin-1 expression significantly reduced the invasiveness of these highly invasive breast cancer cells in vitro. Decrease in the invasion of MDA-MB-231 cells upon ablation of profilin expression could not be rescued by addition of PDBu indicating that phosphoprofilin could be involved in enhanced migratory ability of breast cancer cells. The distribution of endogenous profilin in breast cell lines and how phosphorylation affects its localization in cells was studied by immunofluorescent analysis with anti-profilin antibodies in breast cancer and normal cell lines. This revealed distinct nuclear localization of profilin in cancer cell lines (compared to normal where profilin is uniformly distributed). Phosphoprofilin (by PDBu treatment) showed clear membrane localization. Transfection and overexpression of profilin-1 and mutants showed nuclear localization of only actin mutant (R74E) suggesting that actin might be the factor helping profilin’s cytoplasmic localization. Summary: PLP interactions are critical for profilin’s physiological functions. Increased phosphoprofilin in breast cancer indicates that profilin exists in a form that increases protein- protein interactions with specific signaling proteins involved in cell proliferation, differentiation and in the progression. Also, the data point to the fact that profilin phosphorylation regulates its localization with reference to its interaction with actin. Annexure 5 Synopsis of Dept of Science & Technology funded project - Protein-protein interaction mediated by profilin in bovine platelets: During my PhD, I also worked for 2 years on DST funded project where profilin’s interaction with other proteins was evaluated. A brief description of the project is given below. Although profilin is a small protein its biological functions are sufficiently complex. It interacts with many proteins and lots of proteins are yet to be characterized. Numerous cytoskeletal proteins and important signaling proteins such as P53 etc contain poly-L-Proline stretches within their primary structures and it is unknown whether profilin interacts with any of these proteins.
  8. 8. Profilin-protein interactions has important consequences. PLP containing proteins can recruit profilin and phosphoprofilin (high affinity to PLP) to specific cellular sites where rapid cytoskeletal reorganization is in demand. Secondly, profilin by associating with phosphoinositides including PI3,4,5P3, PI3,4P2, could bring in cytosolic proteins to the membrane where these lipids are generated, thus facilitating regulation/ amplification/ transduction of message downstream. Thus a detailed analysis of profilin and phosphoprofilin binding proteins could facilitate better understanding of the role of these novel lipids, their targets and role of cytoskeletal and nuclear proteins in regulation of such interactions. In the present study, an attempt was made to evaluate profilin’s interaction with other proteins that might be regulated by phosphorylation. To achieve this objective, bovine platelet profilin was isolated and phosphorylated with a phorbol ester like phorbol-12,13-dibutirate and then proteins that bind phosphorylated (PDBU) and unphosphorylated profilin was isolated by immunoprecipitation (using profilin antibody coupled matrix), PLP affinity chromatography (PLP coupled matrix), HAT-profilin column (Recombinant profilin with N- terminal histidine). The isolated proteins were separated by 2D electrophoresis and differentially expressed proteins are then identified by mass spectrometry.