a research proposal (r&d project) submitted to department ... · a research proposal (r&d...

Asima Bhattacharyya (PhD) of 30

A Research Proposal (R&D project)

Submitted to

DEPARTMENT OF BIOTECHNOLOGY

Ministry of Science and Technology

Govt. of India

New Delhi 110003

Title

Understanding Posttranslational Modifications and Subcellular Localization of Siah Proteins in the Context of Gastric Cancer

By

Asima Bhattacharyya (PhD) Assistant Professor

School of Biological Sciences National Institute of Science Education & Research (NISER)

Bhubaneswar 751005


PROFORMA – I

PROFORMA FOR SUBMISSION OF PROJECT PROPOSALS ON RESEARCH AND

DEVELOPMENT, PROGRAMME SUPPORT

(To be filled by the applicant)

PART I: GENERAL INFORMATION 1. Name of the Institute/University/Organization submitting the Project Proposal:

National Institute of Science Education and Research (NISER) Institute of Physics Campus, P.O. Sainik School Bhubaneswar

Odisha- 751 005

2. State: Odisha

3. Status of the Institute: An autonomous Institution of Department of Atomic Energy Govt. of India. 4. Name and designation of the Executive Authority of the Institute forwarding the application:

Prof. TK Chandrashekar Director, National Institute of Science Education & Research Bhubaneswar Odisha

5. Project Title:

6. Category of the Project (Please tick): R&D

Understanding Posttranslational Modifications and Subcellular Localization of Siah Proteins in the Context of Gastric Cancer

7. Specific Area (Please see Annexure - II): MEDICAL SCIENCES & ALLIED AREAS

(2.2 Infectious Diseases of Humans)

8. Duration: 3 Years

9. Total Cost (Rs.) 99,78,000

10. Is the project Single Institutional or Multiple-Institutional (S/M)?: Single institute

11. If the project is multi-institutional, please furnish the following: Not applicable

Name of Project Coordinator: ...............................................................................

Affiliation: ........................................................Address: …………………………………..

12. Scope of application indicating anticipated product and processes

Multiple cellular processes such as proliferation, cell cycle, cell migration, apoptosis, tumor suppression are regulated by ubiquitination and proteasomal degradation of various target proteins catalyzed by E3 ubiquitin ligase family of proteins. Recent reports indicate that posttranslational modifications alter subcellular distribution as well as functions of E3 ubiquitin ligases. Siah (seven in


absentia homolog) proteins are members of the E3 ubiquitin ligase family. These proteins have been found to be upregulated in breast, hepatic and colon cancers but their roles in the development of H. pylori-mediated gastric pathophysiology is entirely unexplored. Therefore, understanding posttranslational modifications, their effects on subcellular distribution pattern and function of Siah proteins in the context of gastric cancer could provide important information towards developing new therapies to treat gastric cancer. 13. Project Summary (Not to exceed one page. Please use separate sheet).

Background:

Gastric cancer is a major cause of cancer-related mortality in today’s world. Microaerophilic bacteria residing in human stomach, Helicobacter pylori, have been considered by the World Health Organization as the prime cause for gastric cancer. A number of genetic alterations of tumor suppressor genes, epigenetic and carcinogenic factors also contribute to gastric cancer. Despite efforts, identification of novel molecular markers for gastric cancer is still lacking which is responsible for non-availability of targeted therapies for gastric cancer. Siah proteins are lately emerging as causative factors in several human cancers but their effect on gastric cancer progression and metastasis have not been studied. Siah proteins were initially considered as tumor suppressors but recent reports have shown that Siah inhibition impairs tumor growth and metastasis in mouse models. Preliminary data generated in our laboratory indicates that Siah proteins are upregulated in H. pylori-infected gastric epithelial cells (GECs) and they have different patterns of subcellular distribution in H. pylori-infected versus non-infected GECs (unpublished data). Recent reports indicate that posttranslational modifications might regulate subcellular distribution and function of Siah proteins. Therefore, unraveling the underlying mechanisms that regulate subcellular localization of Siah proteins in H. pylori-infected gastric epithelium and their roles in cell proliferative and metastatic pathways could be of immense significance in the context of gastric cancer. Thus, we would like to study posttranslational modifications, subcellular localization, substrate-interaction pattern and function of Siah proteins in H. pylori-infected human gastric epithelium. As hypoxia is a major player in tumor development as well as metastasis and H. pylori induces a major protein expressed in hypoxic tissue, hypoxia-inducible factor 1α (HIF1α), we would also like to find out how hypoxia affects subcellular distribution and function of Siah proteins. Thus, this study can provide valuable information to design future therapeutic drugs to cure gastric cancer.

Hypothesis:

We hypothesize that H. pylori infection and hypoxia may regulate posttranslational modifications, localization and functions of Siah proteins and that can determine the pathophysiology of gastric cancer progression and metastasis.

Objectives:

1. Identification of types of post-translational modifications of Siah proteins in H. pylori-infected and hypoxic human GECs

2. Investigation of the effects of posttranslational modifications on localization of Siah proteins 3. Identifying interacting partners of Siah proteins at various subcellular compartments and finding

how these interactions affect gastric cancer progression and metastasis


Significance:

Understanding posttranslational modifications would enable us to identify the regulation of subcellular localization of Siah proteins and their effects on gastric cancer. We believe that this study will advance our knowledge about the mechanisms of gastric cancer development by unravelling the molecular regulations involved in Siah protein expression and activity. This study may also provide valuable inputs towards future drug development efforts including personalized therapy for gastric cancer.

PART II: PARTICULARS OF INVESTIGATORS (One or more co-investigators are preferred in every project. Inclusion of co-investigator(s) is mandatory for investigators retiring before completion of the project)

14. Principal Investigator: Name: Asima Bhattacharyya

Date of Birth: 8th September 1975 Sex (M/F): Female Designation: Assistant Professor Department: School of Biological Sciences Institute/University: National Institute of Science Education and Research Address: IOP campus, PO Sainik School, Bhubaneswar 751 005, Odisha, INDIA Telephone: 06742304068 Fax: 06742302436 E-mail: [email protected] Number of research projects being handled at present: 2; DBT RGYI and DST Fast Track

15. Co-Investigator Name: Subhasis Chattopadhyay, PhD

Date of Birth: 4.11.1973 Sex (M/F): M Designation: Reader Department: Biology Institute/University: National Institute of Science Education and Research (NISER) Address: Institute of Physics Campus, P.O. Sainik School, Bhubaneswar, Odisha PIN: 751 005 Telephone: 09338623351 (M); +91 (674) 2304047 Fax: +91 (674) 2302436 E-mail: [email protected] Number of Research projects being handled at present: 3

Co-Investigator

Name: Praful S Singru, PhD Date of Birth: 15.6.1974 Sex (M/F): M Designation: Assistant Professor Department: Biology Institute/University: National Institute of Science Education and Research (NISER) Address: Institute of Physics Campus, P.O. Sainik School, Bhubaneswar, Odisha PIN: 751 005 Telephone: 09556272259 (M); +91 (674) 2304120 Fax: +91 (674) 2302436 E-mail: [email protected] Number of Research projects being handled at present: 1


PART III: TECHNICAL DETAILS OF PROJECT (Under the following heads on separate sheets)

16. Introduction (not to exceed 2 pages or 1000 words)

16.1 Origin of the proposal

Approximately 700,000 people die every year from gastric cancer1. It is the fifth leading cancer in Indian males and seventh in females2. Helicobacter pylori, a slowly growing, microaerophilic, gram-negative bacterium has been identified as the main cause of gastric cancer. H. pylori colonize in stomachs of ~50% of the human population. Infected subjects have at least twofold increased risk of gastric cancer when compared with uninfected individuals3 and H. pylori gastritis is the only known universal precursor state for diffusive gastric cancer. H. pylori are broadly classified in two groups based on the presence or absence of the cytotoxin associated gene (cag) pathogenicity island (PAI). cag+ strains are more virulent, cause increased gastric inflammation, peptic ulcer and gastric cancer4-7. Phagocytes are recruited in infected gastric mucosa and actively secrete inflammatory modulators and generate reactive oxygen species (ROS) that alter gastric epithelial cell growth, and induce apoptosis. H. pylori infection can directly induce oxidative stress in GECs through generation of ROS and regulates proinflammatory cytokine production, inflammation and cell death8-11. Gastric epithelial reactive oxygen species (ROS), either endogenous or induced by H. pylori, enhance expression of hypoxia-inducible factor 1 (HIF1)12. HIF1 is the main transcription factor induced in hypoxia and is essential for survival of higher organisms during hypoxia as it transactivates homeostasis-maintaining genes13. HIF1 has two subunits- a hypoxia-inducible α subunit and a constitutively expressed β subunit. Hypoxia as well as HIF1α plays crucial role in tumor development and carcinogenesis and has been associated with malignant progression of gastric cancer.

H. pylori infection and hypoxia also induce p53 family of transcription factors that are known to suppress tumor growth14. Both Hif1α and p53 have been implicated in regulation of Siah proteins. siah, the vertebrate homologue of the Drosophila seven in absentia (sina) gene, is evolutionarily conserved from Drosophila to mammals. Siah proteins belong to E3 ubiquitin ligase family that is involved in ubiquitination and proteasome-dependent degradation of various target proteins. Humans have two siah genes, siah-1 and siah-2, whereas mice have three siah genes- siah-1a, siah-1b, and siah-2. Siah proteins are involved in various cellular processes including mitosis, neuronal plasticity, angiogenesis, development, inflammation, cell proliferation, migration, cell death, and tumor suppression15,16. Siah proteins have an N-terminal RING domain that has ligase function, two zinc finger domains, and a C-terminal substrate-binding domain. They can promote tumorigenesis by targeting Ras/ERK and hypoxia-inducible factor 1α (HIF 1α) pathways17. As both of these pathways can induce cancer development, it points to the possible use of Siah proteins as targets for treatment of cancer.

Compartmentalization of E3 ubiquitin ligases has been observed in different cell types and biological conditions. Changes in subcellular localization of Siah proteins result in their differential cellular functions. High-level of Siah1 expression in the cytoplasm results in tumor cell apoptosis18,19, and nuclear accumulation20 is associated with tumor cell proliferation. Nuclear accumulation of Siah2 is detectable in majority of hepatic cell carcinomas (HCCs) and correlates with cell proliferation, tumor progression, and distant metastasis21. An inverse relationship exists between nuclear accumulation of Siah1 and Siah2, suggesting independent mechanisms for nuclear enrichment21. What causes differential subcellular localization of E3 ubiquitin ligases is not clearly known but recent data


suggest that posttranslational modification can result in their different subcellular localization and function. There are only a few reports on the effect of phosphorylation on E3 ubiquitin ligases altering their activity. Siah1 phosphorylation by JNK increases its capacity to contribute to JNK-mediated cell death22. Siah2 phosphorylation by p38 MAPK induces its effect on HIF prolyl hydroxylases degradation resulting in inhibition of apoptosis. Such phosphorylation causes exclusion of Siah2 from the nucleus and its localization in the perinuclear region23. Phosphorylation-dependent localization perhaps serves as a molecular switch to determine substrate specificity of Siah2

Given the central role of Siah proteins in oncogenic and angiogenic pathways, Siah proteins are attractive novel therapeutic targets in H. pylori and hypoxia-induced gastric cancer. Research in our lab has indicated that both Siah proteins 1 and 2 are upregulated in gastric epithelium infected with H. pylori as well as GECs that are hypoxic (unpublished data). We have also identified that infection with H. pylori causes nuclear localization of Siah proteins whereas overexpressed Siah1 and Siah2 localizes in cytosolic punctated structures. Therefore, understanding what causes this differential distribution of Siah proteins in gastric cancer could be of immense importance as this property of Siah proteins might have functional significance in the process of gastric carcinogenesis and metastasis. We also predict that as RING domains in E3 ubiquitin ligases are cysteine rich and can bind to DNA, RNA as well as proteins, nuclear Siah proteins can have transcriptional regulatory role which might be crucial in determining the course of gastric cancer.

23. Siah2 also can extensively self auto-ubiquitinate which results in its degradation under certain conditions21,24.

16.2 (a) Rationale of the study supported by cited literature

We are proposing that posttranslational modification and subcellular distribution pattern of Siah proteins determine their course of function in H. pylori-infected GECs. The rationale for the hypothesis comes from a few studies on this family of proteins. It was long been suggested that changes in subcellular localization of E3 ubiquitin ligases are associated with differential cellular functions. This is supported by recent reports which show that an E3 ubiquitin ligase, parkin, translocates into the nucleus after DNA damage where it is involved in DNA repair25. Siah molecules also shuttle with S-nitrosylated GAPDH into the nucleus where they facilitate nuclear protein degradation26. Earlier considered as tumor suppressors, Siah proteins have recently been found to induce tumor growth and metastasis in mouse models27. Our lab has identified upregulation of both Siah1 and Siah2 in H. pylori-infected and hypoxic GECs (unpublished data). Therefore, they are likely to have roles in regulation of gastric cancer development and metastasis and the present study aims to understand how H. pylori or hypoxia-mediated posttranslational modifications affect subcellular distribution pattern and function of Siah proteins in gastric cancer development. Thus, this study may provide useful information to design therapeutic strategies to cure gastric cancer.

16.2 (b) Hypothesis

We hypothesize that post-translational modifications of Siah proteins control their subcellular localization patterns and functions in H. pylori-infected and hypoxic GECs which regulate gastric cancer progression and metastasis.

16.2 (c) Key questions (definition of the problem).


• What are the post-translational modifications of Siah proteins in H. pylori-infected and hypoxic GECs and which signaling pathways regulate these modifications?

• How these modifications affect localization and substrate binding of Siah proteins? • Whether one posttranslational modification changes the effect of other posttranslational

modifications of Siah proteins in gastric cancer cells? • What are the roles of modified Siah proteins in gastric cancer dissemination?

16.5 Current status of research and development in the subject (both international and national status)

International status:

Seven in absentia (Sina) protein was identified as an essential factor for formation of the R7 photoreceptor in Drosophila almost twenty years ago28. Since then, different laboratories from around the world have contributed to enrich our knowledge about divergent cellular functions of Siah proteins and identified their evolutionarily conserved expression pattern. Although Siah proteins have both tumor suppressive and oncogenic functions27, recent reports potentiate the role of Siah proteins as to have tumor-promotive functions. Tang and coworkers showed positive correlation of Siah expression with ERK activation and growth of human pancreatic tumor cells29. Loss of Siah was also linked with impaired Ras function30. So far, there is only one report of a low frequency of inactivating mutation of Siah proteins in gastric cancer31. Surprisingly, studies linking H. pylori infection with Siah-1/2 expression are still lacking. There are a few reports on the effect of posttranslational modifications of E3 ubiquitin ligase function. Donner et al showed that phosphorylation of Mdm2 alters its subcellular localization and function32. Another E3 ubiquitin ligase, parkin, was found to translocate to the nucleus and promote DNA33. High-level of Siah1 expression in the cytoplasm was found to be associated with tumor cell apoptosis, but reduced expression and nuclear accumulation were linked with tumor cell proliferation18,19. This intrigued us to identify how compartmentalization can influence functions of Siah proteins. Dr. Greene’s group has found JNK to phosphorylate Siah1 and cause cell death (JBC, 2006, 281, 303-312). That p38 MAPK causes Siah2 phosphorylation and can induce its effect on PHD3 degradation was confirmed by Ze’ev Ronai lab. The same group also reported that phospho-Siah2 is excluded from the nucleus after its phosphorylation by p38MAPK and is localized in the perinuclear region23. They predicted that phosphorylation-dependent localization perhaps determines substrate specificity of Siah2. Snyder et al found that Siah1 transports GAPDH to the nucleus and takes part in a novel cell death cascade which suggests that Siah proteins may have additional roles in cellular processes34. Although nucleolar structures convert E3 ubiquitin ligases to their inactive forms, nuclear Siah proteins have been found to remain active in hepatic cancer cells and are key factors in regulating tumor cell viability and motility20. Other than its nuclear and cyoplasmic compartmentalization, presence of enzymatically active Siah1 in early endosomes has been reported35. Effect of other posttranslational modifications such as acetylation on Siah proteins has not been studied yet. A review by Dr. E Seto36 informed that acetylation of a protein might crosstalk with phosphorylation. This captures our interest to address the role of multiple posttranslational modifications on subcellular localization, substrate binding and functions of Siah proteins in gastric cancer. National status: Several research groups in India are currently working in the field of gastric pathologies associated with H. pylori and the PI recognizes many strong H. pylori groups working in India. Dr. AK Mukhopadhyay of National Institute of Cholera and Enteric Diseases, Kolkata are working on genomics and molecular biological aspects of H. pylori infection. Dr. S Swarnakar of Indian Institute of Chemical Biology, Kolkata is actively working on molecular mechanisms of H. pylori-associated gastroenteropathies and pathogenicity factors of H. pylori. Prof. GB Nair of THSTI, has several research articles on genomics and molecular aspects of H. pylori infection. Dr. J Basu and Dr. M


Kundu of Bose Institute, Kolkata are working on regulation of host cell signaling pathways by H. pylori. Physicians, Dr. SP Singh of SCB Medical College Cuttack and Dr. S Nayak of AHRCC, Cuttack are actively engaged in research on gastric cancer and will provide us gastric biopsy samples. We will be highly obliged to all of these experts for their valuable inputs for establishing this project and in all our future endeavors. . Recently Dr. AB Datta of Bose Institute, Kolkata has started research to obtain atomic resolution structures of E3 ligases in complex with their substrates and partner E2s but apparently no other Indian group is working on Siah proteins. Thus we are feeling enthusiastic to start our research on the molecular regulation and role of Siah proteins in H. pylori-mediated gastric carcinogenesis. 16.6 The relevance and expected outcome of the proposed study

The proposed study would help us understand the regulation of subcellular distribution of Siah 1/2 and their effect on cellular functions of Siah proteins in H. pylori-infected and hypoxic gastric tissue. As H. pylori gastritis resembles early stages of gastric cancer and hypoxia is induced in the infected gastric epithelium, this study would particularly reveal the role of unmodified and posttranslationally-modified Siah proteins in gastric carcinogenesis. Subsequently, it has the potential to uncover the effect of combinations of post-translational modifications on the basic properties and functions of Siah proteins. This study will also provide a good step towards understanding how post-translational modifications can be used as molecular switches for controlling Siah functions which can be useful for drug-designing attempts.

16.7 Preliminary work done so far:

As mentioned in the “Origin of the Proposal” section, we have found that both Siah 1 and Siah2 are induced in H. pylori-infected or hypoxic GECs (unpublished data). We have also identified that infection with H. pylori causes nuclear localization of Siah proteins whereas overexpressed Siah1 and Siah2 are localized in cytosolic vesicular structures. Siah-1 and Siah-2 plasmid constructs have been generated. The PI has extensive knowledge in molecular biology, biochemical techniques and signalling events in host-pathogen interactions as reflected from peer reviewed publications. After joining NISER in July 2010, the PI has already acquired various gastric cancer cell lines and H. pylori strains from India and abroad and building up laboratory facilities for advanced research. 17. Specific objectives (should be written in bulleted form, a short paragraph indicating the methods to be followed for achieving the objective and verifiable indicators of progress should follow each specific objective)

• Identification of types of post-translational modifications of Siah proteins in H. pylori-infected and hypoxic human GECs Phosphorylation is the most prevalent post-translational modification associated with regulation of cellular function. It has been reported to alter subcellular localization of Siah proteins. Therefore, the phosphorylation status of Siah1/2 in gastric cancer would be assessed by various methods. For this purpose, GECs exposed to hypoxia or infected with H. pylori will be used or epithelial cells isolated from H. pylori-infected or gastric cancer patient biopsies will be assessed for differential localization of Siah 1/2. We will employ western blotting, fluorescent microscopy and immunohistochemistry for this purpose. If phosphorylation is detected then the residues phosphorylated will be identified by using phospho-specific antibodies. To understand whether these proteins have other modifications (such as ubiquitination or acetylation) or not, similar method will be employed. To understand the signaling pathways that induce posttranslational modification(s), we would use analyzing tools and databases and various techniques mentioned above.


• Investigation of the effects of posttranslational modifications on localization of Siah proteins We would like to determine subcellular distribution of Siah proteins in the cultured GECs under various experimental conditions as well as in gastric biopsies obtained from gastric cancer patients. Experimental conditions will include induction of chronic and acute hypoxia as well as H. pylori infection and changes in Siah 1/2 distribution under these conditions will be assessed by fluorescence and confocal microscopy or immunohistochemistry. Point mutations at involved residues will be done to address the effect of a particular modification on cellular distribution of the proteins. Coimmunoprecipitation, in situ proximity ligation assays and in vitro degradation assays will be undertaken to identify binding partners. Whether nuclear Siah proteins have any DNA-binding and transcriptional regulatory role or not will be detected by luciferase assays. Multisite posttranslational modifications appear to act in sequential and/or combinatorial ways. Besides affecting neighboring modifiable residues, one posttranslational modification has the potential to crosstalk with surrounding modifiable sites. Effect of multiple posttranslational modifications of Siah proteins will be identified. Phospho and acetylation prediction programs will be utilized for this purpose to predict effect of acetylation on phosphorylation and vice versa and that will be matched in our experimental conditions. Once the types of modifications and residues involved are identified, the effect of these modifications would be tested. For this purpose, both wild type and the mutant proteins (lacking the modifiable residues) would be expressed ectopically and their localization and interaction pattern with substrates would be monitored in H. pylori-infected or hypoxic gastric tissues.

• Identifying interacting partners of Siah proteins at various subcellular compartments and finding how these interactions affect gastric cancer progression and metastasis Effect of posttranslational modifications of Siah proteins in H. pylori-infected or hypoxic gastric cancer epithelium will be identified by overexpressing WT and mutant constructs and stable transfected cell lines generated thereafter. Interaction pattern of Siah 1/2 substrates with the post-translationally modified proteins and unmodified proteins will also be identified employing wild type overexpression plasmid constructs and mutated constructs. Wound healing assay and cell migration assays will be performed to identify any alteration of invasiveness in the stable cells. We would also like to perform experiments in mice once our institute gets CPCSEA approval. Final approval of CPCSEA is pending.

18.1 Work Plan: should not exceed 3-4 pages (the section can be divided according to the specific aims and under each specific aim, the following should be stated clearly as sub headings)

FOR OBJECTIVE 1- Phosphorylation is reported to regulate function and subcellular localization of Siah proteins. We would assess phosphorylation status of Siah1/2 in GECs. For this purpose, GECs exposed to hypoxia or infected with H. pylori will be used or epithelial cells isolated from H. pylori-infected or gastric cancer patient biopsies will be assessed for differential localization of Siah 1/2. We would employ western blotting, fluorescence microscopy or immunohistochemistry for this purpose. If phosphorylation is detected then the residues phosphorylated would be identified by using phospho-specific antibodies. We would identify the kinases and pathways responsible for phosphorylation. To understand if the protein has other modifications (such as ubiquitination or


acetylation) similar methods would be employed or ectopically expressed proteins would be analyzed by mass-spectrometry. Once the modifications are identified in the over-expressed proteins, we would employ site-directed mutagenesis to understand the effect of any particular posttranslational modification on protein localization. Signaling pathways responsible for posttranslational modification(s) will be identified by using analyzing tools and databases and various techniques mentioned above.

FOR OBJECTIVE 2- Subcellular distribution of Siah proteins will be analyzed under various experimental conditions in cultured GECs as well as in gastric biopsies obtained from gastric cancer patients. For this, cells will be exposed to hypoxia and H. pylori infection and changes in Siah 1/2 distribution under these conditions will be assessed by fluorescence and confocal microscopy, immunohistochemistry. Post-translational modifications will be detected by using specific antibodies. Once the modifications are identified in the over-expressed proteins, we would employ site-directed mutagenesis to understand the effect of any particular posttranslational modification on protein localization. Coimmunoprecipitation, in situ proximity ligation assays and in vitro degradation assays will be undertaken to identify binding substrates. One posttranslational modification has the potential to crosstalk with surrounding modifiable sites. So, we would like to understand the effect of multiple posttranslational modifications of Siah proteins. Once the types of modifications and residues involved are identified, the effect of these modifications would be tested. For this purpose, both wild type and the mutant proteins lacking the modifications would be expressed ectopically and their localization and interaction pattern with substrates would be monitored. Phosphorylation and acetylation prediction programs will be utilized to predict effect of acetylation on phosphorylation and vice versa. FOR OBJECTIVE 3- Effect of posttranslational modifications of Siah proteins in H. pylori-infected or hypoxic gastric cancer epithelium will be identified by overexpressing WT and mutant constructs and stable transfected cell lines generated thereafter. Interaction pattern of Siah 1/2 substrates with the post-translationally modified proteins and unmodified proteins will also be identified employing wild type overexpression plasmid constructs and mutated constructs. Wound healing assay and soft agar assays will be performed to identify any alteration of invasiveness in the stable cells. If any profound change is noticed, xenografts in mouse will be done to assess metastatic effect of the posttranslational modifications.

18.2 Connectivity of the participating institutions and investigators (in case of multi-institutional projects only) 18.3 Alternate strategies (if the proposed experimental design or method does not work what is the alternate strategy) Involvement of Siah-1 and Siah-2 in various cancers is already established. Recently gastric cancer has been linked with Siah31. High-level of Siah1 accumulation in the nucleus are associated with tumor cell proliferation20 and nuclear accumulation of Siah2 is detectable in majority of hepatic cell carcinomas (HCCs) and correlates with tumor progression, and metastasi21. Our preliminary data show nuclear localization of Siah proteins but the mechanisms and effect of that are yet to be identified. Accordingly, we hypothesized and designed the aforementioned experiments. The strategy mentioned here is routinely used for identifying post-translational modifications. The PI is having extensive knowledge of all mentioned techniques and is confident about the proposed assay methods.


REFERENCES

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23. Khurana A, Nakayama K, Williams S et al. Regulation of the Ring Finger E3 Ligase Siah2 by p38 MAPK. J Biol Chem. 2006;281;35316–35326. 24. Yego EC, and Mohr S. Siah-1 Protein is necessary for high glucose-induced glyceraldehyde-3-phosphate dehydrogenase nuclear accumulation and cell death in Muller cells. J Biol Chem. 2010;285;3181-3190. 25. Kao SY. DNA damage induces nuclear translocation of parkin. J Biomed Sci 2009;16: 67-75. 26. Hara MR, Agarwal N, Kim SF et al. S-nitrosylated GAPDH initiates apoptotic cell death by nuclear translocation following Siah1 binding. Nat Cell Biol 2005;7:665–674. 27. House CM, Möller A, Bowtell DD. Siah proteins: novel drug targets in the Ras and hypoxia pathways. Cancer Res. 2009;69:8835-8838. 28. Carthew RW, Rubin GM. Seven in absentia, a gene required for specification of R7 cell fate in the Drosophila eye. Cell 1990;63;561-577. 29. Schmidt RL, Park CH, Ahmed AU et al.. Inhibition of RAS-mediated transformation and tumorigenesis by targeting the downstream E3 ubiquitin ligase seven in absentia homologue. Cancer Res 2007;67:11798–11810. 30. Nadeau RJ, Toher JL, Yang X et al. Regulation of Sprouty2 stability by mammalian Seven-in-Absentia homolog 2.J Cell Biochem. 2007;100: 151–160. 31. Kim CJ, Cho YG, Park CH, et al. Inactivating mutations of he Siah-1 gene in gastric cancer. Oncogene. 2004;23:8591–8596. 32. Mayo LD and Donner DB. A phosphatidylinositol 3-kinase/Akt pathway promotes translocation of Mdm2 from the cytoplasm to the nucleus.Proc. Natl. Acad. Sci. U. S. A 2001;98:11598–11603. 33. Hu G, Zhang S, Vidal M et al. Mammalian homologs of seven in absentia regulate DCC via the ubiquitin-proteasome pathway.Genes Dev 1997;15:2701–2714. 34. Hara MR and Snyder SH. Nitric oxide-GAPDH-Siah: a novel cell death cascade. Cell Mol Neurobiol 2006;26;527-538. 35. Wheeler TC, Chin LS, Li Y et al. Regulation of synaptophysin degradation by mammalian homologues of seven in absentia. J Biol Chem 2002;277;10273-10282. 36. Yang XJ and Seto E. Lysine acetylation: codified crosstalk with other posttranslational modifications. Mol Cell, 2008, 31, 449-61.

19. Timelines: (Please provide quantifiable outputs)

Period of study

Achievable targets

6 Months

• Purchase of equipment and chemicals

• Identification of posttranslational modifications of Siah proteins

• Prediction of probable signaling pathways responsible for those modifications


12 Months • Generation of mutants lacking modifications

• Identification of residues involved in post-translational modifications and generation of stable cell lines containing mutations lacking those modifications

• Assessing signaling events leading to the posttranslational modifications by using inhibitors and shRNA

18 Months • Identifying effect of posttranslational modifications on localization of Siah proteins

24 Months • Siah substrates will be identified by proximity ligation assay, confocal microscopy, coimmunoprecipitation studies

• Biopsy specimen from gastric cancer patients and patients with H. pylori infection will be examined for evidence of in vivo modifications. After we get CPCSEA approval, mice experiments will be done

30 Months

• Effect of posttranslational modifications of Siah proteins on invasiveness and EMT will be identified by soft agar assay or scratch assay

• Biopsy specimen from gastric cancer patients and patients with H. pylori infection will be examined for evidence of in vivo modifications. After we get CPCSEA approval, mice experiments will be done

36 Months

• Preparation of manuscripts and submission to appropriate journal for publication

20. Name and address of 5 experts in the field:

Sr.No. Name Designation Address

1 Dr. AK Mukhopadhyay

Scientist D National Institute of Cholera and Enteric Diseases P-33, CIT Road Scheme XM P.O. Box 177 Beliaghata Kolkata- 700010 Tel: 033-23633865 Email: [email protected]

2 Dr. S Swarnakar Senior Scientist

Drug Development Diagnostics & Biotechnology Division Indian Institute of Chemical Biology Raja SC Mullick Road Jadavpur, Kolkata- 700032 Fax: 033-24735197 Email: [email protected], [email protected]

3 Dr. M Kundu Senior Department of Chemistry Bose Institute


Professor 93/1, Acharya Prafulla Chandra Road

Kolkata-700009 Tel: 033-23506619 Email: [email protected]

4. Dr. J Basu Senior Professor

Department of Chemistry Bose Institute 93/1, Acharya Prafulla Chandra Road Kolkata-700009 Tel: 033-23506619 Email: [email protected]

5. Dr. GB Nair Professor Executive director Translational Health Science and Technology Institute Plot No. 496, Phase-III, Udyog Vihar Gurgaon - 122016, Haryana Tel: 124-2876400 Email: [email protected]

PART IV: BUDGET PARTICULARS

Budget (In Rupees)

A. Non-Recurring (e.g. equipments, accessories, etc.)

S. No. Item Year 1 Year 2 Year 3 Total

1 Semi dry blotting apparatus with power pack

2,00,000 0 0 2,00,000

2 Vertical tetra electrophoresis system 90,000 0 0 90,000

3 DNA gel electrophoresis system 45,000 0 0 45,000

4 Luminometer with multilabel tester 9,00,000 0 0 9,00,000

5 Inverted fluorescence microscope with ccd camera, software, and attachments

15,00,000 0 0 15,00,000

6 Cytocentrifuge 9,00,000 0 0 9,00,000

7 Liquid N2 dewars, refrigerator, small laboratory equipment

2,00,000 0 0 2,00,000

Sub-Total (A) 38,35,000

B. Recurring


B.1 Manpower (See guidelines at Annexure-III)

S. No.

Position No. Consolidated Emolument

Year 1 Year 2 Year 3 Total

1 Not required N/A

Sub-Total (B.1) = 0

B.2 Consumables S. No.

Items Quantity Year 1 Year 2 Year 3 Total

1 DNA isolation and cloning kit, mutagenesis kit and other kits as the work will demand. Oligo synthesis, sequencing, mass spectrometry and analysis

As required 5,00,000 5,00,000 5,00,000 15,00,000

2 Custom antibody preparation, consumables for animal experiments

As required 2,00,000 2,00,000 2,00,000 6,00,000

3. Culture plates, cell culture media, flasks and other plastic wares

As required 3,00,000 3,00,000 3,00,000 9,00,000

4 Antibodies and fine chemicals

As required 3,00,000 3,00,000 3,00,000 9,00,000

Sub-Total (B.2) = 39,00,000

Other items Consolidated Emolument

Year 1 Year 2 Year 3 Total

B.3 Travel 30,000 50,000 50,000 1,30,000

B.4 Contingency 1,50,000 1,50,000 1,50,000 4,50,000

B.5 Overhead @20% 10,63,000 3,00,000 3,00,000 16,63,000

Sub-total of B (B.1+B.2+B.3+B.4+B.5)

25,43,000 18,00,000 18,00,000 61,43,000

Grand Total (A + B) 63,78,000 18,00,000 18,00,000 99,78,000

Note : Please give justification for each head and sub-head separately mentioned in the above table.


Justification for Equipment (A)

Semi dry blotting apparatus with power pack: This equipment will be heavily used for western blotting

Vertical tetra electrophoresis system: This project involves detection of protein by SDS-PAGE analysis. For detection of post-translational modifications, preparation of samples for mass-spectrometry we require SDS-PAGE apparatus. We need to run multiple gels at a time for faster progress. For this, a vertical tetra electrophoresis unit is absolute requirement

DNA gel electrophoresis system: To analyze PCR reactions, mutataions and DNA constructs, we need a DNA gel electrophoresis unit

Luminometer with multilabel tester: Whether nuclear Siah proteins have any DNA-binding and transcriptional regulatory role or not will be detected by luciferase assays. For this luminometer will be useful

Inverted fluorescence microscope with ccd camera and attachments: Subcellular localizations will be identified by fluorescence microscopy and this equipment will be extensively used for this research

Cytocentrifuge: Thin layer of scantily available GECs from biopsy samples will be prepared by using cytospin. This instrument will ensure better slide preparation for immunohistochemistry or fluorescence microscopy.

Liquid N2 dewars, refrigerator, small laboratory equipment: We need money to buy liquid N2 dewars to store biopsy samples as well as gastric cancer cell lines. A refrigerator is required for storage of costly chemicals and kits. Other than these, small equipments like pH meter, shaker, vortex, low speed spin equipment for PCR reactions, pipetteman are required to conduct this work. We will buy depending on need.

Justification for manpower (B): I would like to take institute fellows for this work and so no salary support for JRF/SRF is required.

Justification for Consumables (C): It would be spent for expensive commercial kits for isolating DNA and for site directed mutagenesis kit. Also purification of expressed protein would require expensive reagents. Part of the money would be used for purchasing costly kits and chemicals, cell culture reagents, plastic wares, glasswares, consumables for sequencing and mass spectrometry analysis. Custom-made antibodies and required items for maintenance of animals will be purchased from this head.

Justification for travel (B3)

The fund will be used to collect gastric biopsy samples from nearby hospitals, cover the expenditure for attending symposia/conferences related to the project as well as to present research data by the PI and students.

Justification for contingency (B4)

This fund will be used for purchasing stationary items, computer and its peripherals, to cover the cost for postage and preparation, submission and publication charges of project reports and


manuscripts. Some money will be utilized for repairing and procurement of spare parts and service warranty for the laboratory instruments. Cost of analysis of data from external facilities/ laboratories will be paid from contingency. Purchasing reagents on an emergency basis can also utilize contingency money.

Justification for overhead charges (B5)

National Institute of Science Education and Research charges overhead at the rate of 20% of the total recurring and non-recurring grant value requested. This amount will go to the institute.

In case of multi-institutional project, the budget estimate to be given separately for each institution.

None

PART V: EXISTING FACILITIES

Resources and additional information

1. Laboratory: State-of-the-art research instruments and laboratory facilities required for this project are available at NISER, Bhubaneswar.

a. Manpower PhD students: 2, and another student is doing lab rotation.

Three Lab attendants are provided from the Institute to help in washing glass wares, plastic wares, cleaning instruments, autoclaving, making buffers etc. There are good numbers of experienced and young scientists having expertise in Immunology, Microbiology, Molecular Biology, Cell Biology and other disciplines in Biology in the School of Biological Sciences at NISER who are willing to support as required.

b. Equipment: At present, School of Biology, NISER has the following major instruments:

No Item Status

1 Centrifuge and Ultracentrifuge Yes

2 Fluorescence Activated Cell Sorter (FACS) Yes

3 Surface Plasmon Resonance (SPR) Yes

4 DNA sequencer Yes

5 Real time PCR, PCR machine Yes

6 High Performance Liquid Chromatography (HPLC) Yes

7 Cell culture incubator Yes

8 (Typhoon) Phospho-imaging and imaging of fluorescent dyes and Chemiluminiscent

Yes


9 Confocal Microscope Yes

10 Microarray reader Yes

11 BSL II cell culture hood Yes

12 CO2 incubator Yes

13 Animal house Yes

c. Other resources:

Sl. No. Item Present

1. Workshop Yes

2. Water and electricity Yes

3. Laboratory space and furnitures

Yes

4. Air-conditioned equipment room

Yes

5. Telecommunication Yes

6. Administrative and secretarial support

Yes

7. Library Yes


PART VII: PROFORMA FOR BIOGRAPHICAL SKETCH OF INVESTIGATORS

Provide the following information for the key personnel in the order listed on PART II. Follow this format for each person. DO NOT EXCEED THREE PAGES

Name: Asima Bhattacharyya, PhD

Designation: Assistant Professor

Department/Institute/University: Biology, National Institute of Science Education and Research

Date of Birth: 8.9.1975 Sex (M/F): F SC/ST: No

Education (Post-Graduation onwards & Professional Career)

Sl No.

Institution Place

Degree Awarded

Year Field of Study

1. University of Calcutta MSc 1998 Physiology

2. Bose Institute PhD, conferred by Jadavpur University

2000-2005, degree awarded in January 2006

Science

3. Department of Gastroenterology, University of Virginia

Postdoctoral Research

June 2005- June 2006

Microbial Pathogenesis

Position and Honors:

Position and Employment (Starting with the most recent employment)

Sl No. Institution

Place

Position From (Date) To (date)

1. National Institute of Science Education and Research, Bhubaneswar

Asst. Professor 6th June

2010

Till date

Honors/Awards: • National Scholarship from Government of India, 1993-1996

• Cleared the National Eligibility Test (NET) for Lectureship conducted by CSIR/UGC, June 1999

• Sir Nil Ratan Sircar Prize from Bose Institute for outstanding research work, 2002

• Crohn’s and Colitis Travel Scholarship to attend Microbes and Mucosal Immunity Conference, VA, 2006


• AGA travel award to attend the symposium on “Host-microbial interactions in digestive

health and disease” held on March 24-26, Marina del Rey, CA, 2006

• 1st place award for the poster “Alterations of Oxygen Balance Associated with Gastric Inflammation and the Regulation of Gastric Epithelial Function” presented at Division of Gastroenterology & Hepatology, Department of Medicine, University of Virginia, 2006

• AGA-travel award to attend GI Response to Injury conference in Montebello, Canada (Oct 3-7), 2008

• Poster of Distinction by the American Gastroenterological Association (AGA) for the poster “Apurinic/apyrimidinic endonuclease (APE)-1/redox factor (Ref)-1 enhances hypoxia inducible factor (HIF)-1α activation in H. pylori-infected gastric epithelium but inhibits HIF-1α-mediated apoptotic events” presented at Digestive Diseases Week, San Diego, May 17-22, 2008

Professional Experience and Training relevant to the Project PI has expertise in studying host-pathogen interaction, apoptosis and cell signaling events, immunopathogenesis of infection, inflammation and cancer.

Animal and human physiology and techniques were learnt during MSc in Physiology. Helicobacter pylori and Mycobacterium avium-mediated host responses were studied and during that time various molecular biological, biochemical and microbiological techniques were learnt. As a research associate at University of Virginia, the PI gained further knowledge in gastric cell biology, hypoxia and infection. Postdoctoral research has enabled the PI to gain further hands on experience in handling BSL1 and BSL2 pathogens, biochemical assays and techniques, siRNA and shRNA-based studies, RNA and protein purifications as well as several cell culture techniques. With all these trainings, the PI is confident that she has the experience, knowledge and scientific concept required to carry out this project towards success.

B. Publications (Numbers only)

Books: 1 Research Papers, Reports: 20 General articles: Patents: Others (Please specify): one article submitted and another one is under preparation

Selected peer-reviewed publications (Ten best publications in chronological order)

1. Bhattacharyya, A, Pathak, S, Datta, S, Chattopadhyay, S, Basu, J, Kundu, M, Mitogen-activated protein kinases and nuclear factor-kappaB regulate Helicobacter pylori-mediated interleukin-8 release from macrophages. Biochem J. 368:121-129, 2002

2. Bhattacharyya, A, Pathak, S, Kundu, M, Basu, J, Mitogen-activated protein kinases regulate Mycobacterium avium-induced tumor necrosis factor-alpha release from macrophages. FEMS Immunol Med Microbiol.34:73-80, 2002

3. Bhattacharyya, A, Pathak, S, Basak, C, Law, S, Kundu, M, Basu, J, Execution of macrophage apoptosis by Mycobacterium avium through apoptosis signal-regulating kinase 1/p38 mitogen-activated protein kinase signaling and caspase 8 activation. J. Biol Chem. 278:26517-525, 2003

4. Pathak, SK, Bhattacharyya, A, Pathak, S, Basak, C, Mandal, D, Kundu, M, Basu, J, Toll-like receptor 2 and mitogen- and stress-activated kinase 1 are effectors of Mycobacterium avium-induced cyclooxygenase-2 expression in macrophages. J. Biol Chem. 279:55127-55136, 2004


5. O’Hara, AM, Bhattacharyya, A, Mifflin, RC, Smith, MF, Ryan, KA, Scott, KG-E, Naganuma, M, Casola, A, Izumi, T, Mitra, S, Ernst, PB, and Crowe, SE, Interleukin-8 induction by Helicobacter pylori in gastric epithelial cells is dependent on apurinic/apyrimidinic endonuclease-1/redox factor-1. J. Immunol. 2006 177: 7990-7999, 2006

6. Pathak, SK, Basu, S, Basu, KK, Banerjee, A, Pathak, S, Bhattacharyya, A, Kaisho, T, Kundu, M, Basu, J, Direct extracellular interaction between the early secreted antigen ESAT-6 of Mycobacterium tuberculosis and TLR2 inhibits TLR signaling in macrophages. Nat Immunol.: 8: 610-8, 2007

7. Basu, S, Pathak, S, Pathak, SK, Bhattacharyya, A, Banerjee, A, Kundu, M, Basu, J, Mycobacterium avium-induced matrix metalloproteinase-9 expression occurs in a cyclooxygenase-2-dependent manner and involves phosphorylation- and acetylation-dependent chromatin modification. Cell Microbiol: 9: 2804-16, 2007

8. O’Hara, AM, Bhattacharyya, A, Bai, J, Mifflin, RC, Ernst, PB, Mitra, S, Crowe, SE, TNF-α-induced IL-8 expression in gastric epithelial cells: role of reactive oxygen species and AP endonuclease-1/redox factor (REF)-1. Cytokine: 46: 359-369, 2009

9. Bhattacharyya, A, Chattopadhyay, R, Cross, JV, Mitra, S, Ernst, PB, Bhakat, KK and Crowe, SE, Acetylation of Apurinic/Apyrimidinic Endonuclease-1 Regulates Helicobacter pylori-Mediated Gastric Epithelial Cell Apoptosis. Gastroenterology: 136: 2258-2269, 2009

10. Chattopadhyay, R, Bhattacharyya, A, Crowe SE, Dual regulation by AP endonuclease-1 inhibits gastric epithelial cell apoptosis during Helicobacter pylori infection. Cancer Research: 70: 2799-808, 2010

List maximum of five recent publications relevant to the proposed area of work.

1. Bhattacharyya, A, Pathak, S, Datta, S, Chattopadhyay, S, Basu, J, Kundu, M, Mitogen-activated protein kinases and nuclear factor-kappaB regulate Helicobacter pylori-mediated interleukin-8 release from macrophages. Biochem J. 368:121-129, 2002

2. O’Hara, AM, Bhattacharyya, A, Mifflin, RC, Smith, MF, Ryan, KA, Scott, KG-E, Naganuma, M, Casola, A, Izumi, T, Mitra, S, Ernst, PB, and Crowe, SE, Interleukin-8 induction by Helicobacter pylori in gastric epithelial cells is dependent on apurinic/apyrimidinic endonuclease-1/redox factor-1. J. Immunol. 2006 177: 7990-7999, 2006

3. Bhattacharyya, A, Chattopadhyay, R, Cross, JV, Mitra, S, Ernst, PB, Bhakat, KK and Crowe, SE, Acetylation of Apurinic/Apyrimidinic Endonuclease-1 Regulates Helicobacter pylori-Mediated Gastric Epithelial Cell Apoptosis. Gastroenterology: 136: 2258-2269, 2009

4. Chattopadhyay, R, Bhattacharyya, A, Crowe SE, Dual regulation by AP endonuclease-1 inhibits gastric epithelial cell apoptosis during Helicobacter pylori infection. Cancer Research: 70: 2799-808, 2010

5. Bhattacharyya, A, Chattopadhyay, R, Hall, EH, Membrahtu, ST, Ernst, PB, Crowe, SE, Mechanism of hypoxia-inducible factor 1 alpha-mediated Mcl1 regulation in Helicobacter pylori-infected human gastric epithelium. Am J Physiol Gastrointest Liver Physiol: 299: G1177-86, 2010



Provide the following information for the key personnel in the order listed on PART II. Follow this format for each person. DO NOT EXCEED THREE PAGES

Name: Subhasis Chattopadhyay Designation: Reader-F

Department/Institute/University:

School of Biological Sciences, National Institute of Science Education and Research

Date of Birth: 04 Nov. 1973 Sex (M/F) : M SC/ST: No


Sl No.

Institution Place

Degree Awarded

Year Field of Study

1 University of Calcutta,

INDIA

M.Sc. 1995-1997 Zoology

2 Indian Institute of Chemical

Biology/ Jadavpur University, India

Ph.D. 1998-2003

Immunology

3 Department of Medicine,

University of Connecticut Health

Center, USA

Post-

Doc: 1st

2003-2006

Cancer

Immunology

4. Department of Immunology,

University of Connecticut Health

Center, USA

Post-

Doc: 2nd

2006-2008

Ocular Immune

privilege and

Cellular

Immunology

Position and Honors

Position and Employment (Starting with the most recent employment)

Sl No. Institution/Place Position From (Date) To (date)

1 NISER, School of Biological Sciences, India

Assistant Professor

Research and Teaching (UG and PG students)

June 2009- March 2012

2 NISER, School of Biological Sciences, India

Reader-F Research and Teaching (UG and PG students)

March 2012- till date


Honors/Awards

• Sudev Bhusan Ghosh Young Scientist Award: conducted by The Zoological society, Kolkata, India, in 2002.

• “Rapid Grant for Young Investigators” (as PI) for the grant proposal entitled “Studies on Toll Like Receptor (TLR) response in T cells” by DBT, Ministry of Science and Technology, India, 2010.

Professional Experience and Training relevant to the Project: Co-PI (Dr. S. Chattopadhyay) has the expertise in cellular assays especially in Infection Immunity and Cancer Immunology. The co-PI has worked with in vitro system in human and mice cell lines and primary cells and also in vivo work in mice model. Moreover, the co-PI has the expertise to analyze the tumor and infection-associated pathobiology and immunology.

B. Publications Numbers only: 22

Books: 1 Research Papers, Reports: 21 General articles: N/A

Patents: NIL Others (Please specify): N/A


1. Pais R , Bhowmick S, Chattopadhyay S, Lemire Y, Sharafieh R, Yadav R, O’Rourke J, Cone RE An Intracameral Injection of Antigen induces in situ Chemokines and Cytokines required for the generation of circulating immunoregulaory monocytes. PlosOne, 7: 2012, e43182

2. Cone RE, Chattopadhyay S, Sharafieh R, Lemire Y, O’ Rourke J, Flavell RA, and Clark RB. T cell sensitivity to TGF- β is required for the effector function but not the generation of splenic CD8+ regulatory T cells induced via the injection of antigen into the Anterior Chamber. Int Immunol. 2009, 21:567-74.

3. Cone RE, Chattopadhyay S, Sharafieh R, Lemire Y and O’ Rourke J. The suppression of hypersensitivity by ocular–induced CD8+ T cells requires compatibility in the Qa-1 haplotype. Immunol Cell Biol. 2009 87:241-248.

4. Cone RE, Chattopadhyay S and O’Rourke J. Suppressor T cells Redux: Ocular Activation and Function in Delayed Type Hypersensitivity. Chemical Allergy and Immunol. 2008. 94: 138-149

5. Chattopadhyay S, O’Rourke J and Cone RE. Implication for the CD94/NKG2A-Qa-1 System in the Generation and Function of Ocular-induced Splenic CD8+ Regulatory T cells. Int Immunol. 2008, 20:509-516

6. Chattopadhyay S, Mehrotra S, Chhabra A, Hegde U, Mukherji B and Chakraborty NG. Effect of CD4+CD25+ and CD4+CD25- T Regulatory Cells on the Generation of Cytolytic T Cell Response to a Self but Human Tumor-Associated Epitope In Vitro. J. Immunol. 2006, 176: 984- 990.

7. Chattopadhyay S, Chakraborty NG, Mukherji B. Regulatory T cells and tumor immunity. Cancer Immunol Immunother. 2005, 54: 1153- 1161.

8. Chattopadhyay S and. Chakraborty N. G. Continuous Presence of TH1 Conditions is Necessary for Longer Lasting Tumor-specific CTL Activity in Stimulation Cultures with PBL. Hum Immunol. 2005, 66: 884- 891.

9. Chakraborty NG, Chattopadhyay S, Mehrotra S, Chhabra A and Mukherji B. Regulatory T-cell response and tumor vaccine-induced cytotoxic T lymphocytes in human melanoma. Hum Immunol. 2004, 65: 794-802.

10. Sen E, Chattopadhyay S, Bandopadhyay S, De T, Roy S. Macrophage Heterogeneity, Antigen Presentation, and Membrane Fluidity; Implication in murine Visceral Leishmaniasis. Scand. J. Immunol, 2001, 53, 111-120.



Name : Praful S. Singru

Designation: Assistant Professor

Department/Institute/University : School of Biological Sciences, National Institute of Science Education and Research (NISER), Institute of Physics Campus, Sachivalaya Marg, Bhubaneswar-751 005

Date of Birth : June 15, 1974 Sex (M/F): M SC/ST : None


Sl No.

Institution

Place

Degree

Awarded

Year Field of Study

1 Post-Graduate Teaching Department of Zoology, Nagpur University, Nagpur

M.Sc. (Zoology)

1997 Animal Physiology, Cell and Molecular Biology, Specialization in Mammalian Reproductive Physiology

2 Dept. Pharma. Sci., RTM Nagpur University, Nagpur

Ph.D. 2004 Neurobiology, Neuroendocrine regulation

3 Division of Endocrinology, Diabetes and Metabolism, Tufts Medical Center, Boston, USA

Research Associate

2009-2010

Neurobiology, Neuroendocrine regulation

Position and Honors

Position and Employment (Starting with the most recent employment) Sl No.

Institution Place

Position From (Date)

To (date)

1 School of Biological Sciences, National Institute of Science Education and Research (NISER), Bhubaneswar

Assistant Professor

26-07-2010

Present


Research Associate

01-04-2009

18-07-2010


Postdoctoral fellow

31-03-2004

31-03-2009

4 Dept. Pharma. Sci., RTM Nagpur University, Nagpur

Senior Research Fellow

27-04-1998

26-10-2001


Honors/Awards 1998: Recipient of Dr. Kamar Banu Karim Medal for first rank in the specialization mammalian reproductive physiology in M. Sc. examination. 2006: Prof. P. C. Dandiya Gold Medal for best poster presentation entitled “Importance of cocaine- and amphetamine-regulated transcript peptide (CARTp) in the hypothalamus and amygdala to anxiogenic responses induced by ethanol withdrawal” by Dandekar MP, Singru PS, Kokare DM, Lechan RM, Thim L, Clausen JT, Subhedar NK, during the 39th Annual Conference of Indian Pharmacological Society, Jaipur. 2006: Prof. Govind Achari Gold Medal for Best Research Paper presentation entitled “Selective melanocortin-4 receptor antagonist HS014 prevented morphine tolerance and withdrawal hyperalgesia” by Upadhya MA, Kalange AS, Kokare DM, Singru PS, Chopde CT, Subhedar NK., during the at 39th Annual Conference of Indian Pharmacological Society, Jaipur. 2006: The findings of the article entitled “Atypical expression of type 2 iodothyronine deiodinase in thyrotrophs explains the thyroxine-mediated pituitary thyrotropin feedback mechanism” published in Endocrinology [2006; 147:1735-43] was given as news in the Endocrine News published by the Endocrine Society, USA. 2008: Conferred with the prestigious “Jaipur Prize” gold medal for the paper entitled “Importance of cocaine-and amphetamine-regulated transcript (CART) peptide in the central nucleus of amygdala in anxiogenic responses induced by ethanol withdrawal” published in Neuropsychopharmacology 2008, 33: 1127–113. This award was given during the 41st Annual Conference of the Indian Pharmacological Society (IPS) and International Conference on Translational Pharmacology, All India Institute of Medical Sciences (AIIMS), New Delhi, India for the best paper published in Pharmacology from India in 2008. 2009: Article “Tanycyte pyroglutamyl aminopeptidase II contributes to regulation of the hypothalamic-pituitary-thyroid axis through glial-axonal associations in the hypothalamic median eminence” Endocrinology [2009; 150:2283-2291] was selected for the News and Views Editorial Comment section of Endocrinology.

Professional Experience and Training relevant to the Project Proficiency in immunohistochemistry, immunofluorescence, cryomicrotomy, western blotting, in-situ hybridization, confocal microscopy.

Publications (Numbers only): 49 (International Publications); Books: Nil; Research Papers, Reports : 49; General articles : Nil; Patents : Nil; Others (Please specify) : 53 (Presentations).


1. Singru PS, Fekete C, Lechan RM. (2005). Neuroanatomical evidence for participation of the hypothalamic dorsomedial nucleus (DMN) in the regulation of the hypothalamic paraventricular nucleus (PVN) by alpha-melanocyte stimulating hormone. Brain Research 1064: 42-51. 2. Singru PS, Sánchez E, Fekete C, Lechan RM. (2007). Importance of melanocortin signaling system in refeeding-induced neuronal activation and satiety. Endocrinology 148:638-646. 3. Singru PS, Mazumdar M, Sakharkar AJ, Lechan RM, Thim L, Clausen J, Subhedar N. (2007). Immunohistochemical localization of cocaine-and amphetamine- regulated transcript peptide in the brain of the catfish, Clarias batrachus (Linn). Journal of Comparative Neurology 502: 215-235. 4. Singru PS, Sánchez E, Acharya R, Fekete C, Lechan RM. (2008). Mitogen-activated protein kinase contributes to lipopolysaccharide-induced activation of corticotropin-releasing hormone synthesizing neurons in the hypothalamic paraventricular nucleus. Endocrinology 149: 2283-2292. 5. Singru PS, Mazumdar M, Barsagade V, Lechan RM, Clausen JT, Thim L, Subhedar N. (2008). Association of cocaine-and amphetamine related transcript peptide and neuropeptide Y in the forebrain and pituitary of the catfish, Clarias batrachus: A double immunofluorescent labeling study. Journal of Chemical Neuroanatomy 36 (3-4): 239-250.


Annexure - I


Status of the Institute submitting the Project Proposal 1. Institution of the Department of Atomic Energy (DAE) 2. Autonomous Institute/Society (please specify the parent Ministry/Department)

Annexure – II

Illustrative List of Subject Areas for the consideration of the Project Proposal MEDICAL SCIENCES & ALLIED AREAS

2.2 Infectious Diseases of Humans

a research proposal (r&d project) submitted to department ... · a research proposal (r&d...

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