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Name : |
Farhad |
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Surname : |
Bonakdarhashemi |
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From : |
Friday, March 1, 2013 |
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To : |
Monday, September 1, 2014 |
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Position : |
Associate professor |
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School/Research center : |
School of Medicine |
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E-mail : |
farhadbhashemi@gmail.com |
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subject : |
Virology/Microbiology |
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Venue : |
University of British Columbia |
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Country : |
Canada |
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Sabbatical Leave Period(...Month) : |
17 |
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Certification : |
http://gsia.tums.ac.ir/images/UserFiles/21428/Forms/280/FBHashemi_12month_Report_3.pdf |
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1.Brief summary of Leave : |
Background: HIV disease is fatal, if not treated. Antiretroviral therapy is effective in suppressing HIV to undetectable levels. However, a cure is not acheivable since viral rebound occurs a few days after ART ceases in infected individuals. The chief barrier to a cure for HIV disease is the stable latent cellular reseviour that harbors integrated proviral HIV DNA. Understanding mechanisms of HIV cellular latency is an essential step towards a cure for HIV disease by devising strategies to purge the latent HIV and achieve viral eradication in infected indiviudals. In this study, we have characterized HIV latency in clones of HIV-infected Jurkat-Tat T cell line by identification of HIV integration sites, and assessment of the long-term stability of HIV expression phenotype of these clones after single-cell live sorting followed by weeks of culture. Materials & Methods: We have utilized a recombinant double-labeled mini-HIV (dmHIV) and flow cytometric analysis as an in vitro HIV latency model that reliablly identifies HIV latent cells using a BD-LSR II 561. Genomic DNA digestion by Mse-I and Bcl-II enzymes followed by a nested PCR, specific HIV-LTR , was used to identify HIV-DNA amplicons that were sequenced to locate the genomic sites of HIV integration using the 4Peaks software. FACS analysis was also utilized to establish clonal cultures of latently HIV infected Jurkat-Tat cells, which were derived from a single cell and cultured for 8 weeks. Subclonal HIV latent cultures were treated with PMA/Ionomycin to reactivate latent HIV and expression profile of HIV was analyzed by flow cytometry using FlowJo software (v 9.8.1).
Results: We show that HIV expression profile of latent cell population, derived from a single HIV-infected T cell is quite stable; and it can recaptulate the HIV expression phenotype of its parental pouplation. The ability of latent HIV-infected clones to regenerate the parental population HIV phenotype was observed in stimulated, as well as untreated cultures. The latenly-infected clone’s capability to generate cellular populations displaying parental phenotypes in all states of HIV expression was independent of genomic HIV integration site and the clone’s HIV-Tat protein expression levels. Our findings indicate that HIV-infected latent cells can regenerate the HIV latent reseviour, which demostrates its resiliance and stablility. Conclusions: We conclude that HIV expression profile during latent infection of Jurkat-tat clones is highly stable, and is probably under control of epigenetic processes at the site of HIV integration. Chromatin architecture at proviral HIV integration site plays a dominant role in HIV latency and proviral transcriptional activity among T cells. As a result, cellular reseviours of HIV latent cells in vivo that survive ART, may present a far greater barrier to achieving a cure to HIV diserase than formerly anticipitated. Therefore, the immense challenge of ultimate eradication of HIV in infected individuals will necessitate a multifaceted approach, which should include ART as well as targeting the chromosomal architecture and epigentic factors that regulates HIV latency and expression. |
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2.List the objectives of your sabbatical leave as listed in your proposal and indicate how completely they were met : |
We show that
1. HIV expression profile of latent cell population, derived from a single HIV-infected T cell is quite stable; and it can recaptulate the HIV expression phenotype of its parental pouplation.
2. The latenly-infected clone’s capability to generate cellular populations displaying parental phenotypes in all states of HIV expression was independent of genomic HIV integration site and the clone’s HIV-Tat protein expression levels.
Our findings indicate that HIV-infected latent cells can regenerate the HIV latent reseviour, which demostrates its resiliance and stablility. Conclusions: We conclude that HIV expression profile during latent infection of Jurkat-tat clones is highly stable, and is probably under control of epigenetic processes at the site of HIV integration. Chromatin architecture at proviral HIV integration site plays a dominant role in HIV latency and proviral transcriptional activity among T cells. As a result, cellular reseviours of HIV latent cells in vivo that survive ART, may present a far greater barrier to achieving a cure to HIV diserase than formerly anticipitated. Therefore, the immense challenge of ultimate eradication of HIV in infected individuals will necessitate a multifaceted approach, which should include ART as well as targeting the chromosomal architecture and epigentic factors that regulates HIV latency and expression. |
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3.Acheivements(Publications,research,et.al.) : |
1. Poster presentation (UBC, Dept. Molecular Biology and Biochemistry)
2. Oral presentation (at St. John College , UBC)
3. Departmental seminar presentation (Dept. of Microbiology, TUMS, Tehran)
4. AIDS international conference presentation (Shahid Beheshti University of Medical Sciences, Tehran)
5. A manuscript article (in preparation for Journal of Virology 2014/2015) |
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4.Assessment of Value of Sabbatical leave(benefits,faculty development,future professional activities,...) : |
During my 18-month sabbatical leave I attained several academic achievements, of which some are listed below (please see documents enclosed):
1. Poster presentation at UBC, Dept. Molecular Biology and Biochemistry (copy enclosed)
2. Oral presentation at St. John College , UBC. (copy of cerifiacte enclosed)
3. Departmental seminar presentation (Dept. of Microbiology, TUMS, Tehran; Mehr 1393)
4. The 5th HIV/AIDS international conference presentation (Shahid Beheshti University of Medical Sciences, Tehran, Aban 1393)
5. A manuscript article (in preparation: Journal of Virology 2014/2015)
In addition to above accomplishments during my sabbatical training; I was able to advance my knowledge and experience in the scientific and administrative in UBC’s academic settings as outlined below:
1. Scientific:
a. Practical scientific expertise: Earned invaluable hands-on experience regarding current technology in performing the following cellular and molecular laboratory techniques:
a. Flow Cytometry using flow cytometer Model BD-LSRII 561
b. Software analysis using FlowJo (version 10.0)
c. Culture of recombinant double-labeled HIV vectors in Jurkat-Tat cells, and primary cells
d. Cellular Transfections assays, followed by Luciferase and Beta-galactosidase experiments
e. Several DNA technologies, including HIV integration site identification by restriction enzyme treatment combined with PCR, and sequencing
b. Theoretical scientific expertise:
Through my interactions with a few Vancouver General Hospital infectious disease specialists and UBC faculty members in the department of Molecular Biology, I also developed in-depth understanding of current topics regarding aspects such as;
HIV/ AIDS pathogenesis,
HIV clinical latency
Epigenetic regulation of eukaryotic gene expression
Fluorescence activated cell sorting (FACS) and
Flow Cytometry
Genomics/ Bioinformatics
2. Administrative:
I also earned valuable hands-on administrative knowledge and experience pertaining to:
Use of electronic office tools for many services, such as material ordering, safety certification, and application for human ethics board approval in UBC system
Graduate student training at M.Sc. and PhD.
Administrative process of grant issuance at CIHR and UBC system
Approval of training process for of chemical and biological safety procedures.
Future plans: My future plans includes short-and Long-term -plans
Short term:
Complete the submission process for my manuscript(s)
Incorporate my knowledge and experience while teaching medical and graduate courses for my Masters and PhD students.
Apply my knowledge in design of experiments.
Long term plans: Initiate collaborations with other centers and universities inside and inside Iran. |
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Additional material may be attached in response to the above summary : |
http://gsia.tums.ac.ir/images/UserFiles/21428/Forms/280/3rd-Progress_IJS.pdf |
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Department Head/Research Center Chair : |