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The use of herpes simplex virus-1 vectors in nociceptive biology

Srinivasan, Rahul (2006) The use of herpes simplex virus-1 vectors in nociceptive biology. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Public Health Relevance: The United States has 80 million employees with chronic pain resulting in annual losses of 61.2 billion dollars due to pain-related productive time lost. In addition, pain-related depression and inactivity reduce the quality of life. The development of effective analgesics is therefore important from a public health perspective. In this dissertation, the natural properties of herpes simplex virus (HSV-1) vectors are exploited to (i) develop an HSV-1 vector-based selection system that can potentially identify natural or chemical inhibitors of chronic pain and (ii) to test HSV-1 vector-expressed dominant negative PKCε (DNP) as a strategy to treat chronic pain. The vanilloid/capsaicin receptor (TRPV1) is a pro-nociceptive calcium ion channel that is upregulated in chronic pain. This occurs partly due to protein kinase C epsilon (PKCε)−mediated receptor phosphorylation. An HSV-1 vector expressing TRPV1 (vTT) was engineered and vTT-expressed TRPV1 functionality was confirmed.Treatment of vTT-infected cells with capsaicin or resiniferatoxin caused concentration-dependent Ca+2 influx, leading to cell-death and a dramatic reduction in infectious particle yield. TRPV1 antagonists, ruthenium red and SB-366791 reversed agonist-induced cell-death and rescued vTT growth, providing a basis for selection. Selection for antagonists was modeled using a mixed infection of vTT and vHG (capsaicin resistant control vector) and virus passage in the presence capsaicin. These experiments demonstrated that a single control vector particle was readily isolated from a population of 10^5 vTT particles. This approach can be used to identify antagonists from chemical or gene libraries and offers advantages of (i) a platform assay applicable to other ion channels and (ii) adaptability to high throughput formats. Dominant negative PKCε (DNP) was engineered into HSV-1 to create the vector, vHDNP. Following functional confirmation of vHDNP in U2OS, Vero cells and neurons, cobalt uptake showed a reduction of capsaicin sensitive vHDNP-transduced neurons. Electrophysiology confirmed this and also demonstrated a knockdown of TRPV1-PKCε coupling in nociceptive neurons. In-vivo studies of noxious heat-induced nocisponsive behavior in vHDNP-inoculated rats showed a subtle inhibition of withdrawal responses when compared with controls. In conclusion, HSV-1 expressed dominant negative PKCε is a viable strategy to specifically inhibit TRPV1 function in order to treat chronic pain.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee CoChairGlorioso, Joseph Cglorioso@pitt.eduGLORIOSO
Committee CoChairFerrell, Robert Erobert.ferrell@hgen.pitt.eduRFERRELL
Committee MemberDeFranco, Donald Bdod1@pitt.eduDOD1
Committee MemberKhan, Saleem Akhan@pitt.eduKHAN
Date: 25 September 2006
Date Type: Completion
Defense Date: 19 July 2006
Approval Date: 25 September 2006
Submission Date: 30 July 2006
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Institution: University of Pittsburgh
Schools and Programs: School of Public Health > Human Genetics
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: dorsal root gaglion neurons; high throughput screen; desensitization; drug discovery; dephosphorylation; fluorescence; overexpression; phosphorylation; electrophysiology; receptor; superfamily; FURA-2; vector-transduced neurons; patch clamp; transient receptor potential
Other ID:, etd-07302006-164356
Date Deposited: 10 Nov 2011 19:55
Last Modified: 15 Nov 2016 13:47


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