Finally, NF-kappaB and another transcriptional regulator p53 are usually tightly related in their control of cell survival. Opposite to NF-kappaB, p53 mediates cell death signals, usually activated under DNA damage and subsequently involved in cell growth control, DNA damage repair or apoptosis. It was found in this study, DNA damage and cell cycle arrest responses tended to participate with the anti-HSV-1 activity. Although in HEp-2 cells, TCS induced more DNA damage ratio and S and G2/M phase arrest proportion than HSV-1 infected cells, but more p53 was expressed and activated by phosphorylating at Ser 15 by TCS in HSV-1 infected HEp-2 cells than uninfected ones. In the same time the activation of BAX, which promotes the apoptotic function of p53, increased during TCS treatment when infected with HSV-1, the p53 therefore regulates apoptosis in HSV-1 infected cell during TCS treatment. / Firstly, we demonstrated that TCS reduced HSV-1 antigen and DNA content, The IC50 (half maximal inhibitory concentration) of TCS on HSV-1 replication was 2.5 +/- 0.23 mug/ml. The anti-HSV-1 effect of TCS was related to interfering with viral replication during 3 to 15 hours after infection which coincide with early to late viral replication period. TCS had no effect on HSV-1 attachment, penetration or immediate early gene expression. However, the expression of early gene, late gene and virion release were diminished. / Fourthly, the role of the nuclear factor-kappaB (NF-kappaB) in the anti-HSV-1 effect of TCS was explored. NF-kappaB initiates cell survival pathways. It is widely involved in viral infection and replication to make sure virus overcomes the host cells immune response. We found HSV-1 enhanced the activity of NF-kappaB in HEp-2 cells by triggering its translocation from cytoplasm to nuclear. However, during the anti-HSV-1 effect of TCS, TCS suppressed HSV-1-aroused NF-kappaB translocation in HEp-2 cells, the inhibition of NF-kappaB activity in HSV-1-infected cells by TCS treatment tend to abolish the anti-apoptosis effect developed by HSV-1, so that the host cells suffered more extracellular stress and showed more apoptosis ratio than uninfected ones. / Taken together, this study demonstrated TCS interfered with HSV-1 early to late infection period. TCS selectively induced more HSV-1 infected HEp-2 cells to apoptosis than uninfected ones, the selectivity of TCS was due to apoptotic signaling pathway switching from CD95 (Fas/Apo-1)-mediated type I to type II apoptotic pathway. Furthermore, during TCS induced-apoptosis in HSV-1 infected cells, TCS suppressed NF-kappaB activation that triggered by HSV-1 infection. At the meanwhile, p53 participated in the TCS-induced apoptosis regulation in infected cell. / TCS is toxic to cell because its RIP activity, killing of the viral host cells certainly inhibits virus expansion, only when it kills more infected cells, the material could be considered as an anti-viral agent. It was found TCS induced losing of cell viability and enhancing in apoptosis in HEp-2 cells and HSV-1 infected HEp-2 cells. The decrease of cell viability and increase of apoptosis ratio were enhanced when HEp-2 cells were infected with HSV-1 compared with uninfected ones. The 50% of effect concentration (EC50 ) in cytotoxicity and apoptosis were decreased from 24.64 +/- 1.17 mug/ml and 37.57 +/- 1.47 mug/ml in uninfected HEp-2 cells to 3.01 +/- 1.30 mug/ml and 3.89 +/- 1.31 mug/ml in HSV-1 infected HEp-2 cells respectively. / The reason of type I to type II apoptosis pathway transition might due to the activity change of death receptor on HEp-2 cells. The type I apoptotic pathway induced by TCS was related to CD95 (Fas/Apo-1) system activation and signaling pathway. When HEp-2 cells were infected with HSV-1, the CD95 (Fas/Apo-1) was suppressed by HSV-1 infection. As a result, TCS triggered a less CD95 (Fas/Apo-1) dependent type II apoptotic pathway in the infected cells. / Thirdly, TCS activated different apoptotic pathways, namely type I and type II apoptotic pathways, between uninfected and infected cells. The type I apoptotic pathway bypasses the dependence on the mitochondrial but quickly activates a large amount of caspase-8 at the CD95 (Fas/Apo-1) formed death inducing signaling complex (DISC), which amplifies the signal. By contrast, the formation of the DISC in the type II apoptotic pathway is strongly reduced. It depends on loss of the mitochondrial transmembrane potential (DeltaPsi m) and release of cytochrome c and capase-9 activation to mediate apoptosis signal transduction. We found in HSV-1 infected an uninfected HEp-2 cells, TCS induced the loss of DeltaPsim, this DeltaPsim losing was increased when HEp-2 cells were infected with HSV-1. Furthermore, when there were no HSV-1 infection, TCS induced caspase-dependent type I apoptosis pathway that quickly activated large amount of caspase-8 after TCS treatment. However, when infected with HSV-1, this pathway turned into mitochondrial dependent type II pathway involving caspase-9 response, whose apoptosis ratio was diminished by over expressed Bcl-2, which is a hallmark defining type I or type II apoptosis. / Trichosanthin (TCS) is a type I ribosome inactivating protein (RIP), it was found to inhibit human simplex virus type 1 (HSV-I) but the anti-HSV-I mechanism is unclear. HSV-1 is a widely distributed DNA virus, it causes large range of human diseases. During the lytic life cycle of HSV-1, highly regulated cascade of genes are expressed to interfere with host cell metabolism and immune response. In this context the anti-HSV-1 mechanism of TCS in human epithelial carcinoma HEp-2 cells was studied. / He, Dongxu. / Advisers: Wing Ho Yung; Siu Cheung Tam. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 124-138). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

No description available.

The human c-myc gene is a cellular proto-oncogene composed of three exons and two introns. Transcription of c-myc is controlled by two promoters, Pl and P2. The activity of these promoters is regulated by many factors, such as cellular transcription factors E2F, YYl, and HSV-1 immediate-early proteins, ICPO, ICP4. Many regulatory elements located both upstream of and between P 1 and P2 have been identified, and some of these are required for optimum expression of c-myc. In this thesis research, a region downstream from P2 in the c-myc exon 1 was identified by its response to transactivation by HSV-1 immediate-early proteins, ICPO and ICP4. The purpose of this research was to examine this region for regulatory sites that respond to HSV-1 infection. I hypothesized that after HSV-1 infection, ICPO and ICP4 activate c-myc expression, in part, through regulatory sequences present in exon 1. To test for this hypothesis, reporter plasmids containing (I) the c-myc promoter (from - 101 bp relative to Pl) and exon 1 coupled to the bacterial CAT gene were constructed. (ii) The c-myc exon sequences used were either intact (wild-type) or they were constructed with various deletions. The activities of these plasmids were examined in transient expression assays. To analyze protein binding, electrophoretic mobility shift assay (EMSA) and completion EMSAs were carried out. The results from these experiments lead to the following conclusions: (i) ICP4 and ICPO serve as activators, whereas ICP27 inhibits c-myc gene expression. (ii) The region from +332 to +513 within the c-myc exon 1 contains an important element required for transactivation of the c-myc gene by HSV-1 proteins. (iii) Cellular proteins, including factor YYl, bind to the region from +332 to +513 in the c-myc exon 1. Although the exact mechanism by which HSV-1 immediate-early proteins regulate cmyc gene expression is still not clear, it gives rise to a possibility that this regulation is caused by turning on or activation of the cellular regulatory proteins by ICP4 and ICPO. The cellular proteins in turn activate the c-myc gene expression by interacting with the ciselement downstream from P2.

A regulatory element involved in the transcriptional activation of the major capsid protein (VP5) of herpes simplex virus type 1 was identified and characterized in this research project. Gel mobility shift assay with nuclear extracts from both uninfected and HSV-1 infected HeLa cells identified two major protein-DNA complexes involving the VP5 promoter. No viral specific complex found. DNase I and orthophenanthroline-cu+ footprint analyses in the same laboratory revealed that the two complexes involve a single binding site, GGCCATCTTGAA, located between -64 and -75 bp relative to the VP5 cap site. To determine the function of this leaky-late binding site (LBS) in VP5 gene activation, mutated VP5 promoters with deletion and insertion around LBS were constructed and linked to a reporter gene, bacterial chloramphenicol acetyltransferase gene. The effect of mutations were tested in transient expression assay. Deletion of LBS resulted in seven to eight-fold reduction in the level of transactivation of the chloramphenicol acetyltransferase gene by superinfection with HSV-1 or by cotransfection of HSV immediate-early genes. These results indicated LBS is involved in the maximal transactivation of the VPS gene. A search of published gene sequences found the homologs of LBS exist in a number of HSV-1 By promoters, and other viral promoters, as well as cellar promoters. Some of these homologs have found involved in the transcription regulation.

The herpes simplex virus 1 (HSVl) VP5 gene codes for the major viral capsid protein. Understanding of the mechanism of how the VP5 gene is regulated in host cells will help us to understand the molecular action of the HSV 1 life cycle and its interplay with the host cell gene expression machinery (transcription and translation). This may ultimately provide scientific bases for both better prevention and cure of HSV 1 caused diseases. Previous work from Dr. Robert L. Millette' s laboratory has indicated that a 164 base pair region of the VP5 promoter gene could activate the transcription of an attached reporter gene (bacteria CAT gene). Furthermore, a 12 bp (GGCCATCTTGAA) cis-acting element situated within the 164 bp promoter region was required for the promoter activity. To understand the function of this cis-element in the regulation of VP5 transcription and to identify the trans-acting factors interacting with this element, gel mobility shift assays were first carried out using the fragment containing the 12 bp site as the probe. A cellular factor, YY 1, was found to bind to this site in a sequence specific manner. Based on the oligonucleotide competition assays, partial protease digestions, and antibody supershift assays, it became clear that two cellular factors bound to the VP5 promoter. These were related, if not identical, to the previously identified Yin-Yang- 1 factor (YY 1), and transcription factor the SPl. Site-directed mutagenesis studies indicated that these two factors bind to distinct sites on the 164 bp fragment. Point mutations studies on the 12 bp YYl binding site demonstrated that seven of the 12 bp were required for YY 1-DNA complex formation and the first four bp in the 12 bp were very important for VP5 gene regulation. Also, it was found that YY 1 performs both positive and negative regulator function in VP5 gene regulation. In conclusion, two cellular transcription factors, YY 1 and SPl, play a major role in VP5 gene expression.

Copies of author's previously published articles inserted inside back cover. / Bibliography: leaves 137-179. / x, 179, [26] leaves, [28] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The aims of this study are to determine whether latent HSV genomes are a result of viral DNA amplification in the PNS during the establishment phase and to investigate the relationship between HSV DNA copy number and viral transcriptional activity during latent infection of the PNS. In order to map the distribution of viral nucleic acid sequences in latently infected sensory ganglia, experiments are undertaken using a mouse model that makes novel use of the segmental sensory innervation of flank skin. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1995?

No description available.

Thesis (Ph.D.) -- McMaster University, 1998. / Includes bibliographical references (leaves 195-230). Also available via World Wide Web.

Thesis (Ph.D.)--McMaster University (Canada), 1996. / Source: Dissertation Abstracts International, Volume: 57-10, Section: B, page: 6096. Adviser: J. P. Capone.

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2006. / Härtill 4 uppsatser.