Enveloped viruses acquire their membrane coat from the plasma membrane or intracellular organelles and rely on cellular machinery to facilitate envelopment and egress of virus progeny. This thesis examines egress-related interactions between host cell factors and proteins of two different enveloped viruses: hepatitis D virus (HDV) and herpes simplex virus 1 (HSV-1). HDV is a small RNA virus causing fulminant hepatitis or severely aggravating cirrhosis and hepatocellular carcinoma. HSV-1 is a large DNA virus infecting epithelial and neuronal cells. Infection with HSV-1 not only triggers the development of recurring sores on oral or genital mucosa, but can also cause severe disease in neonates and immunocompromised patients. The interaction between the large antigen of HDV (HDAg-L) and the N-terminal domain (NTD) of clathrin, a protein crucial for endocytosis and intracellular vesicular trafficking, was examined by structural, biochemical and biophysical techniques. Co-crystal structures of NTD bound to HDAg-L peptides derived from different HDV genotypes revealed that HDV interacts with multiple binding sites on NTD promiscuously, prompting re-evaluation of the binding between cellular peptides and NTD. Surprisingly, co-crystal structures and pull-down capture assays showed that cellular peptides containing clathrin-binding motifs can also bind multiple sites on the surface of NTD simultaneously. In addition, the structures of viral and cellular peptides bound to NTD enabled the molecular characterization of the fourth peptide binding site on NTD, the “Royle box”, and led to the identification of a novel binding mode at the “arrestin box” peptide binding site on NTD. The work in this thesis therefore not only identifies the molecular basis of HDV:clathrin interactions, but also furthers our understanding of basic clathrin biology. Even though many HSV-1 proteins have been implicated in the envelopment and egress of viral particles, only few interactions between HSV-1 and cellular proteins promoting these processes have been described. Therefore, the HSV-1 proteins gE, UL21 and UL56 were selected and characterized bioinformatically and/or biochemically. Cellular proteins interacting with UL56 were identified by yeast two-hybrid screening and quantitative mass spectrometry. Co-immunoprecipitation and pull-down experiments confirmed the Golgi-trafficking protein GOPC, components of the mammalian trafficking protein particle complex, and the ubiquitin ligase NEDD4 as novel binding partners of UL56, thereby suggesting exciting new avenues for the investigation of cellular mechanisms contributing to HSV-1 envelopment and egress.

The non-increasing rearrangement of a measurable real function defined on an appropriate measure space is of the enormous significance in disciplines such as theory of function spaces or interpolation theory and their applications in PDEs. Unfortunately, while it has good and widely applicable mapping properties, it is virtually impossible to calculate the non-increasing rearrangement of a concrete given function precisely. Numerical algorithms for approximation are desirable for this reason. Such method of approximation, based on interpolation by a linear spline, is presented in this thesis. In the first half of this thesis, the developed method is described, while the error estimates of the method are subject to the second part.

Orientador: Rubén Augusto Romero Lázaro / Banca: Jose Roberto Sanches Mantovani / Banca: Sergio Azevedo de Oliveira / Banca: Antônio César Baleeiro Alves / Banca: Eduardo Nobuhiro Asada / Resumo: O problema de planejamento de sistemas de transmissão é um problema de programação não-linear inteira mista (PNLIM) quando é usado o modelo DC. Praticamente todos os algoritmos usados para resolver este problema utilizam uma subrotina de programação linear (PL) para resolver problemas de PL resultantes do algoritmo de solução do problema de planejamento. Às vezes a resolução desses problemas de PL representa o maior esforço computacional dos algoritmos de planejamento. A particularidade desses problemas de PL é que, na solução ótima, apenas algumas restrições de desigualdade estão ativas. Este trabalho considera a formulação e a implementação computacional de vários algoritmos de PL resultantes, ou seja, os algoritmos propostos fazem modificações nos problemas de PL resultantes dos modelos de transportes e DC de maneira que apresentam uma única restrição de igualdade, a equação de balanço de potência ativa, e muitas restrições de desigualdade. É usado um algoritmo dual simplex canalizado e uma estratégia de relaxação para resolver esses problemas de PL que inicia o processo de otimização com uma única restrição de igualdade e, em cada passo, é adicionada a restrição mais violada. Portanto, a lógica de trabalho é parecida com a proposta apresentada por Brian Stott para o planejamento da operação de sistemas elétricos. Os resultados encontrados mostram um desempenho superior do algoritmo quando comparados com métodos do tipo primal simplex. / Abstract: The transmission network planning problem is a non linear integer mixed programming problem (NLIMP) which used the DC model. Most of the algorithms used to solve this problem use a linear programming subroutine (LP) to solve LP problems resulting from planning algorithms. Sometimes the resolution of these LP problems represents a major computational effort of planning algorithms. The particularity of these LP problems in optimal solution is that only some inequality constraints are binding. This work considers the formulation and the computational implementation of algorithms several of the LP problems resulting, i.e., the proposed algorithms make modifications in the LP problems resulting of the transports and DC models so that present an only equality constraint, only one equality constraint, the power flow equation, and many inequality constraints. It is used a dual simplex algorithm and a relaxation strategy to solve these LP problems_ which start the optimization process with only one equality constraint and, in each step, the most unfeasible constraint is added. Then, the logic used is similar to the proposal presented in Brian Stott for electric systems operation planning. The results show a higher performance of the algorithm when compared to primal simplex methods. / Doutor

Yau, Kwok Hei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 64-71). / Abstracts in English and Chinese. / Chapter Chapter 1: --- Introduction --- p.1 / Trichosanthin --- p.2 / Apoptosis --- p.10 / Herpes Simplex Virus --- p.16 / Conclusion --- p.28 / Chapter Chapter 2: --- Materials and Methods --- p.29 / Cell lines and virus --- p.30 / Infectivity assay --- p.30 / Treatment of cells and virus infection --- p.32 / MTT assay for cytotoxicity --- p.34 / Preparation of cell lysate --- p.35 / Bradford assay for protein concentration --- p.36 / Western blot analysis --- p.37 / ELISA for quantification of HSV-1 antigen --- p.38 / Statistical analyses --- p.39 / Chapter Chapter 3: --- Results --- p.40 / Cytotoxicity and anti-herpetic activity of TCS and CHX --- p.41 / Selective cytotoxicity of TCS toward HSV-1 infected cells --- p.44 / Selective cytotoxicity is implicated in the antiviral activity of TCS --- p.50 / The effect of selective cytotoxicity on TI value --- p.53 / Chapter Chapter 4: --- Discussion --- p.55 / References --- p.64

Many insects are important agricultural pests, and active control is necessary to keep them at abeyance. The naturally occurring entomopathogenic fungus Metarhizium is a promising tool to control pest insects, and its use avoids the well-known harmful side effects of chemical pesticides. Thousands of unique isolates of Metarhizium exist throughout the world. These isolates vary widely in their ability to cause infection and to tolerate stressful habitats. The research reported here tests the THESIS: A laboratory-based system can be devised that identifies, from among many Metarhizium isolates, those isolates with the greatest potential for successful biological control of pest insects in the field. The study was built on the testing of four hypotheses: (1) Laboratory bioassays using target pest insects will distinguish highly virulent strains of Metarhizium from less virulent strains, (2) Quantity and quality of mass-produced pathogenic fungi will vary among species and strains of Metarhizium, (3) The tolerance to ultraviolet radiation will vary among species and strains of Metarhizium, (4) The effect of temperature on growth rates and survival of both Metarhizium spores and hyphae will vary among isolates and species. These hypotheses test four field-relevant traits using a panel of ten isolates of Metarhizium isolates. Seven sets of laboratory experiments were devised to define the range of responses within the traits covered by the hypotheses. This series of general laboratory tests was developed to assist in identifying fungal isolates with high potential for field use. These tests included evaluation of each isolate's (a) insect pathogenicity, (b) mass–production capabilities, (c) tolerance to high temperatures, (d) tolerance to UV-B radiation, (e) rate of vegetative growth, (f) rate of spore germination, and (g) an evaluation of presence or absence of a post–stress growth inhibition. The application of this protocol to the isolates used in this study indicates that four isolates have high field potential, i.e., DWR 203, DWR 346, DWR 356 and ARSEF 324, and three of these were tested in a field trial. By following the procedures outlined in this thesis, selection of “good” isolates can be accomplished in the laboratory, and a successful isolate can be identified from the abundance of isolates present in nature.

Background: Genital herpes is one of the most common sexually transmitted infections in the United States. As genital herpes is incurable and contagious, individuals with genital herpes face the decision to disclose their status to potential sexual partners with each new relationship formed. Such disclosure places individuals with genital herpes in a position to face rejection, which is commonly reported as one of the most concerning aspects of having genital herpes. The present study seeks to further understand the nature of genital herpes disclosure by addressing two core aims: 1) to understand determinants of and reasons for disclosure and non-disclosure and 2) to explore the relationship between past partner reactions to a disclosure and future intentions to disclose. Methods: Data on genital herpes disclosure experiences were collected via an online questionnaire, which was distributed through a variety of online channels including social media websites and email lists. Individuals who self-identified as having genital herpes and were 18 years and older were eligible for participation. Results: In examining Aim 1, the majority of participants (80.4%) disclosed to their last sexual partner. Age, relationship length, type of relationship, and expectations of a partner's response were significantly associated with the decision to disclose at the bivariate level. Expectations of a partner's reaction (AOR = .20, 95% CI .074-.539) and relationship type (AOR = 8.31, 95% CI 1.96-35.32) remained significant in multivariable modeling, explaining 45.2% of the variance in disclosure. Respondents who reported being in socially committed relationships and those who expected more positive partner reactions to a disclosure were more likely to disclose. Disclosure was also significantly associated with many romantic relationship building activities (e.g., establishing an exclusive relationship) but largely not associated with the sexual progression of a relationship. The decision to disclose was commonly multi-faceted, with the majority of participants reporting more than one reason that they did or did not disclose. Primary reasons for disclosure included "I wanted to be honest", "To protect my partner from getting herpes", and "It's my partner's right to know", while the most common reasons for non-disclosure were "I was concerned my partner would react badly", "I was ashamed", and "I was concerned that my partner would have rejected me". Regarding Aim 2, participants reported low levels of negative reactions and perceived rejection in response to their last disclosure experience. Intentions to disclose in the future were high among those who anticipated future sex partners. Discussion: The decision to disclose is often multi-faceted, and relationship characteristics play a key role in the decision to disclose. Among those who did disclose in this study, the majority did not report negative repercussions, including bad partner reactions and rejection. Future studies should examine if individuals are able to accurately assess potential partner reactions in order to better understand the differences between those who choose not to disclose and those who choose to disclose but experience a negative partner reaction or rejection.

Herpes simplex virus type 1 (HSV-1) is a common human pathogen that establishes life-long latent infection in sensory neurones. This makes it potentially useful as a gene therapy vector to target neuronal cells. HSV-1 enters cells by membrane fusion, the viral envelope and most tegument proteins dissociate, and the capsid is transported to the cell nucleus to establish infection. There is increasing evidence that the retrograde transport of HSV-1 along sensory axons is mediated by cytoplasmic dynein, but the viral and cellular proteins involved are not known. Cytoplasmic dynein is the major molecular motor involved in minus-end-directed cellular transport along microtubules. It is a large complex molecule, with heavy chains providing motility, while intermediate and light chains are involved in specific cargo binding. A library of HSV-1 capsid and tegument structural genes was constructed and tested for interaction with dynein subunits in a yeast two-hybrid system. A strong interaction was demonstrated between the HSV-1 outer capsid protein VP26 (UL35), as well as the tegument protein VP11/12 (UL46), with the homologous 14 kDa dynein light chains rp3 and Tctex1. In vitro pull-down assays confirmed binding of VP26 to rp3, Tctex1 and cytoplasmic dynein complexes. Recombinant HSV-1 capsids +/- VP26 were used in similar pull-down assays. Only VP26+ capsids bound to rp3. Recombinant HSV-1 capsids were microinjected into living cells and incubated at 37�C. After 1 h capsids were observed to co-localise with rp3, Tctex1 and microtubules. After 2 or 4 h VP26+ capsids had moved closer to the cell nucleus, while VP26- capsids remained in a random distribution. Our results suggest that the HSV-1 outer capsid protein VP26 mediates binding of incoming capsids to the retrograde motor cytoplasmic dynein during cellular infection, through interactions with dynein light chains. It is hoped that these findings will help in the development of a synthetic viral vector, which may allow targeted gene therapy in patients with neurological diseases.

Bibliography: leaves 92-118. / xii, 118, [20] leaves, [12] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Reports a method for the study of HSV-1 transcripts during latency. High resolution non-isotopic in situ hybridization (ISH) is used to study the intracellular location of HSV-1 latency associated transcripts (LATs) in primary sensory neurons of latently infected mice and humans. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1995?

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<p>Viruses depend on their hosts for the production and spread of new virus particles. For efficient virus replication, the viral genes have adapted the strategy of being recognized and processed by the cellular biosynthetic machineries. Viruses therefore provide an important tool to study the cellular machinery regulating gene expression. In this thesis, we have used two model DNA viruses; herpes simplex virus (HSV) and adenovirus, to study RNA processing at the level of pre-mRNA splicing in mammalian cells. </p><p>During a lytic infection, HSV cause an almost complete shut-off of host cell gene expression. Importantly, HSV infection cause inhibition of pre-mRNA splicing which is possibly advantageous to the virus, as only four HSV genes contain introns. </p><p>The HSV immediate early protein, ICP27, has been shown to modulate several post-transcriptional processes such as polyadenylation and pre-mRNA splicing. We have studied the role of ICP27 as an inhibitor of pre-mRNA splicing.</p><p>We show that ICP27 inhibits pre-mRNA splicing <i>in vitro</i> in the absence of other HSV proteins. We further show that ICP27 inhibits splicing at the level of spliceosome assembly. Importantly, ICP27 induced inhibition of splicing can be reversed, either by the addition of purified SR proteins or by the addition of an SR protein specific kinase, SRPK1. We propose that SR proteins are prime candidates as mediators of the inhibitory effect of ICP27 on pre-mRNA splicing. </p><p>In order to learn more about how splicing is organized in the cell nucleus <i>in vivo</i>, we investigated how cellular splicing factors are recruited to sites of transcription and splicing in adenovirus infected cells using confocal microscopy. Our results showed that the SR proteins, ASF/SF2 and SC35, are efficiently recruited to sites in the nucleus where adenovirus genes are transcribed and the resulting pre-mRNAs are processed. Our results demonstrate that only one of the two RNA recognition motifs (RRMs) present in the ASF/SF2 protein is required for its recruitment to active sites of splicing. The arginine/serine rich (RS) domain in ASF/SF2 is redundant and insufficient for the translocation of the protein to active viral polymerase II genes in adenovirus infected cells.</p>