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Infection of most cell types with herpes simplex virus (HSV) mutants lacking the activation functions of VP16 and/or ICP0 results in repression of viral gene expression. However, the human osteosarcoma cell line U2OS supports the replication of VP16 and ICP0 mutants to nearly wild type levels. Prior to the studies presented in this thesis, the basis for the permissivity of U2OS cells to VP16 and ICP0 mutants had not been explored. Here, somatic cell fusion assays were used to determine that U2OS cells support the replication of VP16 and ICP0 mutants due to a defect in an innate gene silencing mechanism. The artificial induction of interferon stimulated genes that occurs during the somatic cell fusion assays is not the basis for the observed repression of viral gene expression. As one means of identifying components of the antiviral pathway defective in U2OS cells, restrictive cell types were treated with kinase inhibitors and infected with VP16 and/or ICP0 mutants. Although several compounds were identified which compensate for the defect in gene expression of VP16 mutants, these drugs also stimulate mutant virus gene expression in U2OS. Thus, U2OS are most likely not defective in the cellular signalling pathway(s) targeted by these compound(s). Finally, the importance of VP16 and ICP0 in modulating chromatin structure on the viral genome in both restrictive and permissive cells was examined, uncovering an essential role for both proteins in altering histone occupancy and acetylation levels. Importantly, U2OS cells have a defect in the chromatin-based pathway targeted by ICP0. However, evidence suggests that the ability of VP16 and ICP0 to affect histone occupancy and acetylation levels is not required for viral gene expression. Taken together, the results of this thesis demonstrate that U2OS cells support the replication of VP16 and ICP0 mutants due to a defect in an innate antiviral mechanism which does not involve the targets of several well characterized kinase inhibitors. The significance of the defect in a chromatin-based pathway targeted by ICP0 in U2OS cells remains to be elucidated. / Virology

Intratumoral injection of oncolytic viruses provides a direct means of tumor cell elimination for inoperable tumors. Unfortunately, oncolytic vectors based on human adenovirus (HAdV) typically do not spread efficiently throughout the tumor mass, reducing the efficacy of treatment. In this thesis, I explore the efficacy of conditionally replicating HAdV vectors expressing either the p14 Fusion Associated Small Transmembrane (FAST) protein (CRAdFAST) or p14 FAST protein in combination with the adenovirus death protein (CRAdFAST-ADP). The p14 FAST protein mediates cell-cell fusion, which may enhance spread of the virus-mediated, tumor cell-killing effect, while ADP aids in cell lysis and HAdV spread at late times in infection. I first explored the efficacy of CRAdFAST in the 4T1 immune competent mouse model of cancer. Treatment with CRAdFAST resulted in enhanced cell death compared to vector lacking the p14 FAST gene in vitro, but did not reduce the tumor growth rate in vivo. The 4T1 model was significantly resistant to HAdV infection and propagation, so I next explored CRAdFAST efficacy in human A549 cell culture and a xenograft mouse model of cancer. In the human A549 lung adenocarcinoma model of cancer, CRAdFAST showed significantly improved oncolytic efficacy in vitro and in vivo. In an A549 xenograft tumor model in vivo, CRAdFAST induced tumor cell fusion which led to the formation of large acellular regions within the tumor, and significantly reduced the tumor growth rate compared to control vector. Finally, to assess the use of a newly constructed CRAdFAST vector co-expressing the adenovirus death protein (ADP), a new model was explored comprised of CMT-64.6 mouse lung carcinoma cells which are syngeneic with Balb/C mice. This model was significantly more sensitive to HAdV infection and CRAdFAST induced fusion than the 4T1 cell line. In this model, expression of ADP and p14 FAST from a CRAdFAST-like vector (CRAdFAST-ADP) resulted in significant oncolytic synergy in vitro but not in vivo. My results indicate that expression of p14 FAST protein, and potentially ADP, from an oncolytic HAdV can improve vector efficacy for the treatment of cancer, but improved in vivo models will be required to analyze the full preclinical potential of these oncolytic HAdV vectors.

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg in partial fulfillment of the requirements for the degree of Master of Medicine in the Branch of Medical Genetic. Johannesburg, South Africa, 2012 / Fanconi anaemia (FA) is a genotypically and phenotypically heterogeneous genetic condition , characterized microscopically by chromosomal breakage and instability and usually inherited in an autosomal recessive manner. Affected individuals often present with a diverse variety of physical congenital abnormalities and most progress to haematological disease including bone marrow aplasia and myelodysplasia in early childhood. In South Africa, Black individuals with FA share a common causative founder deletion mutation in the Fanconi G gene (FANCG del) in 82% cases. They are thus an ideal patient cohort for a genotype-phenotype correlation study. Thirty Black patients, homozygous for FANCG del, were ascertained from haematology/oncology clinics in Johannesburg and Bloemfontein. They were subjected to a comprehensive clinical examination to a document their physical features. A concurrent review of each participant's hospital file allowed data to be collected regarding disease presentation and haematological progression . Significant growth abnormalities and a high frequency of skin of skin pigmentary anomalies were found in the research cohort. Although subtle, anomalies of the eye, ears, and hands were noted in a high frequency. The overall physical phenotype does not appear to be appreciably different from that described in other Fanconi anaemia cohorts; however, affected Black individuals may present with more severe haematological indices and have poorer outcome that FA individuals of heterogeneous genotype. Further, it would appear that haematological disease progression cannot be predicted by the presence of abnormalities.

Prostate cancer (PCa) accounts for an estimated 20% of new cancer cases and 10% of deaths in just US males in 2020. Despite this prevalence, the molecular basis of its development and initiation remains unclear. To help identify the molecular basis of PCa progression, it is important to generate a collection of human prostate epithelial cells (hPrEC) that remain karyotypically normal and represent the epithelial cell types present in the human prostate. hPrEC can only go through a limited number of passages before they become senescent. Immortalization prevents senescence and enables continuous cell division. Our lab previously immortalized hPrEC cells by the expression of human telomerase (hTERT) with concomitant CRISPR inactivation of the CDKN2A locus, which directs the expression of both p16INK4A and p14ARF genes.Characterization of the two clonal cell lines that were generated showed that they maintained normal cell growth characteristics with intact p53 and pRb pathways, near normal karyotypes and have characteristics of basal cell origin. Subsequently, our lab sought to determine if expression of hTERT with knockout of just p16INK4A alone was also sufficient for immortalization, using CRISPR technology to inactivate exon 1α of the CDKN2A locus along with ectopic expression of the hTERT transgene. Knockout of p16INK4A but not p14ARF along with exogenous expression of hTERT resulted in the generation of a new immortal clone. Using these immortalized clones, along with primary hPrEC from ATCC our goal is to further characterize these cells to aid in future attempts aimed at immortalizing normal PrEC from multiple individuals and for the efficient establishment of a primary prostate cancer cell line. Our first approach included immunophenotyping our generated immortal hPrEC clones and ATCC hPrEC’s to identify the cell populations defining each of our clones and the different cell populations present in the primary hPrEC. We also characterized the expression of cells using 3D cell culture to determine their morphology and the expression of relevant markers. Finally, we identified the differentially expressed genes by RNA-seq in our immortalized hPrEC clones and ATCC hPrEC to determine their closest lineage identity as well as find suitable markers to use for future studies. These cell lines will also serve as a model to study transformation of PrEC in culture and xenograft tumorigenesis in mice. / Biomedical Sciences

O presente trabalho analisa a relação entre os jovens e a cena contemporânea tendo como objetos de estudo o grupo P14 Jugendtheater der Volksbühne e o espetáculo Favor beber o leite, senão estraga, do Coletivo Cronópio. O objetivo é refletir sobre obras do teatro pós-dramático ou performático para avaliar em que medida elas sugerem possibilidades formativas por meio da experiência. A pesquisa sobre o trabalho do grupo alemão foi feita a partir da observação in loco, no período de abril a junho de 2014, na cidade de Berlim, e de sua contextualização na perspectiva histórica do teatro jovem alemão (Jugendtheater) e do teatro Volksbühne. A peça do grupo brasileiro, por sua vez, nasceu a partir da premissa dessa dissertação - a experiência como aprendizado - e foi construída entre junho de 2013 e maio de 2015, período em que o grupo se debruçou sobre treinamentos pré-expressivos e elaborou a construção da dramaturgia em processo. / This paper analyzes the relationship between the youth and the contemporary scene, having as objects of study the P14 group Jugendtheater der Volksbühne and the theatre play Favor beber o leite, senão estraga, by the Cronópio collective theatre company. The aim is to reflect on the works of postdramatic and performative theater to assess to what extent they suggest formative possibilities through the experience. The research on the work of the German group was made from on-site observation in the period from April to June 2014, in Berlin, and its context in the historical perspective of young German theater (Jugendtheater) and the Volksbühne theatre. The Brazilian theatre group play, in turn, comes from the premise of this paper - the experience as learning - and was built between June 2013 and May 2015, when the group focused on pre-expressive trainings and prepared the construction of the drama in the process.

O presente trabalho analisa a relação entre os jovens e a cena contemporânea tendo como objetos de estudo o grupo P14 Jugendtheater der Volksbühne e o espetáculo Favor beber o leite, senão estraga, do Coletivo Cronópio. O objetivo é refletir sobre obras do teatro pós-dramático ou performático para avaliar em que medida elas sugerem possibilidades formativas por meio da experiência. A pesquisa sobre o trabalho do grupo alemão foi feita a partir da observação in loco, no período de abril a junho de 2014, na cidade de Berlim, e de sua contextualização na perspectiva histórica do teatro jovem alemão (Jugendtheater) e do teatro Volksbühne. A peça do grupo brasileiro, por sua vez, nasceu a partir da premissa dessa dissertação - a experiência como aprendizado - e foi construída entre junho de 2013 e maio de 2015, período em que o grupo se debruçou sobre treinamentos pré-expressivos e elaborou a construção da dramaturgia em processo. / This paper analyzes the relationship between the youth and the contemporary scene, having as objects of study the P14 group Jugendtheater der Volksbühne and the theatre play Favor beber o leite, senão estraga, by the Cronópio collective theatre company. The aim is to reflect on the works of postdramatic and performative theater to assess to what extent they suggest formative possibilities through the experience. The research on the work of the German group was made from on-site observation in the period from April to June 2014, in Berlin, and its context in the historical perspective of young German theater (Jugendtheater) and the Volksbühne theatre. The Brazilian theatre group play, in turn, comes from the premise of this paper - the experience as learning - and was built between June 2013 and May 2015, when the group focused on pre-expressive trainings and prepared the construction of the drama in the process.

<p>Despite improvements in detection and treatment, cancer is the leading cause of death worldwide. Current treatment modalities have not been able to improve the mortality rates and significant toxicities limit efficacy. Therefore there is a need for development of novel therapeutics.</p> <p>Oncolytic viruses have the ability to efficiently replicate in and destroy tumours while leaving normal tissues unharmed. These treatment platforms have been gaining momentum in recent years due to pre-clinical and clinical successes. Oncolytic viruses are extremely safe with limited toxicity observed in phase I/II clinical trials, and objective responses have been observed in some patients treated with oncolytic viruses. However, there is still room to improve on these therapeutic platforms.</p> <p>Recently, the importance of the induction of anti-tumour immunity during oncolytic virotherapy has been realized and harnessing this immune response can be used to improve current oncolytic virus platforms. To this end we have conducted numerous studies assessing our ability to improve oncolytic VSV through the addition of transgenes to enhance the immunostimulatory properties of oncolytic VSV treatment. These studies showed that only the addition of a highly secreted form of human IL-15 was able to improve VSV therapy through enhanced anti-tumour immunity. However, expressing cell-autonomous transgenes from oncolytic VSV was unable to modify the therapeutic efficacy of VSV due to limited replication, both temporally and geographically within the tumour, and the indirect vascular shutdown induced by VSV infection of tumours. We believe that the drastic vascular shutdown observed following VSV therapy is an important component to the success of VSV and we have investigated which steps in this process are critical for induction of anti-tumour immunity.</p> <p>The research presented in this thesis further enforces the requirement for induction of anti-tumour immune responses in the success of OV therapy. Our findings also indicate that manipulating the tumour as a whole, rather than the virus, will lead to improved oncolytic therapeutics.</p> / Doctor of Philosophy (PhD)

Introdução: Anormalidades nos supressores tumorais p14, p16 e p53 são relatadas em diversos tipos de câncer em humanos. Na carcinogênese ovariana, p16 e p53 foram extensivamente estudados, mas p14 foi analisado somente em carcinomas. Objetivo: O estudo visa determinar a expressão imuno-histoquímica de p14, p16 e p53 em tumores ovarianos epiteliais benignos, borderline e malignos. Método: Estudo transversal utilizando imuno-histoquímica em amostras de tumores epiteliais ovarianos emblocados em parafina do Hospital de Clínicas de Porto Alegre. Resultados: p14 foi positivo em 93% dos tumores benignos, 94% dos borderline e 60% dos malignos. A perda de expressão foi estatisticamente associada com carcinomas. p16 foi positivo em 94,6% dos carcinomas, 75% dos tumores borderline e 45,7% dos benignos. p53 foi positivo em 29,7%, 16,7% e 2,9% dos tumores malignos, borderline e benignos, respectivamente. Os subtipos de carcinoma não mostraram diferenças de expressão. Conclusão: Nosso estudo foi o primeiro a descrever a expressão de p14 em tumores benignos e borderline. Ela permanece estável nos benignos e borderline, enquanto os carcinomas exibem uma perda de expressão significativa. Isso pode indicar que anormalidades de p14 acontecem tardiamente na carcinogênese. As taxas de expressão de p16 e p53 foram semelhantes a estudos anteriores. Estudos futuros devem investigar anormalidades genéticas nas sequencias codificadoras de p14 e incluir todos os tipos de tumor epitelial ovariano. / Background: Abnormalities in tumor suppressors p14, p16 and p53 are reported in several human cancers. In ovarian carcinogenesis, p16 and p53 have been extensively studied, but p14 was only analyzed in carcinomas. Aim: This study seeks to determine p14, p16 and p53 immunohistochemical expression in benign, borderline and malignant ovarian epithelial tumors and correlate them with survival and clinical variables. Methods: Cross-sectional study utilizing immunohistochemical staining of p14, p16 and p53 in paraffin-embedded tissue samples from ovarian epithelial tumors obtained from Hospital de Clinicas de Porto Alegre. Results: p14 was positive in 93% of benign, 94% of borderline and 60% of malignant tumors. Loss of expression was statistically associated with carcinomas. p16 was positive in 94.6% of carcinomas, 75% of borderline and 45.7% of benign tumors. p53 was positive in 29.7%, 16.7% and 2.9% of malignant, borderline and benign tumors, respectively. Carcinoma subtypes showed no difference in expression. Conclusions: To our knowledge, this is the first description of p14 expression in benign and borderline tumors. It remains stable in benign and borderline tumors, while carcinomas show a significant absence of staining. This may indicate p14 abnormalities occur later in carcinogenesis. p16 and p53 expression rates show similar results to previous reports. Future studies should investigate genetic abnormalities in p14 coding sequences and include all types of ovarian epithelial tumors.

The genus Benyvirus includes the most important and widespread sugar beet viruses transmitted through the soil by the plasmodiophorid Polymyxa betae. In particular Beet necrotic yellow vein virus (BNYVV), the leading infectious agent that affects sugar beet, causes an abnormal rootlet proliferation known as rhizomania. Beet soil-borne mosaic virus (BSBMV) is widely distributed in the United States and, up to date has not been reported in others countries. My PhD project aims to investigate molecular interactions between BNYVV and BSBMV and the mechanisms involved in the pathogenesis of these viruses.BNYVV full-length infectious cDNA clones were available as well as full-length cDNA clones of BSBMV RNA-1, -2, -3 and -4. Handling of these cDNA clones in order to produce in vitro infectious transcripts need sensitive and expensive steps, so Ideveloped agroclones of BNYVV and BSBMV RNAs, as well as viral replicons allowing the expression of different proteins.Chenopodium quinoa and Nicotiana benthamiana plants have been infected with in vitro transcripts and agroclones to investigate the interaction between BNYVV and BSBMV RNA-1 and -2 and the behavior of artificial viral chimeras. Simultaneously I characterized BSBMV p14 and demonstrated that it is a suppressor of posttranscriptional gene silencing sharing common features with BNYVV p14.