Made available in DSpace on 2014-06-11T19:32:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-12-08Bitstream added on 2014-06-13T21:04:09Z : No. of bitstreams: 1 felipe_lb_dr_rcla.pdf: 4814352 bytes, checksum: 82366956a2d6f6b06cf31ba0bb697761 (MD5) / Este trabalho aborda os aspectos tectono-estruturais e geomorfológicos dos sedimentos miocênicos na região de Marabá, no Estado do Pará. As unidades geomofológicas desenvolveram-se, predominantemente, sobre os sedimentos areno-argilosos e mal consolidados da Formação Barreiras. Essa formação repousa de forma discordante, de oeste para leste, sobre o embasamento cristalino (Província Transamazonas) e sobre as rochas metamórficas do Cinturão Araguaia, depositados na extensão sul da Sub-bacia de Mocajuba. A partir de interpretações de imagens SAR e SRTM, dados de campo, análises de perfis topográficos, da integração e interpretação das informações obtidas, pode-se concluir que as reativações tectônicas pós-cretácicas constituem o fator mais importante na configuração geomorfológica da área de estudo. Essas reativações foram responsáveis por soerguimentos distintos e basculamentos de superfícies geomorfológicas, além de criar inúmeros e importantes falhamentos que orientaram entalhes e dissecações, definindo a compartimentação e a configuração atual dos terraços fluviais e das principais anomalias de drenagem na área de estudo: o Paleo-Canal do Tocantins (bacia romboédrica) e o Bico do Papagaio (encontro dos rios Tocantins e Araguaia). Portanto, a interpretação mostra uma importante movimentação neotectônica controlando o relevo e a drenagem atual com ajuste ao quadro regional, ou seja, um sistema distensivo com falhas NNE – SSW a NS, seguido pela sedimentação da Formação Barreiras (Mioceno), e notável distensão ENE-SSW a EW / The geomorphological units developed predominantly on sandy clay sediments and poorly consolidated of Barreiras Formation. This formation rests in discordant form, from west to east, on the crystalline basement (Transamazonas Province) and on the metamorphic rocks of the Araguaia Belt, deposited in the southern extension of the Mocajuba Sub-basin. From interpretations of SAR images and SRTM, field observations, analysis of topographic profiles, integration and interpretation of information obtained, it can be concluded that the post-Cretaceous tectonic reactivations are the most important factor in morphological configuration of the study area. These reactivations were responsible for uplifts and slant distinct geomorphic surfaces, and create numerous and general faulting that supported cuts and guided dissections, defining the partitioning and the current configuration of the river terraces and major drainage anomalies in the study area: the Paleo-channel Tocantins (romboedric basin) and the Bico do Papagaio (confluence of Tocantins and Araguaia rivers). Therefore, the data shows a significant neotectonic movement controlling the relief and current drainage, in adjustment with the regional framework, a system with extensional faults NNE - SSW to NS, followed by sedimentation of Barreiras Formation (Miocene), and noticeable distension ENE- SSW to EW

The rising demand for powerful oncolytic virotherapy agents has led to the identification of Maraba virus, one of the most potent oncolytic viruses from Rhabdoviridae family which displays high selectivity for killing malignant cells and low cytotoxicity in normal cells. Although the virus is readied to be used for clinical trials, the interactions between the virus and the host cells is still unclear. Using a newly developed interferon-sensitive mutant Maraba virus (MG1), we have identified two key regulators of global translation (4E-BP1 and eIF2α) responsible for the inhibition of protein synthesis in the infected cells. Despite the translational arrest upon viral stress, we showed an up-regulation of anti-apoptotic Bcl-xL protein that provides a survival benefit for the host cell, yet facilitates effective viral propagation. Given the fact that eIF5B canonically regulates 60S ribosome subunit end joining, and is able to replace the role of eIF2 in delivering initiator tRNA to the 40S ribosome subunit upon the phosphorylation of eIF2α, we have tested whether eIF5B mediates the translation of target mRNAs during MG1 infection. Our results show that the inhibition of eIF5B significantly down-regulates the level of Bcl-xL steady-state mRNA, thus indirectly attenuates viral propagation.

Oncolytic viruses (OVs) are effective anti-cancer agents, however their abilities to induce anti-tumor immunity are not yet optimal. Mutanome epitopes are a novel source of tumor antigen formed as a result of mutations within the tumor genome. Within this project we attempted to combine B16F10 mutanome vaccination with OV therapy. We confirmed previous findings that significant immune responses to these epitopes can be generated. Furthermore, we designed and cloned a multi-epitope mutanome construct into MG1 Maraba virus and E1-/E3- deleted type 5 Adenovirus to use for heterologous prime-boost vaccination. While we demonstrated that these viruses induced T-cell responses to one mutanome epitope, we failed to detect responses to the other epitopes. Furthermore there was no effect seen on overall survival. This approach warrants further investigation because coupling mutanome vaccination with OV therapy has the potential to exploit the therapeutic effects of the OV while inducing anti-tumor immunity to tumor-unique antigens.

HPV-associated cancer and carcinoma of the prostate are responsible for significant worldwide morbidity and mortality. The viral transforming proteins E6 and E7 make human papilloma virus positive (HPV+) malignancies an attractive target for cancer immunotherapy however, therapeutic vaccination exerts limited efficacy in the setting of advanced disease. In prostatic carcinoma therapeutic vaccination shows some therapeutic activity but is infrequently curative. A strategy to induce substantial specific immune responses against multiple epitopes of E6 and E7 proteins based on an attenuated transgene from HPV serotypes 16 and 18, that is incorporated into MG1-Maraba virotherapy (MG1-E6E7), was designed. MG1-E6E7 is able to boost specific immunity following priming with either an adenoviral vector (Ad-E6E7) or customised synthetic peptide vaccines resulting in multifunctional CD8+ T cell responses of an enormous magnitude. MG1-E6E7 vaccination in the HPV+ murine model TC1 is curative against large tumours in a CD8+ dependent manner and results in durable immunologic memory. Using the same adenoviral prime and MG1 boosting strategy targeting the prostatic antigen, STEAP, immunity against multiple CD8+ STEAP epitopes was induced. In a murine prostate cancer model, STEAP specific oncolytic virotherapy significantly improved the survival of mice bearing advanced TRAMP-C2 tumours. One significant obstacle to therapeutic cancer vaccination is an immunosuppressive tumour microenvironment. MG1 Maraba is able to lethally infect HPV-associated and prostate cancer cells, increase the immunologic activity within the tumour microenvironment in vivo and exploit molecular hallmarks of HPV-positive cancer and prostatic carcinoma enabling infection of bulky tumours. Pre-clinical data generated within this thesis has been instrumental in securing funding for future clinical trials assessing the safety and activity of MG1 Maraba virotherapy for HPV-associated cancer and prostatic carcinoma. This promising approach has the potential to be directly translatable to human clinical oncology to tackle these two highly prevalent and frequently lethal groups of epithelial neoplasia. / Thesis / Doctor of Philosophy (PhD) / Carcinoma (epithelial cancer) is the most common form of human cancer and two frequently encountered types, namely HPV-associated and prostatic carcinoma are responsible for a substantial worldwide cancer burden. Current therapeutic options show limited clinical benefit and/ or significant long-term side effects for advanced carcinomas, therefore new treatments are urgently required. Oncolytic viruses represent an exciting new form of anti-tumour immunotherapy capable of infecting and killing cancerous cells; here we present a virus called MG1 Maraba that is able to exploit molecular characteristics of these cancers. When MG1 Maraba is engineered to target proteins from HPV-associated cancer and prostatic carcinoma, specific immune attack against these tumours occur in mouse cancer models. MG1 Maraba offers a novel, selective, safe and highly promising therapeutic approach against advanced carcinomas. Based on the information within this thesis human clinical trials assessing MG1 Maraba are due to take place for both HPV-associated and prostate cancer.

Peritoneal carcinomatosis (PC), the intra-abdominal dissemination of malignancy, is equated with a 5-year survival of 15%, depending on the source. Appendiceal PC is a challenge to treat as cancer cells are embedded in copious amounts of mucin and are difficult to target. Oncolytic viruses (OVs) preferentially replicate and lyse cancer cells and present a targeted, novel strategy for PC. The hypothesis of this study is that appendiceal PC will show variable susceptibility to OVs and that protein expression in these tumours will predict OV replication efficiency. Human appendiceal PC infected ex-vivo with 4 different OVs displayed variable infectivity and replication by fluorescence microscopy and plaque assay. Immunohistochemistry analysis revealed differential expression of IRF3, pERK and TK in tumour compared to normal appendix. No correlation of protein expression with viral replication was observed. Personalizing OV therapy will be critical in the optimization of future care of patients treated with this modality.

Peritoneal carcinomatosis (PC), the intra-abdominal dissemination of malignancy, is equated with a 5-year survival of 15%, depending on the source. Appendiceal PC is a challenge to treat as cancer cells are embedded in copious amounts of mucin and are difficult to target. Oncolytic viruses (OVs) preferentially replicate and lyse cancer cells and present a targeted, novel strategy for PC. The hypothesis of this study is that appendiceal PC will show variable susceptibility to OVs and that protein expression in these tumours will predict OV replication efficiency. Human appendiceal PC infected ex-vivo with 4 different OVs displayed variable infectivity and replication by fluorescence microscopy and plaque assay. Immunohistochemistry analysis revealed differential expression of IRF3, pERK and TK in tumour compared to normal appendix. No correlation of protein expression with viral replication was observed. Personalizing OV therapy will be critical in the optimization of future care of patients treated with this modality.

A major obstacle in the implementation of adoptive cell therapy (ACT) for solid tumors is CD8+ T cell quantity and functional quality. In order to address this issue, the ACT field has directed considerable effort toward the generation of less-differentiated memory T cells (Tm), which demonstrate superior effector function and engraftment over effector T cells. An obstacle in using Tm for ACT is their requirement for in vivo activation before full effector function can be acquired. We sought to determine if a rhabdovirus expressing a defined tumor antigen (i.e. a rhabdoviral oncolytic vaccine) could activate adoptively-transferred Tm in vivo and eliminate established tumors. We used ex vivo cultured DUC18 TCR-transgenic Tm combined with a rhabdoviral oncolytic vaccine to target established CMS5 fibrosarcomas in both balb/c and NRG mice, and we compared the efficacy of the combination treatment versus monotherapies. Our data demonstrate that the rhabdoviral oncolytic vaccine was capable of expanding adoptively-transferred Tm in order to eliminate established tumors. Furthermore, synergy between ACT and oncolytic vaccination was required for optimal therapeutic outcome. Interestingly, we observed a population of endogenous, tumor-primed lymphocytes which appeared to be required for complete tumor elimination and subsequent memory formation. This was in contrast to the current consensus in the ACT field which is that endogenous lymphocytes are detrimental to therapeutic outcome, thus necessitating lymphodepletion prior to the commencement of therapy. Our data suggest that endogenous lymphocytes may be a beneficial cell population which is overlooked by current approaches to ACT. / Thesis / Master of Science (MSc) / Current approaches to the T cell therapy of cancer are hindered by poor cell quality. It is simple to grow higher quality T cells, but it is difficult to grow very large numbers of them. Furthermore, higher quality T cells need a signal in order to “switch on” before they can start killing cancer cells. Here, we use a cancer-targeting virus as a signal for these cells to activate, grow to very large numbers in the patient, and destroy their tumor. Our vaccine also switches on other immune cells in the patient, which help guarantee the destruction of the tumor. The significance of this work is that it will improve T cell therapy for cancer by opening the possibility of using higher-quality T cells which are much better at killing cancer than the currently used type of T cells.