Ovine herpesvirus 2 (OvHV-2) is the causative agent of sheep-associated malignant catarrhal fever (MCF) in susceptible ruminants. Through an unknown mechanism, presence of the virus leads to proliferation of NK-like T cells that are not targetrestricted by the MHC class molecules. These host cells cause the symptoms found in MCF; fever, swollen lymph nodes, and necrotic lesions of the nasal, conjunctival, and oral mucosa, which usually leads to death of the host. MicroRNAs (miRNAs) are ~22 nt RNA molecules expressed by eukaryotes and viruses that regulate genes post-transcriptionally. Viral miRNAs have been found to regulate cellular genes to control the cell cycle and have a role in pathogenesis. It was hypothesised that OvHV-2 expresses miRNAs and these play a role in MCF pathogenesis. The aim of this project was to determine if OvHV-2 encodes miRNAs. Bioinformatic analysis was conducted on deep sequencing data from RNA of OvHV-2- immortalised T cells. Candidate miRNAs were selected if they adhered to miRNA secondary structure. 46 candidate miRNAs were found, with three clusters on the minus strand; one at the 5’ end and the other two in a 9.3 kb region that contains no predicted open reading frames. The 8 most abundant candidates were successfully validated by northern hybridisation for small RNAs. The majority of the predicted targets for the 8 validated OvHV-2 miRNAs were from the OvHV-2 genome. This study adds OvHV-2 to the list of herpesviruses that encode miRNAs and provides another tool for studying the pathogenesis of MCF.

Cellular life is highly complex. In order to expand our understanding of the workings of human cells, in particular in the context of health and disease, detailed knowledge about the underlying molecular systems is needed. The unifying theme of this thesis concerns the use of data derived from sequencing of RNA, both within the field of transcriptomics itself and as a guide for further studies at the level of protein expression. In paper I, we showed that publicly available RNA-seq datasets are consistent across different studies, requiring only light processing for the data to cluster according to biological, rather than technical characteristics. This suggests that RNA-seq has developed into a reliable and highly reproducible technology, and that the increasing amount of publicly available RNA-seq data constitutes a valuable resource for meta-analyses. In paper II, we explored the ability to extrapolate protein concentrations by the use of RNA expression levels. We showed that mRNA and corresponding steady-state protein concentrations correlate well by introducing a gene-specific RNA-to-protein conversion factor that is stable across various cell types and tissues. The results from this study indicate the utility of RNA-seq also within the field of proteomics. The second part of the thesis starts with a paper in which we used transcriptomics to guide subsequent protein studies of the molecular mechanisms underlying malignant transformation. In paper III, we applied a transcriptomics approach to a cell model for defined steps of malignant transformation, and identified several genes with interesting expression patterns whose corresponding proteins were further analyzed with subcellular spatial resolution. Several of these proteins were further studied in clinical tumor samples, confirming that this cell model provides a relevant system for studying cancer mechanisms. In paper IV, we continued to explore the transcriptional landscape in the same cell model under moderate hypoxic conditions. To conclude, this thesis demonstrates the usefulness of RNA-seq data, from a transcriptomics perspective and beyond; to guide in analyses of protein expression, with the ultimate goal to unravel the complexity of the human cell, from a holistic point of view. / <p>QC 20161121</p>

O tratamento dos gliomas malignos cerebrais é um dos grandes desafios da medicina atualmente, pois, apesar do grande avanço no conhecimento destes tumores, o prognóstico de vida dos portadores desta doença é muito ruim. Foram estudados retrospectivamente 61 pacientes com diagnóstico de glioblastoma multiforme ou astrocitoma anaplásico, no período de 1998 a 2002, com o objetivo de avaliar o uso do Paclitaxel como potencializador do tratamento radioterápico destes tumores. Todos os pacientes foram tratados inicialmente com cirurgia para retirada ampla do volume tumoral (mínimo de 80%) seguido de tratamento com radioterapia fracionada e reforço com radiocirurgia estereotáxica. Em caso de crescimento tumoral, após o tratamento inicial dos pacientes com KPS > 70, novo tratamento cirúrgico e nova radiocirurgia foram indicados. Destes 61 pacientes, 32 receberam tratamento com Paclitaxel, na dose de 100mg/m2 e 29 pacientes não receberam nenhum tipo de quimioterápico. Os grupos foram comparados, em relação ao tipo histológico, faixa etária, sexo e localização tumoral, não havendo diferenças estatisticamente significantes entre os mesmos. Os pacientes de ambos os grupos tiveram acompanhamento laboratorial antes, durante e após o tratamento com paclitaxel e foram acompanhados até o óbito causado pela doença. Foram excluídos do estudo os portadores de tumor que foram a óbito por outras causas. A análise dos resultados mostrou que não houve diferenças estatísticas em relação à sobrevida média do grupo tratado com Paclitaxel e o grupo sem o tratamento (p=1,000). Da mesma forma, a comparação entre os pacientes com glioblastomas (p=0,8933) e com astrocitomas anaplásicos (p=0,5920) de ambos os grupos não mostrou diferença estatística em relação à sobrevida. O número de craniotomias( p=0,5268) e o número de radiocirurgias (p=0,3666) foram semelhantes estatisticamente. Os estudos laboratoriais realizados durante o tratamento no grupo que recebeu o paclitaxel, não mostraram alterações que levassem à suspensão do tratamento. A análise dos resultados deste estudo permitiu concluir que o uso do paclitaxel, concomitante ao tratamento radioterápico dos gliomas malignos cerebrais, não mostrou nenhum ganho adicional na sobrevida dos pacientes portadores destes tumores e, pela análise da necessidade de novo tratamento durante o curso da doença, não potencializou os efeitos da radioterapia. Palavras-chave: Paclitaxel, gliomas malignos, radioterapia / Nowadays, the treatment of the malignant cerebral gliomas is one of the greatest challenges for neurosurgons. Despite of the advance regarding these tumors? knowledge expectance of life for these patients is very bad. The main purpose of this study was to evaluate the use of paclitaxel to enhance the radiotherapy treatment in those tumors. Sixty-one patients with diagnosis for glioblastoma multiforme or anaplastic astrocytoma in the period of 1998 to 2002 were, retrospectively, studied. All patients were initially treated with surgery in order to remove a wide portion of the tumor?s volume (minimum of 80%). Then, the patients were treated with fractionated radiotherapy and reinforcement with stereotactic radiosurgery. If there was an increase in the tumor after the initial treatment, the patients with a KPS higher than 70 had new treatment with surgery as well as with radiosurgery. Among the 61 patients, 32 were treated with a 100 mg/m2 dose of paclitaxel, and 29 of then did not have any kind of chemotherapy treatment. Comparisons bettwen both regardhg to the histological type, age, gender and location of the tumor showed no differences . Patients of both groups had a laboratory follow-up before, during and after the treatment with paclitaxel. All of them were followed until their death, which was caused by the disease. Patients that died from other diseases were not included in the study. The analysis of the results indicated that there were no statistics differences regarding the mean survival time between the groups treated or nor treated with paclitaxel ( p=1,000). Likewise, a comparison between the glioblastomas (p=0,8933) and the anaplastic astrocytomas (p=0,5920) of both groups did not indicate any statistic difference regarding to the survival time. The was no statistic difference between the number of craniotomies (p=0,5268) as well as between the number of radiosurgeries. (p=0,3666). The laboratory studies held during the treatment of the group that received the paclitaxel did not show any changes which could lead to cease the treatment. Hence the results led to the conclusion that the treatment of malignant cerebral gliomas with paclitaxel and radiotherapy treatment at the same time did not give any additional gain in the patients?survival. Regarding to the demand for new treatment throughout the disease, there was no enhance of the radiotherapy effects with the Paclitaxel. Key words: Paclitaxel, malignant gliomas, radiotherapy.

A avaliação da presença de destruição óssea provocada por neoplasias malignas de boca e orofaringe é um fator de fundamental importância no estabelecimento da terapêutica adequada para o caso, como também, para a determinação do prognóstico do paciente. O presente estudo teve por objetivo determinar os aspectos clínicos (localização, forma de apresentação e estadiamento) que podem estar associadas com o potencial de infiltração do osso subjacente a lesão, como também determinar a sensibilidade e especificidade do exame físico. A população de estudo consistio de vinte e cinco pacientes (17 homens e 8 mulheres, média de idade de 57,88 anos) portadores de neoplasias malignas de boca e orofaringe atendidos no Ambulatório de Semiologia da Faculdade de Odontologia da Universidade de São Paulo – campus São Paulo, no período de agosto de 2003 a agosto de 2004, os quais foram submetidos ao exame clínico e a tomografia computadorizada (TC). A TC foi considerada o padrão ouro para a avaliação da presença de destruição óssea. Foi observada a presença de infiltração neoplásica para o tecido ósseo adjacente em 68% dos casos (17 pacientes). O exame físico dos pacientes revelou uma sensibilidade de 80% e especificidade de 87,50% na análise de comprometimento do osso, além de uma acurácia de 84%. As lesões que se apresentavam clinicamente como uma úlcera do tipo infiltrativa e lesões do tipo nodulares, não ulceradas, foram as que apresentaram maior potencial de infiltrar-se para o osso, 68,75% e 100% respectivamente. A localização do tumor em determinados sítios, também influenciou diretamente na presença de invasão óssea,principalmente lesões localizadas em região de gengiva, trígono retromolar, palato duro e orofaringe. O estadiamento das lesões revelou relação existente entre o tamanho do tumor e a presença de metástases à distância com a presença de infiltração da neoplasia para o tecido ósseo. Concluindo, observou-se que a identificação de determinados parâmetros clínicos como localização, forma de apresentação clinica, tamanho da lesão e a presença de metástases à distância, associado a um criterioso exame físico regional podem servir como valiosas ferramentas para a análise de envolvimento ósseo por neoplasias malignas de boca e orofaringe. / The assessment of bone destruction by oral and oropharynx malignant neoplasms is a critical factor in the therapeutic planning and to determine the patient prognostic. The aim of this study was to determine the clinical aspects (localization, clinical manifestation and stage) that can be associated with the potential of bone infiltration, and also determine the physical exam sensibility and specificity. The study population consisted of twenty five patients (17 men and 8 women, mean age 57.88 years-old), with malignant neoplasms of the mouth and oropharynx, of the Stomatology Clinic of the College of Dentistry at the Sao Paulo University - campus Sao Paulo, in the period of august 2003 to august 2004, who were submitted to a clinical and computed tomography (CT) examinations. CT was considered the gold standard to evaluate the presence of bone involvement. The presence of bone destruction by the tumor was observed in 68% of the cases (17 patients). The physical examination of the patients revealed 82% of sensibility, 87.50% of specificity, and 84% of accuracy in the assessment of bone invasion by these diseases. The lesions that were clinical considered to be infiltrative ulcer and nodular lesions, non-ulcerated, presented the highest potential to cause bone destruction, 68.75% and 100% respectively. The tumor localization in specific sites also influenced the presence of bone invasion, meanly with the lesions localized in the gingival, retromolar trigone, hard palate and oropharynx. The stage of the lesions revealed a relation between the size and the presence of distant metastasis, with the presence of invasion by the neoplasm. In conclusion, it was determined that the identification of some clinical parameters such localization, clinical presentation, lesion size and the presence of distant metastasis, associated with a perceptive regional physical exam must be use as a value tool is the assessment of bone destruction by oral and oropharynx malignant neoplasms.

Background: Matrix metalloproteinases (MMPs) have been shown to be involved in cancers and atherosclerosis, the leading causes of present day mortality. The objectives of the cancer element of this project were to investigate single nucleotide polymorphisms (SNPs) in MMP1 and MMP8 regarding breast cancer and malignant melanoma, and a functional characterisation of the genetic variants, including the MMP1 polymorphism rs19799750, previously associated with multiple cancers. The objective of the second part of this project was to investigate whether MMP8 played a role in the development of atherosclerotic lesions and if so, the underlying mechanisms. Methods/Results: Genetic investigations found the MMP8 SNP rs11225395 to be associated with the occurrence of both breast cancer and malignant melanoma; furthermore it was also associated with reduced lymph node metastasis, reduced cancer relapse and greater survival. Functional luciferase assays showed that the minor allele of the polymorphism has higher promoter activity in breast cancer and melanoma cell lines. They also showed haplotypic effects on MMP1 promoter activity in several cancer cell lines by the 2G allele of polymorphism rs1799750 and one or more MMP1 promoter SNPS. The second part of the study found an association between a MMP8 SNP and the extent of coronary atherosclerosis; additionally a relationship among MMP8 gene variation, plasma VCAM-1 level, and atherosclerosis progression was observed in a prospective study. Murine studies showed reduced atherosclerosis in MMP8/ApoE knockout mice compared with ApoE knockout littermate controls. Biochemical studies confirmed that MMP8 can convert angiotensin I to angiotensin II. Conclusions: The data of the first part of this project support the notion that genetic polymorphisms in the MMP1 and MMP8 influence the expression of these genes and the development and progression of cancer. The results of the second part of this project indicate an important role of MMP8 in the pathogenesis of atherosclerosis.

Approximately 50% of all malignant pleural mesotheliomas (MPM) are deficient in argininosuccinate synthetase (ASS1), the rate-limiting enzyme in arginine biosynthesis, and are sensitive to arginine deprivation. This discovery in MPM has been translated into the clinic using the arginine depletor pegylated arginine deiminase (ADI-PEG20), which showed a halving in the risk of disease progression in a randomised phase II study. However, unstudied to date, stromal resistance to ADI-PEG20 may reduce its efficacy. Here, I studied the effect of macrophages, abundant in mesothelioma, on the tumour cytotoxicity of ADI-PEG20. A distinct pro-inflammatory cytokine gene expression signature involved in macrophage recruitment and activation was identified and validated in ADI-PEG20-treated ASS1 negative MPM cell lines. In vivo induction of pro-inflammatory cytokines was also seen in ADI-PEG20-treated patient plasma. Notably, in vitro co-culture experiments demonstrated a significant increase in ASS1 negative MPM cell viability upon co-culture with macrophages in the presence of ADI-PEG20. This was accompanied by a significant increase in ASS1 expression in co-cultured macrophages, with a corresponding increase in argininosuccinate lyase (ASL) expression in co-cultured tumour cells and a doubling in levels of the arginine precursor, argininosuccinate, in cell supernatant. The addition of argininosuccinate to tumour cell media rescued ASS1 negative MPM cells from ADI-PEG20 cytotoxicity, while the macrophage-mediated resistance to ADI-PEG20 was abrogated following ASL knockdown in MPM cells. Finally, xenograft studies demonstrated a significant reduction in tumour volume in mice treated with ADI-PEG20 in combination with macrophage depletion, compared with ADI-PEG20 alone. Collectively, the data indicate that as a result of metabolic 'cross-talk' between macrophages and ASS1 negative MPM cells, macrophages mediate MPM resistance to ADI-PEG20 via the provision of argininosuccinate. My studies provide a rationale for combining ADI-PEG20 with an inhibitor of macrophage recruitment in the treatment of ASS1-deficient mesothelioma.

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg; in partial fulfillment for the degree of Master of Medicine (Anatomical Pathology) 2015. / Cytological criteria distinguishing phyllodes tumour of the breast from fibroadenoma. Fibroepithelial lesions of the breast include fibroadenomas and phyllodes tumours. Fibroadenomas are benign tumours, whereas phyllodes tumours range from benign, indolent neoplasms to malignant tumours capable of distant metastasis and occasionally resulting in death. The aim of this study was to determine whether there are statistically significant differences between fibroadenomas and phyllodes tumours with regard to selected cytological features. A ten year retrospective review was performed of patients who had an excision of a fibroadenoma or phyllodes tumour, and on whom a pre-operative fine needle aspirate was performed. The following cytological criteria were assessed: number of stromal and epithelial fragments, stromal to epithelial ratio, stromal cellularity, Stromal borders, stromal atypia and proportion of background wavy spindled cells. The patient age, tumour laterality and tumour size were recorded. Fifty fibroadenomas and 17 phyllodes tumours were included. When compared with phyllodes tumours, fibroadenomas had a larger number of epithelial fragments, a smaller number of stromal fragments and a lower stromal to epithelial ratio. The stroma tended to be less cellular and less atypical compared with phyllodes tumours and the background cellular population contained less spindled cells. Fibroadenomas and phyllodes tumours differ with regard to various cytological features, possibly aiding in their distinction on fine needle aspiration biopsy. / AC2016

Malignant mesothelioma (MM) is an aggressive tumour, which is highly associated with previous asbestos exposure and is resistant to most conventional anticancer therapies. Previous studies have used a mouse model of to 01 p effective approaches to induction of anti-tumour immunity using modification of tumour cells by the introduction of genetic constructs expressing genes such as that for B7-1 so that tumour growth can be inhibited in vivo. Transfectant clones, AC29 B7-7 and AC29 B7-6, which showed equal levels of expression of B7-1 but were markedly different in tumorigenicity were assessed using suppression subtractive hybridization (SSH) in order to isolate transcripts which may have been differentially expressed in the two clones. SSH allowed isolation of a number of cDNAs which were apparently differentially expressed in the cell lines. These required characterisation in order to determine their possible relevance to tumorigenicity. Two cDNAs designated as 7-7-76 and 7-7-43 had been isolated previously and the aim of this project was to characterise these cDNAs by sequencing, searching for their homology relationships and investigating gene expression profiles. Preliminary searches revealed that clone 7-7-43 had homology to cyclin-dependent kinase regulatory subunit 1 which plays a role in the cell cycle. On the other hand, clone 77-76 showed only homology to an EST of hypertension related protein and therefore, further investigation was required to obtain the identity of clone 7-7-76. The first part of this project was to in investigate and evaluate gene expression on clone 7-7-43, using both relative RT-PCR and Northern blotting.' In the second part of this project, a more intense study of clone 7-7-76 was conducted. Clone 7-7-76 was investigated for its homology relationships and its gene expression profile. / Results obtained from relative RT-PCR suggested no difference in the expression of the either eDNA clone (7-7-43 and 7-7-76) between the MM clones AC29 B7-6 and AC29 B7-7, the cells used to derive these clones by SSH. Therefore, it was concluded that neither clone 7-7-43 nor 7-7-76 was differentially expressed in MM cells of differing immuno enicit RACE was employed in order to derive a longer sequence of clone 7-7-76 and the newly derived sequence of 7-7-76 was again used to search for homologies using a wider range of sequences for human and other species. These investigations on clone 7-7-76 showed it to correspond to the sequence of human mitofusin 2 which is involved in determining mitochondrial morphology The results determined in this project suggest that clones 7-7-43 and 7-7-76 are not differentially expressed in the range of MM cell lines tested. The data have however highlighted the potential of the SSH technique to easily derive cDNA clones worthy of investigation, but underline the possibility of false positive clones being isolated. The need for an efficient, accurate screening procedure such as real-time PCR is acknowledged.

Malignant mesothelioma (MM) is an aggressive malignancy that originates from mesothelial cells and is highly resistant to conventional forms of anti-cancer therapy. Defects in apoptotic pathways are believed to play a major role in determining resistance to chemotherapy. The characterization of these pathways in mesothelioma is essential in order to develop more effective therapies. The inhibitor of apoptosis proteins (IAPs) are a family of proteins that regulate apoptosis and have been implicated in the resistance of malignant cells. There is evidence that upregulation of specific IAP molecules can influence tumour progression and response to chemotherapy. In this study we examined the apoptotic signalling in MM cells and the potential role of IAPs in both cell proliferation and chemosensitivity. We examined expression of six IAP genes or isoforms in both malignant and normal mesothelial cells. Results demonstrated that XIAP, IAP-1, IAP-2, survivin and Bruce were expressed in all four MM cell lines and four primary mesothelial cultures. There was no evidence for differential expression of these genes between MM and mesothelial cultures. Livin expression was detected in only one MM cell line. Various aspects of apoptotic signalling pathways in response to the chemotherapeutic drug cisplatin were also analysed including: a) the mitochondrial integrity, b) caspase activation, c) cell viability and d) phosphatidylserine translocation. In order to further characterize the role of IAPs, the transcriptional regulation of these genes in response to cisplatin was investigated using real-time RT-PCR. The results of these experiments indicated that there was no significant regulation of IAPs at the transcriptional level in the cells examined during cisplatin-induced apoptosis. Overall the data was consistent with cisplatin inducing apoptosis in MM cells via intrinsic signalling pathways in a dose dependent manner. Regulation of IAP expression was not seen at the RNA transcription level as has been described in other tumour types but may occur through protein posttranslational events. In order to further investigate IAP function we performed analyses of two IAPs which had previously been proposed as having a role in mesothelioma: XIAP and survivin. Protocols for RNAi knockdown at the protein expression level were established. Although the data indicated significant reduction in protein expression, the effects on cell survival after treatment with cisplatin were moderate. These studies were then extended to other molecules that are known to interact with and modulate the function of IAPs. We characterized the expression of the proteins: XAF1, HTRA2, ARTS in MM cells. RT-PCR data showed that HTRA2 and XAF1 genes were expressed in MM cell lines, however we did not see expression of ARTS. On the basis of recently published data we examined the XAF1 splice variants expressed in MM cell lines by sequence determination and PCR screening.

A model with human melanoma xenografts transplanted to athymic mice has been adopted for in vivo studies of 5-S-cysteinyldopa (an intermediate pigment metabolite), glutathione, and cysteine. L-Dopa is an intermediate metabolite in pigment formation and is also important in the treatment of Parkinson's disease, and therefore 1 have also studied the pharmacokinetics of this compound. We were first to describe in vivo microdialysis in melanoma tissue and showed that dialysis membranes of cuprophane or polyamide are suitable for studies of interstitial 5-S-cysteinyldopa and selected thiols. Analytical procedures were also improved for quantitation of 5-S-cysteinyldopa, L-dopa, glutathione, cysteine, and N-acetylcysteine (NAC). In the melanoma xenografts the interstitial concentration of 5-S-cysteinyldopa reflected the high intracellular production of this intermediate metabolite. For in vivo manipulation of glutathione in the melanoma tissue we gave intraperitoneal injection of buthionine sulphoximine to the animals and thus reduced the glutathione concentrations substantially. We showed that restitution of glutathione in melanoma tissue occurs spontaneously and is not much improved by treatment with the cysteine deliverers NAC and L-2-oxothiazolidine-4-carboxylate (OTC). 5-S-Cysteinyldopa was not substantially affected by great variations in glutathione concentrations. Transport of NAC from intraperitoneal injection to melanoma tissue occurred rapidly and deacetylation to cysteine in vivo could be detected soon after NAC injection. In vivo formation of cysteine was slower from OTC than from NAC. Pharmacokinetic studies of L-dopa in human subjects indicated a slight to moderate protein binding. Plasma free L-dopa had similar elimination T½ as interstitial L-dopa, but in some cases the elimination of total L-dopa was slower. Difficulties in intestinal absorption of L-dopa were revealed by microdialysis in blood and subcutaneous tissue. Studies showed that this was due to delayed emptying of the stomach. L-Dopa intake increased 5-S-cysteinyldopa concentrations in blood within 30 min in patients with Parkinson's disease and a history of melanoma. No melanoma activation occurred during long-term treatment with L-dopa. Microdialysis is thus a safe and easily applied method for in vivo studies of both pigment metabolites from human melanoma tissue transplanted to nude mice and for pharmacokinetic studies of L-dopa. / On the day of the public defence the status of the articles IV, V and VI was: Submitted.