The separation, identification, and characterisation of the proteins present in a tissue or biological sample is called ‘proteomics’. This technique can be used for example to identify biomarkers and investigate signalling pathways. Increasingly, proteomics is being applied to the analysis of virus related samples; here two such examples are described. Presently there is no reliable non-invasive way of assessing liver fibrosis. Here a novel 2D-PAGE based proteomics study was used to identify potential fibrosis biomarkers. Serum from patients with varying degrees of hepatic scarring induced by infection with the hepatitis C virus (HCV) was analysed. Several proteins associated with liver scarring and/or viral infection were identified. The most prominent changes were observed when comparing serum samples from cirrhotic patients with healthy controls: Expression of inter-α-trypsin inhibitor heavy chain H4 fragments, α1 antichymotrypsin, apolipoprotein L1 (Apo L1), prealbumin and albumin was decreased in cirrhotic serum, whereas CD5 antigen like protein (CD5L) and β2 glycoprotein I (β2GPI) increased. In general, α2 macroglobulin (a2M) and immunoglobulin components increased with hepatic fibrosis whereas haptoglobin and complement components (C3, C4 and factor H-related protein 1) decreased. Novel proteins associated with HCV-induced fibrosis include the inter-alpha-trypsin inhibitor heavy chain H4 fragments, complement factor H-related protein 1, CD5L, Apo L1, β2GPI and the increase in thiolester cleaved products of a2M. The relationship between these changes is discussed. One of the accessory genes of the HIV viral genome encodes for the Nef protein. Nef is present in lipid rafts and increases viral replication within infected host cells by binding to a guanine nucleotide exchange factor, Vav. This leads to activation of a GTPase, Cdc42, however, the signalling pathway is poorly understood. 2D-PAGE based proteomics was used to identify differentially expressed raft-associated proteins by comparing T cells in the presence and absence of Nef. A ubiquitin conjugating enzyme UbcH7, which acts in conjugation with c-Cbl, was absent from the rafts of Nef-transfected cells. Vav ubiquitination was also absent from these rafts. In collaboration with Dr. Alison Simmons and Prof. Andrew McMichael the absence of UbcH7 in rafts was found to be caused by β-Pix forming a ternary complex with c-Cbl and activated Cdc42. Vav ubiquitination was restored and viral replication was diminished when β-Pix was knocked down providing a new candidate target for inhibiting HIV replication. This thesis demonstrates the use of proteomics in providing novel information for virus related samples. This influential technology benefits in both biomarker discovery to aid clinicians with early diagnosis of diseased individuals and in the elucidation of novel signalling pathways in infected cells to provide new candidate targets.

Introdução: O tumor de canal anal é raro, sendo a histologia epidermóide a mais frequente. A quimio-radioterapia é capaz de curar a maioria dos casos de câncer de canal anal localizado, entretanto, um grupo de pacientes apresenta resistência primária ao tratamento multimodal. Métodos: Estudo de coorte prospectivo desenhado para avaliar a influência de biomarcadores (HIV, Ki-67, PD-L1, HPV e mutações no DNA tumoral) na resposta do câncer de canal anal ao tratamento com quimio-radioterapia. Resposta completa aos 6 meses após tratamento foi o desfecho primário. O DNA tumoral foi avaliado por sequenciamento genético, através do painel TruSight Tumor 26®. HPV foi testado pelo teste PapilloCheck®. Ki-67 e PD-L1 foram avaliados por imunoistoquímica. Sorologia para HIV foi realizada em todos os pacientes antes do início do tratamento. Resultados: Os pacientes foram recrutados de outubro/2011 a dezembro/2015 e 75 foram avaliados para resposta após o tratamento. A idade mediana foi de 57 anos, a maioria dos pacientes apresentou estadio III ao diagnóstico 65% (n=49) e 12% (n=9) tinha sorologia positiva para HIV. Aos 6 meses após término da quimio-radioterapia 62,7% (n=47) dos pacientes apresentou resposta completa, 24% (n=18) resposta parcial e 13,3% (n=10) progressão de doença. HPV foi avaliado em 67 amostras e encontrado em 70,1%, sendo o HPV 16 o tipo mais frequente. A pesquisa de PD-L1 foi realizada em 61 amostras e 16,4% (n=10) apresentou expressão > 1%. Idade, estadio clínico, HIV, expressão de Ki-67, presença de HPV, expressão de PD-L1 e interrupção de tratamento foram avaliados como preditores de resposta, aos 6 meses após o término do tratamento, por meio de regressão logística multivariada. Pacientes com estadio II apresentaram 4,7 vezes mais chance de resposta completa que pacientes com estadio III (OR=4,70; IC95%=1,36-16,30; p=0,015). Quando considerada resposta completa e/ou parcial, a presença do vírus HIV foi associada a pior resposta: pacientes HIV negativo apresentaram 5,7 vezes mais chance de resposta completa e/ou parcial que pacientes HIV positivo (OR=5,72; IC95%=2,5-13,0; p < 0,001). Foi possível a realização de sequenciamento do DNA tumoral em 25 pacientes avaliáveis para resposta, sendo as mutações mais frequentes encontradas nos genes PIK3CA (n=6) e MET (n=6). Não houve diferença em resposta de acordo com presença dessas mutações. Conclusões: Aos 6 meses após término do tratamento com quimio-radioterapia a expressão de Ki-67, PD-L1, presença de HPV e mutações em PIK3CA e MET não foram associadas com resposta ao tratamento. Pacientes com estadio III e pacientes portadores do vírus HIV apresentaram pior resposta ao tratamento / Background: While chemoradiation is the curative treatment for squamous cell carcinoma of the anal canal, some patients present primary resistance. As a rare tumor, predictors of response remain unknown. Patients and Methods: Prospective cohort study aimed at evaluating biomarkers (Ki-67, PD-L1, Human papillomavirus (HPV), tumor mutations and HIV) possibly associated with tumor response to chemoradiation. Complete response at 6 months was the primary endpoint. Tumor DNA was analyzed by next-generation sequencing (TruSight Tumor 26 ®). HPV was tested by PapilloCheck®. KI-67 and PD-L1 were evaluated by immunohistochemistry. Results: Seventyeight patients were recruited from October/2011 to December/2015, and 75 were evaluable for response. Median age was 57 years, 65% (n=49) were stage III, and 12% (n=9) were HIV positive (HIV+). At 6 months 62.7% (n=47) presented complete response, 24% (n=18) partial response and 13.3% (n=10) disease progression. HPV was evaluated in 67 and found in 70.1%, the majority being HPV 16. PD-L1 was tested in 61 being 16.4% (n=10) positive. Age, clinical stage, HIV status, KI-67, HPV, PD-L1 and treatment interruption were tested as predictive factors for complete response at 6 months by logistic regression. On multivariate analyses, stage II patients were 4.7 more likely to achieve complete response than stage III (OR=4.70; IC95%=1.36-16.30; p=0.015). When we considered patients with complete and partial response, HIV+ was associated with a worse response (OR=5.72; IC95%=2.5-13.0; p < 0.001). Twenty-five patients had samples proper for NGS and 17 had at least one mutation, with PIK3CA (n=6) and MET (n=6) being the most common mutated genes. There were no differences in response according to MET or PIK3CA status. Conclusions: At 6 months after chemoradiation Ki-67, PD-L1, HPV and mutations in PIK3CA and MET were not associated with response. Patients with stage III disease and patients HIV+ had a significantly poor response

Introdução: O tumor de canal anal é raro, sendo a histologia epidermóide a mais frequente. A quimio-radioterapia é capaz de curar a maioria dos casos de câncer de canal anal localizado, entretanto, um grupo de pacientes apresenta resistência primária ao tratamento multimodal. Métodos: Estudo de coorte prospectivo desenhado para avaliar a influência de biomarcadores (HIV, Ki-67, PD-L1, HPV e mutações no DNA tumoral) na resposta do câncer de canal anal ao tratamento com quimio-radioterapia. Resposta completa aos 6 meses após tratamento foi o desfecho primário. O DNA tumoral foi avaliado por sequenciamento genético, através do painel TruSight Tumor 26®. HPV foi testado pelo teste PapilloCheck®. Ki-67 e PD-L1 foram avaliados por imunoistoquímica. Sorologia para HIV foi realizada em todos os pacientes antes do início do tratamento. Resultados: Os pacientes foram recrutados de outubro/2011 a dezembro/2015 e 75 foram avaliados para resposta após o tratamento. A idade mediana foi de 57 anos, a maioria dos pacientes apresentou estadio III ao diagnóstico 65% (n=49) e 12% (n=9) tinha sorologia positiva para HIV. Aos 6 meses após término da quimio-radioterapia 62,7% (n=47) dos pacientes apresentou resposta completa, 24% (n=18) resposta parcial e 13,3% (n=10) progressão de doença. HPV foi avaliado em 67 amostras e encontrado em 70,1%, sendo o HPV 16 o tipo mais frequente. A pesquisa de PD-L1 foi realizada em 61 amostras e 16,4% (n=10) apresentou expressão > 1%. Idade, estadio clínico, HIV, expressão de Ki-67, presença de HPV, expressão de PD-L1 e interrupção de tratamento foram avaliados como preditores de resposta, aos 6 meses após o término do tratamento, por meio de regressão logística multivariada. Pacientes com estadio II apresentaram 4,7 vezes mais chance de resposta completa que pacientes com estadio III (OR=4,70; IC95%=1,36-16,30; p=0,015). Quando considerada resposta completa e/ou parcial, a presença do vírus HIV foi associada a pior resposta: pacientes HIV negativo apresentaram 5,7 vezes mais chance de resposta completa e/ou parcial que pacientes HIV positivo (OR=5,72; IC95%=2,5-13,0; p < 0,001). Foi possível a realização de sequenciamento do DNA tumoral em 25 pacientes avaliáveis para resposta, sendo as mutações mais frequentes encontradas nos genes PIK3CA (n=6) e MET (n=6). Não houve diferença em resposta de acordo com presença dessas mutações. Conclusões: Aos 6 meses após término do tratamento com quimio-radioterapia a expressão de Ki-67, PD-L1, presença de HPV e mutações em PIK3CA e MET não foram associadas com resposta ao tratamento. Pacientes com estadio III e pacientes portadores do vírus HIV apresentaram pior resposta ao tratamento / Background: While chemoradiation is the curative treatment for squamous cell carcinoma of the anal canal, some patients present primary resistance. As a rare tumor, predictors of response remain unknown. Patients and Methods: Prospective cohort study aimed at evaluating biomarkers (Ki-67, PD-L1, Human papillomavirus (HPV), tumor mutations and HIV) possibly associated with tumor response to chemoradiation. Complete response at 6 months was the primary endpoint. Tumor DNA was analyzed by next-generation sequencing (TruSight Tumor 26 ®). HPV was tested by PapilloCheck®. KI-67 and PD-L1 were evaluated by immunohistochemistry. Results: Seventyeight patients were recruited from October/2011 to December/2015, and 75 were evaluable for response. Median age was 57 years, 65% (n=49) were stage III, and 12% (n=9) were HIV positive (HIV+). At 6 months 62.7% (n=47) presented complete response, 24% (n=18) partial response and 13.3% (n=10) disease progression. HPV was evaluated in 67 and found in 70.1%, the majority being HPV 16. PD-L1 was tested in 61 being 16.4% (n=10) positive. Age, clinical stage, HIV status, KI-67, HPV, PD-L1 and treatment interruption were tested as predictive factors for complete response at 6 months by logistic regression. On multivariate analyses, stage II patients were 4.7 more likely to achieve complete response than stage III (OR=4.70; IC95%=1.36-16.30; p=0.015). When we considered patients with complete and partial response, HIV+ was associated with a worse response (OR=5.72; IC95%=2.5-13.0; p < 0.001). Twenty-five patients had samples proper for NGS and 17 had at least one mutation, with PIK3CA (n=6) and MET (n=6) being the most common mutated genes. There were no differences in response according to MET or PIK3CA status. Conclusions: At 6 months after chemoradiation Ki-67, PD-L1, HPV and mutations in PIK3CA and MET were not associated with response. Patients with stage III disease and patients HIV+ had a significantly poor response