The dopaminergic neurodegeneration in the substantia nigra pars compacta (SNpc) is responsible for the marked motor impairment observed in Parkinson's disease (PD). However, the molecular mechanisms underlying this are not completely understood. Since by the time of diagnosis, 50-70% of the dopaminergic neurons of the nigrostriatal pathway have already been degenerated, it is difficult to investigate the early-stage events of disease pathogenesis. Due to inaccessibility of the human brain to study initial pathogenic mechanisms of the disease, experimental models have been developed in an attempt to elucidate PD etiology and its progression. Nevertheless, PD models are a controversial issue in neuroscience research since it is challenging to mimic human neuronal complexity. Therefore, the lack of optimal models that recreate disease pathology is one of the causes of failure of clinical trials that have attempted to find new/better PD therapies. Taking this in consideration, the development of more suitable models is necessary to improve our knowledge regarding PD etiological mechanisms. Additionally, the understanding of the advantages and disadvantages of models already established would also be beneficial for PD research, which our group addressed by reviewing this subject. Considering this, we chose SH-SY5Y cells as a PD model for our studies. To investigate the initial stages of PD-induced neurodegeneration, our work focused in the role of cofilin-1, a protein involved in mitochondrial dysfunction caused by oxidant-induced-apoptosis, which are two pathogenic processes strongly related to PD. Hence, in the thesis, we aimed to validate the use of retinoic-acid-(RA)-differentiated SH-SY5Y cells as an in vitro model and use it to investigate the potential role of cofilin-1 in the initial molecular and cellular mechanisms of PD. Although SH-SY5Y cells are widely used in PD research, their major drawback is their lack of important neuronal features, such as low levels of proliferation and stellate morphology. On the other hand, SH-SY5Y cells can acquire a neuronal phenotype when treated with differentiation agents such as RA. Since several protocols have been described, the consequence of which may be the discrepancies observed among studies regarding neuronal and dopaminergic features. In Chapter I, we aimed to validate a RA-differentiation protocol for SH-SY5Y cells previously established by our research group, focusing upon characterization of neuronal features and its subsequent response to 6-hydroxydopamine (6-OHDA), a toxin widely used to induce dopaminergic degeneration. RA-differentiated SH-SY5Y cells have low proliferative rates, a pronounced neuronal morphology and high expression of genes related to synapse vesicle cycle, dopamine synthesis/degradation, and dopamine transporter (DAT). After exploring phenotypic differences between these two models, we verified that RA-differentiated cells were more sensitive to 6-OHDA toxicity than undifferentiated cells, which could be related to an increase of DAT immunocontent. Many lines of evidence have showed that DAT is responsible for 6-OHDA uptake in vivo. Once inside the neuron, 6-OHDA underwent auto-oxidation causing a significant increase in oxidative stress. However, toxin uptake is not an essential step in undifferentiated SH-SY5Y cells, as auto-oxidation occurs extracellularly. We showed here, for the first time, that RA-differentiated SH-SY5Y cells can mimic, at least in part, an important mechanism of the 6-OHDA-induced cell death found in previous in vivo studies. Hence, the cellular model established by our research group presents essential neuronal features, being a suitable model for PD research. In Chapter II, RA-differentiated SH-SY5Y cells were used as cellular model to investigate disease molecular mechanisms, focusing upon cofilin-1. Our previous data have shown that oxidation of non-phosphorylate (activated) cofilin-1 leads to mitochondrial dysfunction and cell death induced by apoptosis in tumour cells. Here we found that cofilin-1 played a role in early stages of neuronal apoptosis induced by 6-OHDA in our cellular model since cofilin-1 mitochondrial translocation precedes organelle dysfunction. Overexpression of wild type CFL1 resulted in increased sensitivity of SH-SY5Y cells to 6-OHDA-induced neuronal cell death. Furthermore, overexpression of non-oxidizable CFL1 containing Cys-to-Ala mutations (positions 39, 80 and 139) increased neuronal resistance to this toxin, suggesting that oxidation is an important step in 6-OHDA toxicity. Follow-up experiments were performed in order to evaluate clinically whether cofilin-1 pathway proteins content is altered in PD post mortem human brain. Our findings showed a significant decrease in p-cofilin-1/cofilin-1 ratio in PD patients, which indicates an increase in the amount of activated cofilin-1 available for oxidation. Moreover, through principal component analysis, the immunodetection of cofilin-1 pathway proteins were able to discriminate controls and PD individuals during the early-stage of neuropathological changings. Hence, we demonstrated, for the first time, a possible role for cofilin-1 in PD pathogenesis and its potential use as biomarker. Taken together, our data showed that RA-differentiated SH-SY5Y cells present terminally-differentiated dopaminergic neuron features, that are essential to mimic dopaminergic neurons. By using this cellular model and post mortem brain tissue, we also demonstrated a possible role for cofilin-1 in early steps of the neurodegeneration process found in PD, which it could impact drug and biomarker discovery researches.

Câncer de Pulmão de Não-pequenas células (CPNPC) é o maior problema de saúde pública atualmente no mundo.Encontramos em nossas pesquisas que os níveis de mRNA da CFL-1 (cofilina) podem ser usados como um biomarcador prognóstico em biópsias de tumores de CPNPC. Em nossos estudos, estabelecemos e optimizamos um método simples de imunohistoquímica semiquantitativa (SQ-IHC) para quantificar em biópsias de tumores sua aplicação em uma coorte retrospectiva de pacientes diagnosticados com CPNPC, para explorar seu papel prognóstico. Tivemos acesso a uma coleção de arquivos médicos bem delineada de 50 tumores de pacientes, juntamente com informações relevantes clínicopatológicas de 5 anos de acompanhamento hospitalar. A análise imunoistoquímica e semiquantitativa da cofilina foi realizada em um estudo cego para confiabilidade do resultado clínico. A associação entre imunoconteúdo de cofilina e resultado clínico foi assegurando utilizando as curvas de mortalidade de Kaplan-Meier e o test log-rank. Pacientes foram agrupados pela expressão do biomarcador ou pelo seu estadiamento. Imunoconteúdo de cofilina (em densidade óptica) em biópsias de tumor foram capazes de determiner entre bom e mau prognóstico, onde altos níveis de cofilina foram correlacionados com baixa razão de sobrevida. Um método simples de imunoistoquímica semiquantitativa foi bem desenvolvido para avaliação quantitativa de cofilina em CPNPC. Nosso método mostrou boa sensibilidade/especificidade para indicar resultado em pacientes e por isso deve ser empregado em estudo prospectivo, de larga escala, com triagens clínicas randomizadas para estabelecer o valor prognóstico do imunoconteúdo de cofilina em CPNPC. / Nonsmall cell lung cancer (NSCLC) is a major public health problem worldwide. Previously we found that CFL1 (cofilin-1) mRNA levels can be used as a prognostic biomarker in NSCLC tumor biopsies. In this study, we established and optimized a simple semi-quantitative immunohistochemistry (SQ-IHC) method for cofilin quantification in tumor biopsies and applied it in a retrospective cohort of NSCLC patients, to exploit prognostic role. We accessed a well-defined archival collection of tumor samples from 50 patients with relevant clinicopathologic information and 5 years follow-up. Immunohistochemistry and semi-quantitative analysis of cofilin were performed blinded to clinical outcome. Association between cofilin immunocontent and clinical outcome was assessed using standard Kaplan-Meier mortality curves and the log-rank test. Patients were clustered according to either biomarker expression level or NSCLC stage grouping. Cofilin immunocontent (in optical densities) in tumor biopsies was able to discriminate between good and bad prognosis, where high cofilin levels are correlated with lower overall survival rate (P < .05). A simple semi-quantitative immunohistochemical method has been developed for quantitative evaluation of cofilin in NSCLC. Our method showed good sensitivity/specificity to indicate the outcome of patients and should be further employed in a prospective, large-scale, randomized clinical trial to establish the prognostic value of cofilin immunocontent in NSCLC.

The dopaminergic neurodegeneration in the substantia nigra pars compacta (SNpc) is responsible for the marked motor impairment observed in Parkinson's disease (PD). However, the molecular mechanisms underlying this are not completely understood. Since by the time of diagnosis, 50-70% of the dopaminergic neurons of the nigrostriatal pathway have already been degenerated, it is difficult to investigate the early-stage events of disease pathogenesis. Due to inaccessibility of the human brain to study initial pathogenic mechanisms of the disease, experimental models have been developed in an attempt to elucidate PD etiology and its progression. Nevertheless, PD models are a controversial issue in neuroscience research since it is challenging to mimic human neuronal complexity. Therefore, the lack of optimal models that recreate disease pathology is one of the causes of failure of clinical trials that have attempted to find new/better PD therapies. Taking this in consideration, the development of more suitable models is necessary to improve our knowledge regarding PD etiological mechanisms. Additionally, the understanding of the advantages and disadvantages of models already established would also be beneficial for PD research, which our group addressed by reviewing this subject. Considering this, we chose SH-SY5Y cells as a PD model for our studies. To investigate the initial stages of PD-induced neurodegeneration, our work focused in the role of cofilin-1, a protein involved in mitochondrial dysfunction caused by oxidant-induced-apoptosis, which are two pathogenic processes strongly related to PD. Hence, in the thesis, we aimed to validate the use of retinoic-acid-(RA)-differentiated SH-SY5Y cells as an in vitro model and use it to investigate the potential role of cofilin-1 in the initial molecular and cellular mechanisms of PD. Although SH-SY5Y cells are widely used in PD research, their major drawback is their lack of important neuronal features, such as low levels of proliferation and stellate morphology. On the other hand, SH-SY5Y cells can acquire a neuronal phenotype when treated with differentiation agents such as RA. Since several protocols have been described, the consequence of which may be the discrepancies observed among studies regarding neuronal and dopaminergic features. In Chapter I, we aimed to validate a RA-differentiation protocol for SH-SY5Y cells previously established by our research group, focusing upon characterization of neuronal features and its subsequent response to 6-hydroxydopamine (6-OHDA), a toxin widely used to induce dopaminergic degeneration. RA-differentiated SH-SY5Y cells have low proliferative rates, a pronounced neuronal morphology and high expression of genes related to synapse vesicle cycle, dopamine synthesis/degradation, and dopamine transporter (DAT). After exploring phenotypic differences between these two models, we verified that RA-differentiated cells were more sensitive to 6-OHDA toxicity than undifferentiated cells, which could be related to an increase of DAT immunocontent. Many lines of evidence have showed that DAT is responsible for 6-OHDA uptake in vivo. Once inside the neuron, 6-OHDA underwent auto-oxidation causing a significant increase in oxidative stress. However, toxin uptake is not an essential step in undifferentiated SH-SY5Y cells, as auto-oxidation occurs extracellularly. We showed here, for the first time, that RA-differentiated SH-SY5Y cells can mimic, at least in part, an important mechanism of the 6-OHDA-induced cell death found in previous in vivo studies. Hence, the cellular model established by our research group presents essential neuronal features, being a suitable model for PD research. In Chapter II, RA-differentiated SH-SY5Y cells were used as cellular model to investigate disease molecular mechanisms, focusing upon cofilin-1. Our previous data have shown that oxidation of non-phosphorylate (activated) cofilin-1 leads to mitochondrial dysfunction and cell death induced by apoptosis in tumour cells. Here we found that cofilin-1 played a role in early stages of neuronal apoptosis induced by 6-OHDA in our cellular model since cofilin-1 mitochondrial translocation precedes organelle dysfunction. Overexpression of wild type CFL1 resulted in increased sensitivity of SH-SY5Y cells to 6-OHDA-induced neuronal cell death. Furthermore, overexpression of non-oxidizable CFL1 containing Cys-to-Ala mutations (positions 39, 80 and 139) increased neuronal resistance to this toxin, suggesting that oxidation is an important step in 6-OHDA toxicity. Follow-up experiments were performed in order to evaluate clinically whether cofilin-1 pathway proteins content is altered in PD post mortem human brain. Our findings showed a significant decrease in p-cofilin-1/cofilin-1 ratio in PD patients, which indicates an increase in the amount of activated cofilin-1 available for oxidation. Moreover, through principal component analysis, the immunodetection of cofilin-1 pathway proteins were able to discriminate controls and PD individuals during the early-stage of neuropathological changings. Hence, we demonstrated, for the first time, a possible role for cofilin-1 in PD pathogenesis and its potential use as biomarker. Taken together, our data showed that RA-differentiated SH-SY5Y cells present terminally-differentiated dopaminergic neuron features, that are essential to mimic dopaminergic neurons. By using this cellular model and post mortem brain tissue, we also demonstrated a possible role for cofilin-1 in early steps of the neurodegeneration process found in PD, which it could impact drug and biomarker discovery researches.

O câncer de pulmão é responsável por aproximadamente 13% do total de casos de neoplasias malignas e por cerca de 1.4 milhões de mortes por ano em todo mundo. Esta neoplasia apresenta-se sob dois principais subtipos: câncer de pulmão de pequenas células (CPPC) e câncer de pulmão de não-pequenas células (CPNPC). Cerca de 85% dos casos de câncer de pulmão são do tipo CPNPC. Os sinais e sintomas são secundários ao crescimento do tumor primário, ao comprometimento lobo-regional, à disseminação à distância, ou são secundários às síndromes paraneoplásicas. Essas características refletem diretamente sobre as taxas de mortalidade; de cada 100 novos casos, 80 são inoperáveis e a maioria morre dentro de 3 anos. Isso significa que, apesar dos diversos avanços no diagnóstico e tratamento, o prognóstico do câncer de pulmão permanece sendo extremamente ruim, com sobrevida média de 10 meses, e cumulativa total em 5 anos de aproximadamente 12%. Atualmente, o prognóstico e a decisão terapêutica de pacientes com câncer de pulmão é baseada no TNM, Embora esse seja o procedimento considerado padrãoouro entre os profissionais de saúde, ele não leva em consideração características biológicas do tumor. Nesse contexto, a identificação de biomarcadores para câncer pode agregar importantes informações ao já estabelecido sistema TNM e resultar em tratamentos mais eficientes e em menores taxas de mortalidade. Existem 5 fases distintas que conceitualizam o desenvolvimento de um biomarcador tumoral. Através dessas fases consecutivas, é possível que se desenvolvam ferramentas úteis para triagem populacional, capazes de serem implementadas na rotina clínica para predição de desfecho do paciente, resposta terapêutica e monitoramento da doença. O presente projeto avaliou o valor prognóstico dos principais genes citados na literatura como potenciais biomarcadores para CPNPC, e verificou-se que nenhum deles apresentou significância na correlação estatística que indica poder prognóstico. Além disso, identificamos e validamos o papel prognóstico da cofilina-1 por meio de dados de microarranjo e quantificação de seu imunoconteúdo em biópsias de CPNPC. Para tanto, fizemos uso de meta-análise de bancos de dados e análise densitométrica das reações imuno-histoquímicas, seguida de correlação com dados de grau de diferenciação tumoral, classificação histológica, sexo, idade e desfecho relativo a cada caso. Além disso, desenvolvemos um método de baixo custo, fácil execução e ampla aplicação e reproducibilidade, capaz de quantificar a proteína em amostras biológicas, com potencial para ser implementado na rotina clínica e aplicamos esse método em uma coorte restrospectiva de CPNPC. Confirmamos assim o papel prognóstico da cofilina-1. Estes achados seguem a lógica das fases de desenvolvimento de um biomarcador e representam um grande passo no seu processo de validação. / Lung cancer accounts for approximately 13% of all malignant tumor cases and for about 1.4 million deaths per year worldwide. This cancer has two main subtypes: Small Cell Lung Cancer (SCLC) and Non-Small Cell Lung Cancer (NSCLC). About 85% of cases of lung cancer are NSCLC type. The signs and symptoms are secondary to the primary tumor growth, to regional lobe commitment and distant spread, or are secondary to paraneoplastic syndromes. These features reflect directly on mortality rates; 80 in every 100 new cases are inoperable and most die within 3 years. This means that, despite many advances in diagnosis treatment, the prognosis of lung cancer remains extremely poor, with median survival of 10 months, and total cumulative survival in 5-year of approximately 12%. Currently, prognosis and therapeutic decisions in patients with lung cancer is based on TNM. Although this procedure is considered gold standard among health professionals, it does not take into account the biological characteristics of the tumor. In this context, the identification of cancer biomarkers may add important information to the already established TNM system and result in better treatments and lower mortality rates. There are five distinct phases that conceptualize a tumor biomarker development of. Through these successive phases, it is possible to develop useful tools for population screening, capable of implementation in clinical practice for prediction of patient outcome, therapeutic response and disease monitoring. This project evaluated the prognostic value of major genes mentioned in literature as potential biomarkers for NSCLC and found that none of them showed statistical significance in the correlation that indicates prognostic power. It also identified and validated the prognostic role of cofilin-1 by microarray data and quantification of their immunocontent in biopsies of NSCLC. For this purpose, we used data metaanalysis and immunohistochemical reactions densitometric analysis, followed by correlation with data from tumor grade, histological classification, sex, age and outcome for each case. In addition, we developed a low-cost protocol, of easy implementation and wide application and reproducibility, able to quantify the protein in biological samples, with the potential to be implemented in clinical practice. We applied this method in a retrospective cohort of NSCLC and confirm the prognostic role of cofilin-1. These findings follow the logical phases of biomarker development and represent a major step in its validation process.

Câncer de Pulmão de Não-pequenas células (CPNPC) é o maior problema de saúde pública atualmente no mundo.Encontramos em nossas pesquisas que os níveis de mRNA da CFL-1 (cofilina) podem ser usados como um biomarcador prognóstico em biópsias de tumores de CPNPC. Em nossos estudos, estabelecemos e optimizamos um método simples de imunohistoquímica semiquantitativa (SQ-IHC) para quantificar em biópsias de tumores sua aplicação em uma coorte retrospectiva de pacientes diagnosticados com CPNPC, para explorar seu papel prognóstico. Tivemos acesso a uma coleção de arquivos médicos bem delineada de 50 tumores de pacientes, juntamente com informações relevantes clínicopatológicas de 5 anos de acompanhamento hospitalar. A análise imunoistoquímica e semiquantitativa da cofilina foi realizada em um estudo cego para confiabilidade do resultado clínico. A associação entre imunoconteúdo de cofilina e resultado clínico foi assegurando utilizando as curvas de mortalidade de Kaplan-Meier e o test log-rank. Pacientes foram agrupados pela expressão do biomarcador ou pelo seu estadiamento. Imunoconteúdo de cofilina (em densidade óptica) em biópsias de tumor foram capazes de determiner entre bom e mau prognóstico, onde altos níveis de cofilina foram correlacionados com baixa razão de sobrevida. Um método simples de imunoistoquímica semiquantitativa foi bem desenvolvido para avaliação quantitativa de cofilina em CPNPC. Nosso método mostrou boa sensibilidade/especificidade para indicar resultado em pacientes e por isso deve ser empregado em estudo prospectivo, de larga escala, com triagens clínicas randomizadas para estabelecer o valor prognóstico do imunoconteúdo de cofilina em CPNPC. / Nonsmall cell lung cancer (NSCLC) is a major public health problem worldwide. Previously we found that CFL1 (cofilin-1) mRNA levels can be used as a prognostic biomarker in NSCLC tumor biopsies. In this study, we established and optimized a simple semi-quantitative immunohistochemistry (SQ-IHC) method for cofilin quantification in tumor biopsies and applied it in a retrospective cohort of NSCLC patients, to exploit prognostic role. We accessed a well-defined archival collection of tumor samples from 50 patients with relevant clinicopathologic information and 5 years follow-up. Immunohistochemistry and semi-quantitative analysis of cofilin were performed blinded to clinical outcome. Association between cofilin immunocontent and clinical outcome was assessed using standard Kaplan-Meier mortality curves and the log-rank test. Patients were clustered according to either biomarker expression level or NSCLC stage grouping. Cofilin immunocontent (in optical densities) in tumor biopsies was able to discriminate between good and bad prognosis, where high cofilin levels are correlated with lower overall survival rate (P < .05). A simple semi-quantitative immunohistochemical method has been developed for quantitative evaluation of cofilin in NSCLC. Our method showed good sensitivity/specificity to indicate the outcome of patients and should be further employed in a prospective, large-scale, randomized clinical trial to establish the prognostic value of cofilin immunocontent in NSCLC.

O câncer de pulmão é responsável por aproximadamente 13% do total de casos de neoplasias malignas e por cerca de 1.4 milhões de mortes por ano em todo mundo. Esta neoplasia apresenta-se sob dois principais subtipos: câncer de pulmão de pequenas células (CPPC) e câncer de pulmão de não-pequenas células (CPNPC). Cerca de 85% dos casos de câncer de pulmão são do tipo CPNPC. Os sinais e sintomas são secundários ao crescimento do tumor primário, ao comprometimento lobo-regional, à disseminação à distância, ou são secundários às síndromes paraneoplásicas. Essas características refletem diretamente sobre as taxas de mortalidade; de cada 100 novos casos, 80 são inoperáveis e a maioria morre dentro de 3 anos. Isso significa que, apesar dos diversos avanços no diagnóstico e tratamento, o prognóstico do câncer de pulmão permanece sendo extremamente ruim, com sobrevida média de 10 meses, e cumulativa total em 5 anos de aproximadamente 12%. Atualmente, o prognóstico e a decisão terapêutica de pacientes com câncer de pulmão é baseada no TNM, Embora esse seja o procedimento considerado padrãoouro entre os profissionais de saúde, ele não leva em consideração características biológicas do tumor. Nesse contexto, a identificação de biomarcadores para câncer pode agregar importantes informações ao já estabelecido sistema TNM e resultar em tratamentos mais eficientes e em menores taxas de mortalidade. Existem 5 fases distintas que conceitualizam o desenvolvimento de um biomarcador tumoral. Através dessas fases consecutivas, é possível que se desenvolvam ferramentas úteis para triagem populacional, capazes de serem implementadas na rotina clínica para predição de desfecho do paciente, resposta terapêutica e monitoramento da doença. O presente projeto avaliou o valor prognóstico dos principais genes citados na literatura como potenciais biomarcadores para CPNPC, e verificou-se que nenhum deles apresentou significância na correlação estatística que indica poder prognóstico. Além disso, identificamos e validamos o papel prognóstico da cofilina-1 por meio de dados de microarranjo e quantificação de seu imunoconteúdo em biópsias de CPNPC. Para tanto, fizemos uso de meta-análise de bancos de dados e análise densitométrica das reações imuno-histoquímicas, seguida de correlação com dados de grau de diferenciação tumoral, classificação histológica, sexo, idade e desfecho relativo a cada caso. Além disso, desenvolvemos um método de baixo custo, fácil execução e ampla aplicação e reproducibilidade, capaz de quantificar a proteína em amostras biológicas, com potencial para ser implementado na rotina clínica e aplicamos esse método em uma coorte restrospectiva de CPNPC. Confirmamos assim o papel prognóstico da cofilina-1. Estes achados seguem a lógica das fases de desenvolvimento de um biomarcador e representam um grande passo no seu processo de validação. / Lung cancer accounts for approximately 13% of all malignant tumor cases and for about 1.4 million deaths per year worldwide. This cancer has two main subtypes: Small Cell Lung Cancer (SCLC) and Non-Small Cell Lung Cancer (NSCLC). About 85% of cases of lung cancer are NSCLC type. The signs and symptoms are secondary to the primary tumor growth, to regional lobe commitment and distant spread, or are secondary to paraneoplastic syndromes. These features reflect directly on mortality rates; 80 in every 100 new cases are inoperable and most die within 3 years. This means that, despite many advances in diagnosis treatment, the prognosis of lung cancer remains extremely poor, with median survival of 10 months, and total cumulative survival in 5-year of approximately 12%. Currently, prognosis and therapeutic decisions in patients with lung cancer is based on TNM. Although this procedure is considered gold standard among health professionals, it does not take into account the biological characteristics of the tumor. In this context, the identification of cancer biomarkers may add important information to the already established TNM system and result in better treatments and lower mortality rates. There are five distinct phases that conceptualize a tumor biomarker development of. Through these successive phases, it is possible to develop useful tools for population screening, capable of implementation in clinical practice for prediction of patient outcome, therapeutic response and disease monitoring. This project evaluated the prognostic value of major genes mentioned in literature as potential biomarkers for NSCLC and found that none of them showed statistical significance in the correlation that indicates prognostic power. It also identified and validated the prognostic role of cofilin-1 by microarray data and quantification of their immunocontent in biopsies of NSCLC. For this purpose, we used data metaanalysis and immunohistochemical reactions densitometric analysis, followed by correlation with data from tumor grade, histological classification, sex, age and outcome for each case. In addition, we developed a low-cost protocol, of easy implementation and wide application and reproducibility, able to quantify the protein in biological samples, with the potential to be implemented in clinical practice. We applied this method in a retrospective cohort of NSCLC and confirm the prognostic role of cofilin-1. These findings follow the logical phases of biomarker development and represent a major step in its validation process.

Base teórica: O câncer de pulmão é uma doença com alta incidência e mortalidade, cujo prognóstico permanece discreto apesar do melhor entendimento da doença nas últimas décadas. A radioterapia tem papel terapêutico em todos os estágios da doença. A expressão da cofilina-1, uma proteína relacionada à mobilidade celular, determinou maior radiossensibilidade a células de adenocarcinoma brônquico em estudos in vitro, porém pior sobrevida em estádios iniciais Objetivo: Avaliar se a expressão da cofilina-1 interfere na sobrevida e no controle local em pacientes com câncer de pulmão submetidos a tratamento com radioterapia definitiva Métodos: Foram avaliados pacientes com câncer de pulmão não pequenas células, com estádios I–IV, que receberam radioterapia exclusiva ou combinada com quimioterapia, dirigida à neoplasia brônquica, na unidade de radioterapia do HCPA, nos anos de 2009 a 2015. Todos os pacientes tiveram a expressão de cofilina-1 aferida e foram distribuídos conforme os níveis de expressão de cofilina-1 utilizando-se de protocolo específico. Os prontuários foram avaliados retrospectivamente para calcular a sobrevida mediana. A progressão foi verificada através de avaliação de tomografias de tórax de controle. Resultados: Foram avaliados 45 pacientes neste estudo. A sobrevida mediana de todos os pacientes foi de 11,3 meses e a sobrevida global em 5 anos de 17,3%. Pacientes com expressão média ou alta de cofilina-1 tiveram maior mortalidade quando comparados com pacientes com baixa expressão (respectivamente, HR 1,628, IC95 1,137-8,287 e HR 1,59 IC95 1,105-7,342). Não houve diferença significantemente estatística entre controle local e expressão de cofilina-1. Conclusão: A expressão de cofilina-1 está associada à sobrevida em pacientes com carcinoma brônquico tratados com radioterapia e existe uma tendência a melhor controle local com baixa expressão. Nossos resultados sugerem um novo campo a ser explorado no manejo do carcinoma de pulmão localmente avançado, utilizando-se dos níveis de cofilina-1. / Background: Lung cancer is a disease with high incidence and mortality, whose prognosis remains poor despite a better understanding of the disease in the last decades. Radiotherapy plays a therapeutic role in all stages of disease. The expression of cofilin-1, a protein related to cellular mobility, determined greater radiosensitivity to lung adenocarcinoma cells in in vitro studies, but worse survival at initial stages. Objective: To evaluate if the expression of cofilin-1 modified survival and local control in lung cancer patients submitted to definitive treatment with radiotherapy. Methods: Patients with non-small cell lung cancer with stage IIV who received radiotherapy alone or combined with chemotherapy for lung cancer at the HCPA radiotherapy unit from 2009 to 2015 were evaluated. All patients had the expression of measured cofilin-1 evaluated and were distributed by cofilin-1 expression according to specific protocol. The medical records were retrospectively evaluated to estimate median survival. The progression was verified through evaluation of control chest tomography. Results: 45 patients were assessed in this study. The median survival of all patients was 11.3 months and the 5-year overall survival was 17.3%. Patients with medium or high expression of cofilin-1 had higher mortality rates when compared to patients with low expression (HR, 1,628, CI95, 1,137-8,287 and HR, 1.59, CI95, 1,105-7,342). There was no statistically significant difference between local control and cofilin-1 expression. Conclusion: cofilin-1 expression is associated with survival in patients with lung cancer treated with radiotherapy and there is a tendency for better local control with low CFL1 expression. Our results suggest a new field to be explored in the management of locally advanced lung carcinoma, using cofilin-1 expression levels.

Base teórica: O câncer de pulmão é uma doença com alta incidência e mortalidade, cujo prognóstico permanece discreto apesar do melhor entendimento da doença nas últimas décadas. A radioterapia tem papel terapêutico em todos os estágios da doença. A expressão da cofilina-1, uma proteína relacionada à mobilidade celular, determinou maior radiossensibilidade a células de adenocarcinoma brônquico em estudos in vitro, porém pior sobrevida em estádios iniciais Objetivo: Avaliar se a expressão da cofilina-1 interfere na sobrevida e no controle local em pacientes com câncer de pulmão submetidos a tratamento com radioterapia definitiva Métodos: Foram avaliados pacientes com câncer de pulmão não pequenas células, com estádios I–IV, que receberam radioterapia exclusiva ou combinada com quimioterapia, dirigida à neoplasia brônquica, na unidade de radioterapia do HCPA, nos anos de 2009 a 2015. Todos os pacientes tiveram a expressão de cofilina-1 aferida e foram distribuídos conforme os níveis de expressão de cofilina-1 utilizando-se de protocolo específico. Os prontuários foram avaliados retrospectivamente para calcular a sobrevida mediana. A progressão foi verificada através de avaliação de tomografias de tórax de controle. Resultados: Foram avaliados 45 pacientes neste estudo. A sobrevida mediana de todos os pacientes foi de 11,3 meses e a sobrevida global em 5 anos de 17,3%. Pacientes com expressão média ou alta de cofilina-1 tiveram maior mortalidade quando comparados com pacientes com baixa expressão (respectivamente, HR 1,628, IC95 1,137-8,287 e HR 1,59 IC95 1,105-7,342). Não houve diferença significantemente estatística entre controle local e expressão de cofilina-1. Conclusão: A expressão de cofilina-1 está associada à sobrevida em pacientes com carcinoma brônquico tratados com radioterapia e existe uma tendência a melhor controle local com baixa expressão. Nossos resultados sugerem um novo campo a ser explorado no manejo do carcinoma de pulmão localmente avançado, utilizando-se dos níveis de cofilina-1. / Background: Lung cancer is a disease with high incidence and mortality, whose prognosis remains poor despite a better understanding of the disease in the last decades. Radiotherapy plays a therapeutic role in all stages of disease. The expression of cofilin-1, a protein related to cellular mobility, determined greater radiosensitivity to lung adenocarcinoma cells in in vitro studies, but worse survival at initial stages. Objective: To evaluate if the expression of cofilin-1 modified survival and local control in lung cancer patients submitted to definitive treatment with radiotherapy. Methods: Patients with non-small cell lung cancer with stage IIV who received radiotherapy alone or combined with chemotherapy for lung cancer at the HCPA radiotherapy unit from 2009 to 2015 were evaluated. All patients had the expression of measured cofilin-1 evaluated and were distributed by cofilin-1 expression according to specific protocol. The medical records were retrospectively evaluated to estimate median survival. The progression was verified through evaluation of control chest tomography. Results: 45 patients were assessed in this study. The median survival of all patients was 11.3 months and the 5-year overall survival was 17.3%. Patients with medium or high expression of cofilin-1 had higher mortality rates when compared to patients with low expression (HR, 1,628, CI95, 1,137-8,287 and HR, 1.59, CI95, 1,105-7,342). There was no statistically significant difference between local control and cofilin-1 expression. Conclusion: cofilin-1 expression is associated with survival in patients with lung cancer treated with radiotherapy and there is a tendency for better local control with low CFL1 expression. Our results suggest a new field to be explored in the management of locally advanced lung carcinoma, using cofilin-1 expression levels.

Base teórica: O câncer de pulmão é uma doença com alta incidência e mortalidade, cujo prognóstico permanece discreto apesar do melhor entendimento da doença nas últimas décadas. A radioterapia tem papel terapêutico em todos os estágios da doença. A expressão da cofilina-1, uma proteína relacionada à mobilidade celular, determinou maior radiossensibilidade a células de adenocarcinoma brônquico em estudos in vitro, porém pior sobrevida em estádios iniciais Objetivo: Avaliar se a expressão da cofilina-1 interfere na sobrevida e no controle local em pacientes com câncer de pulmão submetidos a tratamento com radioterapia definitiva Métodos: Foram avaliados pacientes com câncer de pulmão não pequenas células, com estádios I–IV, que receberam radioterapia exclusiva ou combinada com quimioterapia, dirigida à neoplasia brônquica, na unidade de radioterapia do HCPA, nos anos de 2009 a 2015. Todos os pacientes tiveram a expressão de cofilina-1 aferida e foram distribuídos conforme os níveis de expressão de cofilina-1 utilizando-se de protocolo específico. Os prontuários foram avaliados retrospectivamente para calcular a sobrevida mediana. A progressão foi verificada através de avaliação de tomografias de tórax de controle. Resultados: Foram avaliados 45 pacientes neste estudo. A sobrevida mediana de todos os pacientes foi de 11,3 meses e a sobrevida global em 5 anos de 17,3%. Pacientes com expressão média ou alta de cofilina-1 tiveram maior mortalidade quando comparados com pacientes com baixa expressão (respectivamente, HR 1,628, IC95 1,137-8,287 e HR 1,59 IC95 1,105-7,342). Não houve diferença significantemente estatística entre controle local e expressão de cofilina-1. Conclusão: A expressão de cofilina-1 está associada à sobrevida em pacientes com carcinoma brônquico tratados com radioterapia e existe uma tendência a melhor controle local com baixa expressão. Nossos resultados sugerem um novo campo a ser explorado no manejo do carcinoma de pulmão localmente avançado, utilizando-se dos níveis de cofilina-1. / Background: Lung cancer is a disease with high incidence and mortality, whose prognosis remains poor despite a better understanding of the disease in the last decades. Radiotherapy plays a therapeutic role in all stages of disease. The expression of cofilin-1, a protein related to cellular mobility, determined greater radiosensitivity to lung adenocarcinoma cells in in vitro studies, but worse survival at initial stages. Objective: To evaluate if the expression of cofilin-1 modified survival and local control in lung cancer patients submitted to definitive treatment with radiotherapy. Methods: Patients with non-small cell lung cancer with stage IIV who received radiotherapy alone or combined with chemotherapy for lung cancer at the HCPA radiotherapy unit from 2009 to 2015 were evaluated. All patients had the expression of measured cofilin-1 evaluated and were distributed by cofilin-1 expression according to specific protocol. The medical records were retrospectively evaluated to estimate median survival. The progression was verified through evaluation of control chest tomography. Results: 45 patients were assessed in this study. The median survival of all patients was 11.3 months and the 5-year overall survival was 17.3%. Patients with medium or high expression of cofilin-1 had higher mortality rates when compared to patients with low expression (HR, 1,628, CI95, 1,137-8,287 and HR, 1.59, CI95, 1,105-7,342). There was no statistically significant difference between local control and cofilin-1 expression. Conclusion: cofilin-1 expression is associated with survival in patients with lung cancer treated with radiotherapy and there is a tendency for better local control with low CFL1 expression. Our results suggest a new field to be explored in the management of locally advanced lung carcinoma, using cofilin-1 expression levels.

Neospora caninum é um parasita intracelular obrigatório pertencente ao filo Apicomplexa, conhecido por ser uma das principais causas de aborto parasitário em bovinos e por apresentar transmissão transplacentária. Para locomoverem-se e acessarem o conteúdo intracelular de células hospedeiras, organismos apicomplexas fazem uso de um mecanismo não convencional que se utiliza de uma maquinaria celular cujo papel central é exercido pelo motor actina-miosina, auxiliado por proteínas intermediárias e de acoragem, que realiza a propulsão do parasita na direção do movimento. Para o funcionamento dessa maquinaria, é essencial que actina esteja em sua forma filamentosa (actina-F). Porém, actinas de apicomplexas são conhecidas por serem funcional e estruturalmente não convencionais, formando filamentos pequenos e instáveis in vitro, assim como pelo predomínio de grande maioria de actina monomérica (actin-G) nas células in vivo. Desse modo, para formar e manter actina-F a dinâmica de actina desses organismos requer uma regulação precisa, que, em apicomplexas, é conduzida por um arsenal conhecidamente pequeno de proteínas que se ligam a actina (ABPs). Nosso objetivo neste estudo foi identificar e caracterizar ABPs de N. caninum. Para isso, duas ABPs de N. caninum foram estudadas: fator de despolimerização de actina (NcADF) e proteína associada a ciclase (NcCAP); também, foi gerado e caracterizado soro contra região de actina de N. caninum entre aminoácidos 201 e 310 (anti-NcAct201-310). NcADF (correspondente ao acesso NCLIV_012510 em ToxoDB) foi submetida a caracterização molecular e bioquímica. A sua estrutura terciária foi gerada por modelagem molecular baseada em homologia, apresentando folding conservado, porém com F-loop de menor tamanho, quando comparada a ADF/cofilinas canônicas. A forma recombinante de NcADF foi expressa E. coli BL21 por plasmídeos pET32a(+) e pET28a(+) e solubilizada em tampão desnaturante e nativo, respectivamente. NcADF_pET32 foi purificada e utilizada para geração de soro anti-NcADF, que detectou ambas NcADF recombinantes, assim como proteínas endógenas em western blot 1-D e 2-D com peso molecular e pI próximos aos preditos. O soro anti-NcADF também localizou NcADF difusa no citoplasma, com menos intensidade nos polos de taquizoítas de N. caninum extracelulares. NcADF_pET28 foi purificado na forma nativa e utilizado para caracterização funcional para avaliação de seu papel na dinâmica de actina liofilizada de coelho. Ensaios de cossedimentação, cinética de polimerização e despolimerização, viscosimentria de baixo cisalhamento (queda de bola), estado estacionário e ligação entre actina-G e NcADF, em conjunto, mostraram que NcADF causa despolimerização de actina-F, realiza sequestro de monômeros de actina e quebra de filamentos. NcCAP foi submetida a caracterização molecular e foi identificada como produto de expressão do gene de acesso NCLIV_054140. NcCAP recombinante foi expressa em pET32a(+) e pET28a(+) predominantemente em corpos de inclusão e foi solubilizada em tampão desnaturante. A forma purificada de pET32_NcCAP, identificada por espectrometria de massas, foi utilizada para imunização e o soro resultante detectou NcCAP recombinante e endógena por western blot 1-D e 2-D, apresentando bandas e spots de peso molecular e pI próximos ao esperado. O soro anti-NcCAP também localizou NcCAP em taquizoítas ii extracelulares de N. caninum difusa no citoplasma e/ou com predomínio na região periplasmática da célula. Por fim, o soro anti-NcAct201-310 foi gerado, sendo capaz de detectar proteínas em sua forma nativa e realizar marcação na região periférica e, possivelmente, nuclear de taquizoítas de N. caninum extracelulares. A caracterização de ABPs de N. caninum feita neste trabalho amplia o conhecimento sobre a conservação dessas proteínas ao longo do filo Apicomplexa. Ademais, representa uma contribuição para o entendimento da dinâmica de actina e, por consequência, futuramente, pode colaborar para a elucidação de mecanismos-chave para a sobrevivência e disseminação dos parasitas pelo seu hospedeiro / Neospora caninum is an obligate intracellular parasite that belongs to the phylum Apicomplexa. It is known as one of the main causes of infectious abortion in cows and for its efficient transplacentary transmission. Apicomplexan organisms use a phylum-specific mechanism of invasion and gliding motility, which use an unusual cellular machinery based on an actin myosin motor assisted by intermediary and anchoring proteins that creates the traction force to impulse the parasite forward. Filamentous actin (F-actin) is essential to the appropriate functioning of this machinery, although apicomplexan unconventional actin forms small and unstable filaments in vitro and is found preponderantly as monomer (G-actin) in cells. Thus, the parasites need actin-binding proteins (ABPs) to strictly regulate actin dynamics and to form and maintain F-actin when it is necessary to the cell. Here, we aimed at identifying and characterising ABPs from N. caninum. Two ABPs were characterised: actin-depolymerising factor (NcADF) and cyclase-associated protein (NcCAP) from N. caninum. In addition, a serum against the actin region between amino acids 201 and 310 (anti-NcAct201-310) was raised. NcADF, which corresponds to identification NCLIV_012510 on ToxoDB, was molecular and biochemically characterised. Firstly, the tertiary structure of NcADF was generated by molecular modelling based on homology. Comparing to canonical ADF/cofilins, NcADF presented a conserved folding, albeit its smaller F-loop. The recombinant form of NcADF was expressed in E. coli BL21 using pET32a(+) and pET28a(+) plasmids and solubilized in denaturing and native buffers, respectively. Polyclonal antibodies were raised in mice against purified NcADF_pET32, which was able to detect both forms of recombinant NcADF as well as proteins in 1-D and 2-D western blot with expected molecular weight and isoelectric point (pI). Additionally, NcADF was localised in extracellular N. caninum tachyzoites as a diffuse pattern on cytoplasm with less intensity in both poles. NcADF_pET28 was successfully purified in native form and used for functional characterisation to evaluate the role of recombinant NcADF on lyophilised rabbit actin dynamics. Together, co-sedimentation, polymerisation and depolymerisation kinetic, low shearing viscometry (falling ball), steady state, and G-actin and NcADF binding assays showed that NcADF was able to depolymerise actin-F, sequester actin monomers, and sever filaments. Moreover, NcCAP (identification NCLIV_054140) was also characterised. Recombinant NcCAP was expressed in pET32a(+) and pET28a(+) plasmids predominantly in inclusion bodies and was solubilised in denaturing buffer. NcCAP_pET32 was purified and identified by mass spectrometry. Then, the polyclonal antibodies against this recombinant protein was generated in mice. It was able to detect recombinant and endogenous NcCAP, presenting bands and spots in 1-D and 2-D western blot with molecular weight and pI quite near to the predicted ones. NcCAP was localised as a diffuse pattern on cytoplasm and/or predominantly on periplasmic regions of extracellular taclyzoites of N. caninum. Finally, the serum containing anti-NcAct201-310 polyclonal antibodies was raised in mice. It detected endogenous proteins mainly in native form and localised them on periplasmic and possibly nuclear region in extracellular N. caninum tachyzoites. The characterisation of N. caninum ABPs iv extends our understanding of these proteins conservation and their function throughout the Apicomplexa phylum. Furthrmore, it represents a contribution to the field towards the comprehention of actin dynamics and in the future might provide information for important mechanisms of dissemination and survival of the parasite at its host