O câncer de pâncreas é considerado um dos adenocarcinomas mais agressivos, ocupando o quarto lugar de mortes devidas a câncer, isto é dado principalmente a seu desenvolvimento silencioso e a sua complexidade genética, tornando-o de difícil detecção. Consequentemente, o diagnóstico desta doença ocorre apenas em fase tardia, quando o tratamento é apenas para fins paliativos. As anomalias genéticas mais frequentes no câncer de pâncreas invasivo estão relacionadas à ativação por mutações do gene KRAS e à inativação dos genes supressores de tumor CDKN2A, TP53, SMAD4 e BRCA2. Além destas conhecidas vias de sinalização, fatores de crescimento como o TGF-? e EGF também apresentam papel fundamental na progressão e metástase do câncer de pâncreas. Interessantemente, TGF-? e EGF são também indutores do processo denominado transição epitelial-mesenquimal (EMT), onde células epiteliais normais, durante a embriogênese, ou células cancerosas durante a progressão tumoral e metástase, perdem seus contatos intracelulares e adquirem caráter migratório. Desta forma a EMT, induzida por altos níveis de TGF-? e/ou EGF, é considerada como um dos mecanismos de progressão tumoral em adenocarcinomas. No presente estudo, foram estudados os proteomas e o fosfoproteoma da EMT do PanCa. Assim, células de câncer de pâncreas PANC- 1 foram induzidas à EMT em cultura com os fatores de crescimento TGF-?1 ou TGF-?2 e EGF, e após a indução, marcadores moleculares e propriedades funcionais de migração e invasão foram confirmados. Duas condições de indução da EMT foram estabelecidas, para as quais foram desenvolvidas as análises proteômicas quantitativas. A abordagem foi baseada em marcação isotópica de células em cultura (SILAC), em conjunto com fracionamento celular e de proteínas intactas, e cromatografia líquida acoplada à espectrometria de massas para identificação de proteínas em larga escala. No total, aproximadamente 5.000 proteínas foram identificadas, e a maioria delas quantificadas com precisão nas duplicatas experimentais. Foram selecionadas 37 proteínas com expressão diferencial estatisticamente significativa nos experimentos proteômicos, as quais participam principalmente em processos de biogênese, adesão e apoptóticos. A análise de redes de interação revelou que as proteínas alteradas estavam principalmente localizadas em vias de sinalização que controlam processos de organização da matriz extracelular, splicing alternativo e regulação da apoptose. A análise do fosfoproteoma foi feita usando TGF-?1 como agente indutor da EMT nas células PANC-1, usando a estratégia ERLIC para o enriquecimento de fosfopeptídeos. No total, foram identificados 5.965 fosfopeptídeos não redundantes, correspondendo a um total de 2.250 fosfoproteínas analisadas, sendo quantificadas 2.053 ao menos em duas replicatas, e destas foram identificados 61 fosfopeptídeos regulados pertencentes a 55 fosfoproteínas, relacionados com processos de regulação do mRNA e vias de sinalização ligadas à adesão celular. Em conclusão, nosso estudo elucida potenciais novos alvos para inibição da EMT, controle da metástase, ou para auxiliar no diagnóstico da doença, quando devidamente validada. / Pancreatic cancer kills more than 200 thousand people worldwide every year. Also, pancreatic cancer is considered one of the most aggressive adenocarcinomas and difficult to diagnose since it develops silently and presents a high genetic complexity. Consequently, the diagnostic is often late, when the pancreatic cancer has already metastasized and the treatment has only palliative purposes. The most frequent genetic alterations observed in pancreatic cancer are related to mutations in KRAS oncogene and CDKN2A, TP53, SMAD4 and BRCA2 tumor suppressor genes. In addition to these known frequent mutations, growth factors such as TGB- ? and EGF play important roles in pancreatic cancer progression and metastasis. Interestingly, TGB- ? and EGF are also inducers of the Epithelial to Mesenchymal Transition (EMT), in which epithelial cells lose their intracellular contacts and acquire migratory capacities. Therefore, EMT is considered one of the mechanisms responsible for tumor progression and metastasis in adenocarcinomas in addition of being correlated to the process of generating cancer stem cells. In our present study, pancreatic cancer cell line PANC-1 was induced to EMT by using growth factors TGF-?1 or TGF-?2 and EGF. Both molecular and functional properties, such as invasion and migration, were evaluated in PANC-1 cells undergoing EMT and confirm the induction. Two conditions of EMT induction were properly established and in-depth quantitative proteomic analysis based on stable isotope labeling in cell culture (SILAC) followed by cellular and protein fractionation were assessed. In total, 5.000 proteins were identified and most of them were accurately quantified in duplicate experiments. Thirty-seven proteins were selected as differentially expressed with statistical significance, and were related mainly with biogenesis, adhesion and apoptosis processes. Interaction network analysis showed that regulated proteins were predominantly participating in signaling pathways linked to extracellular matrix organization, alternative splicing and apoptosis regulation. Phosphoproteome analysis was done using TGF-?1 as EMT-inductor agent on PANC-1 cells and ERLIC strategy for phosphopeptides enrichment. In total, were identified 5.965 non-redundant phosphopeptides corresponding to approximately 2.250 analyzed phosphoproteins, thereof 2.053 were quantified in at least two replicates. At comparison, were identified 61 regulated phosphopeptides belonging to 55 phosphoproteins, which were related with mRNA regulation processes and signialing pathways linked to cellular adhesion. In conclusion, our study highlighted potential new targets for EMT inhibition, metastasis control or to help in pancreatic cancer diagnosis, when careful validated.

No description available.

O câncer de pâncreas é considerado um dos adenocarcinomas mais agressivos, ocupando o quarto lugar de mortes devidas a câncer, isto é dado principalmente a seu desenvolvimento silencioso e a sua complexidade genética, tornando-o de difícil detecção. Consequentemente, o diagnóstico desta doença ocorre apenas em fase tardia, quando o tratamento é apenas para fins paliativos. As anomalias genéticas mais frequentes no câncer de pâncreas invasivo estão relacionadas à ativação por mutações do gene KRAS e à inativação dos genes supressores de tumor CDKN2A, TP53, SMAD4 e BRCA2. Além destas conhecidas vias de sinalização, fatores de crescimento como o TGF-? e EGF também apresentam papel fundamental na progressão e metástase do câncer de pâncreas. Interessantemente, TGF-? e EGF são também indutores do processo denominado transição epitelial-mesenquimal (EMT), onde células epiteliais normais, durante a embriogênese, ou células cancerosas durante a progressão tumoral e metástase, perdem seus contatos intracelulares e adquirem caráter migratório. Desta forma a EMT, induzida por altos níveis de TGF-? e/ou EGF, é considerada como um dos mecanismos de progressão tumoral em adenocarcinomas. No presente estudo, foram estudados os proteomas e o fosfoproteoma da EMT do PanCa. Assim, células de câncer de pâncreas PANC- 1 foram induzidas à EMT em cultura com os fatores de crescimento TGF-?1 ou TGF-?2 e EGF, e após a indução, marcadores moleculares e propriedades funcionais de migração e invasão foram confirmados. Duas condições de indução da EMT foram estabelecidas, para as quais foram desenvolvidas as análises proteômicas quantitativas. A abordagem foi baseada em marcação isotópica de células em cultura (SILAC), em conjunto com fracionamento celular e de proteínas intactas, e cromatografia líquida acoplada à espectrometria de massas para identificação de proteínas em larga escala. No total, aproximadamente 5.000 proteínas foram identificadas, e a maioria delas quantificadas com precisão nas duplicatas experimentais. Foram selecionadas 37 proteínas com expressão diferencial estatisticamente significativa nos experimentos proteômicos, as quais participam principalmente em processos de biogênese, adesão e apoptóticos. A análise de redes de interação revelou que as proteínas alteradas estavam principalmente localizadas em vias de sinalização que controlam processos de organização da matriz extracelular, splicing alternativo e regulação da apoptose. A análise do fosfoproteoma foi feita usando TGF-?1 como agente indutor da EMT nas células PANC-1, usando a estratégia ERLIC para o enriquecimento de fosfopeptídeos. No total, foram identificados 5.965 fosfopeptídeos não redundantes, correspondendo a um total de 2.250 fosfoproteínas analisadas, sendo quantificadas 2.053 ao menos em duas replicatas, e destas foram identificados 61 fosfopeptídeos regulados pertencentes a 55 fosfoproteínas, relacionados com processos de regulação do mRNA e vias de sinalização ligadas à adesão celular. Em conclusão, nosso estudo elucida potenciais novos alvos para inibição da EMT, controle da metástase, ou para auxiliar no diagnóstico da doença, quando devidamente validada. / Pancreatic cancer kills more than 200 thousand people worldwide every year. Also, pancreatic cancer is considered one of the most aggressive adenocarcinomas and difficult to diagnose since it develops silently and presents a high genetic complexity. Consequently, the diagnostic is often late, when the pancreatic cancer has already metastasized and the treatment has only palliative purposes. The most frequent genetic alterations observed in pancreatic cancer are related to mutations in KRAS oncogene and CDKN2A, TP53, SMAD4 and BRCA2 tumor suppressor genes. In addition to these known frequent mutations, growth factors such as TGB- ? and EGF play important roles in pancreatic cancer progression and metastasis. Interestingly, TGB- ? and EGF are also inducers of the Epithelial to Mesenchymal Transition (EMT), in which epithelial cells lose their intracellular contacts and acquire migratory capacities. Therefore, EMT is considered one of the mechanisms responsible for tumor progression and metastasis in adenocarcinomas in addition of being correlated to the process of generating cancer stem cells. In our present study, pancreatic cancer cell line PANC-1 was induced to EMT by using growth factors TGF-?1 or TGF-?2 and EGF. Both molecular and functional properties, such as invasion and migration, were evaluated in PANC-1 cells undergoing EMT and confirm the induction. Two conditions of EMT induction were properly established and in-depth quantitative proteomic analysis based on stable isotope labeling in cell culture (SILAC) followed by cellular and protein fractionation were assessed. In total, 5.000 proteins were identified and most of them were accurately quantified in duplicate experiments. Thirty-seven proteins were selected as differentially expressed with statistical significance, and were related mainly with biogenesis, adhesion and apoptosis processes. Interaction network analysis showed that regulated proteins were predominantly participating in signaling pathways linked to extracellular matrix organization, alternative splicing and apoptosis regulation. Phosphoproteome analysis was done using TGF-?1 as EMT-inductor agent on PANC-1 cells and ERLIC strategy for phosphopeptides enrichment. In total, were identified 5.965 non-redundant phosphopeptides corresponding to approximately 2.250 analyzed phosphoproteins, thereof 2.053 were quantified in at least two replicates. At comparison, were identified 61 regulated phosphopeptides belonging to 55 phosphoproteins, which were related with mRNA regulation processes and signialing pathways linked to cellular adhesion. In conclusion, our study highlighted potential new targets for EMT inhibition, metastasis control or to help in pancreatic cancer diagnosis, when careful validated.

No description available.

No description available.

Complex I (NADH:ubiquinone oxidoreductase) couples electron transfer to proton translocation across the inner mitochondrial membrane, to drive the synthesis of ATP. Its distinctive L-shaped structure comprises 45 subunits, encoded by both the mitochondrial and nuclear genomes, which are assembled by a complicated modular pathway. Complex I genetic defects are the most common cause of mitochondrial disorders and often present in early childhood, with high mortality rates. Recent high-resolution electron cryo-microscopy structures of mammalian complex I provide a foundation for both interpreting biochemical and biomedical data and understanding the catalytic mechanism. First, this thesis explores how the flavin cofactor is inserted into the NADH-binding (N-) domain of complex I. Genetic manipulation of cultured human cells, to starve them of flavin, revealed a hierarchal impact on the mitochondrial flavoproteome. High riboflavin content in the growth media ameliorated observed phenotypes, requiring cell conditioning in low riboflavin conditions. CRISPR knockout of the putative mitochondrial flavin transporter SLC25A32 demonstrated the severe impact of decreased flavin on complexes I and II, and mass spectrometry 'complexome' analyses suggest that the N-domain is still assembled onto complex I in the absence of the flavin. Second, the model organism Yarrowia lipolytica was used to assess the importance of residues in the quinone-binding site of complex I. Three residues with proposed roles in binding the quinone head-group were targeted. One variant was catalytically inactive, while two retained some activity. They showed decreased ability to reduce physiologically-relevant, long chain quinones, but their ability to reduce short-chain analogues was affected less severely. The results suggest a complicated picture in which interactions between the protein and both the hydrophilic quinone head-group and hydrophobic isoprenoid chain contribute to quinone-binding affinity and catalysis. Finally, a model for human complex I, generated from a recent high-resolution structure of mouse complex I, was used to investigate whether the pathogenicity of human variants could be predicted. Structural information on variant residues, including their secondary structure, proximity to key features and surface exposure, was collated and the power of each property to predict pathogenicity investigated. The analysis was then extended to the whole structure, to identify potential pathogenic hotspots in the enzyme, inform future studies of functionally important regions in complex I, and aid the diagnosis of clinically relevant pathogenic variants.

Despite therapeutic advances, colorectal cancer still has a 45% mortality rate, and one of the most crucial problems is the development of acquired resistance to treatment with anticancer drugs. Thus the aims of this project are to develop drug-resistant colon cancer cell lines in order to identify mechanisms of resistance for the most commonly drugs used in colorectal cancer: 5-fluorouracil, oxaliplatin, and irinotecan. Following evaluation of drug sensitivity to these agents in an initial panel of eight colorectal cancer cell lines, 3 lines (DLD-1, KM-12 and HT-29) were selected for the development of 5-FU (3 lines), oxaliplatin (2) and irinotecan (1) resistant sublines by continuous drug exposure, with resistance confirmed using the MTT assay. Consistently resistant sublines were subject to a „stable isotope labelling with amino acids in cell culture‟ (SILAC) approach and a MudPIT proteomics strategy, employing 2D LC and Orbitrap Fusion mass spectrometric analysis, to identify novel predictive biomarkers for resistance. An average of 3622 proteins was quantified for each resistant and parent cell line pair, with on average 60-70 proteins up-regulated and 60-70 down-regulated in the drug resistant sublines. The validity of this approach was further confirmed using immunodetection techniques. These studies have provided candidate proteins which can be assessed for their value as predictive biomarkers, or as therapeutic targets for the modulation of acquired drug resistance in colorectal cancer.

Polychlorinated biphenyls (PCBs) are the persistent environmental pollutants with the continuous concerns over adverse human health effects. As semi-volatile compounds, PCBs were found in indoor and outdoor air. The observation of high levels of airborne PCBs in old school buildings raised the concerns of inhalation exposure and toxicity of PCBs. Lower chlorinated PCBs (LC-PCBs), major components of airborne PCBs, are subject to biotranformation. In vitro and in vivo studies revealed that reactive metabolites of LC-PCBs formed covalent adducts on DNA and proteins. The hypothesis of the project is that the reactive metabolites of LC-PCBs are able to form adducts on proteins or even protein crosslinks, and the formation of protein adducts and crosslinks causes the dysfunction of the target proteins. In addition, the objectives of the project are also to identify protein targets by PCB metabolites, which may be related to the mechanism of toxicity of LC-PCBs. The alkaline permethylation (AP) was established and optimized to identify and measure the protein adducts from LC-PCB metabolites. The AP method evidenced PCB metabolites formed protein adducts through the sulfhydryl groups and also one molecule of PCB quinoid metabolites was able to bind to more than one protein. Application of cytochrome c as the model protein revealed PCB quinoid metabolites also formed adducts on lysine and glutamic acid. The adduct formation and crosslinks caused the dysfunction of cytochrome c. In addition, the quinone protein adducts still kept the ability for redox reactions, which may lead to unexpected toxicity. The SILAC method was applied to identify the target proteins in the samples of in vitro proteome incubation. The instability of PCB quinone protein adducts was found by further reaction of quinone protein adducts. This may be the reason why cysteine-PCB quinone adducts were not frequently identified by proteomics method. The further understanding of protein adducts by reactive PCB metabolites helps to identify the target proteins, and ultimately reveal the role of protein adducts impacting on human health.

Identification of differential expressions of proteins in proteomic profiles of biological samples shows great potential as a valuable technique for the early diagnosis of various diseases. An important challenge in modern protein profiling approaches is to reduce the complexity of the samples by limiting the number of proteins that need to be evaluated for distinction in the expression between normal and deceased cells. In this research, an affinity based approach for the enrichment of nucleotide and nucleoside binding proteins from a complex cell proteome has been developed. To achieve this goal, new N6-biotinylated-8-azido-adenosine probes (AdoRs) have been designed and synthesized to photolabel the nucleotide and nucleoside binding proteins. These probes contain a reactive group that forms a covalent bond with the target proteins, as well as a biotin tag for affinity enrichment using avidin chromatography. Further, a mass spectrometric protein profiling approach is employed to quantitatively identify small variations in expression of nucleoside and nucleotide binding proteins in samples of interest. Mouse neuroblastoma N18TG2 cell proteome has been used as a model system for the development of the LC-MS/MS based proteomic analysis of these affinity enriched protein fractions. Upon enrichment, the photolabeled proteome exhibited an approximately four-fold abundance of nucleoside and nucleotide binding proteins over nonlabeled proteome. The approach was extended to compare the proteomic profiles of nucleotide and nucleoside binding proteins in cancerous (Hey) and non-cancerous (T-80) human ovarian cell proteome. Certain proteins that were not detected in cell lysate were also identified in labeled proteome, thereby demonstrating the strength of our approach in enriching low abundant proteins. To substantiate the qualitative analysis, we have employed the Stable Isotope Labeling in Amino Acid Cell Culture (SILAC) for the quantitative study of the protein expression in cancerous and non-cancerous human ovarian cells. A modest panel of proteins with differential expressions in these cell lines was identified, a few of which have been correlated to various forms of cancer. Vimentin, stress induced phosphoprotein-1, and heat shock protein 90 that were identified to have altered expressions in these cell lines are among some of the proteins associated with ovarian cancer.

Viruses are obligate parasites that use the host cellular machinery to produce progeny virions. The host responds to this invading pathogen by induction of the immune system; however, the virus employs a variety of strategies to overcome these attacks. The complexity of the virus-host interaction is of great interest to researchers with aims to both characterize the relationship and target steps of the viral life cycle to hinder infection. Many targeted tactics employ single protein analysis; however, approaches that examine the whole set of virus/host interactions are available. Transcriptional alterations within host cells have been determined for many virus- host interactions by micro-array techniques; however little is known about the effects on cellular proteins. This study uses a quantitative mass spectrometric-based method, SILAC, to study differences in a host cell's proteome with infection by a virus. Mammalian reoviruses and herpes simplex viruses are prototypical viruses commonly studied to determine virus life cycle and interactions with hosts. Using three strains of reoviruses and one HSV1 strain, cells were infected to identify differentially regulated proteins at different times. Thousands of proteins were identified for each virus type, some up or down regulated after infection. Biological functions and network analyses were performed using online networking tools. These pathway analyses indicated numerous processes including cell death and inflammatory response are affected by T1L reovirus infection. Comparing reovirus strains revealed a greater overall proteomic change in host function when infected with the more pathogenic T3DC strain. For the HSV infection, host proteins altered during the different immediate early, earlyand late phases of infection helped characterize the host-virus interaction parallel to the virus life cycle. Overall, my study has characterized proteomic changes in different virus infection systems, identifying numerous novel cellular functional pathways and specific proteins altered during virus infections, specifically the secretogranin II protein that had opposite types of regulation in reoviruses and HSV and was examined for its effects on virus replication. Further studies on the novel proteomic characteristics may provide greater understanding to the complex virus-host interactome, leading to possible antiviral targets. / October 2015