O câncer colorretal (CCR) é a segunda de causa morte relacionada ao câncer, com aproximadamente 500.000 mortes por ano em países desenvolvidos. Os pacientes com doença localmente avançada ou metastática possuem um prognóstico ruim e, desta forma, a detecção precoce é necessária para reduzir a alta mortalidade associada à essa doença. Ainda, os métodos de rastreamento utilizados no momento apresentam boa exatidão, porém são de alto custo, consomem muito tempo e são incômodos para os pacientes. A colonoscopia é o método mais comumente utilizado para a detecção de CCR. Entretanto, apresentam duas limitações importantes: i) necessidade da luz intestinal estar limpa, sem resíduos fecais, o que só é conseguido após um preparo rigoroso, com dieta e laxativos, nos dias que antecedem a realização do exame, e, ii) é examinador-dependente, ou seja, dependem da experiência do profissional em localizar as alterações anatômicas, e ainda da habilidade em selecionar o melhor local para a realização da biópsia. Portanto, o desenvolvimento de um método rápido e sensível para o diagnóstico do câncer colorretal é extremamente importante.<br /> A proposta desta dissertação foi desenvolver um método eficaz para o preparo dos tecidos parafinizados e extração de proteínas para experimentos em eletroforese bidimensional (2DE). Tecidos fixados em formaldeído e embebidos em parafina apresentam como vantagens a preservação da qualidade morfológica, abundância e facilidade de armazenamento, o que permite estudos futuros. Entretanto, para análises proteômicas, a recuperação do material biológico é ineficiente. A partir deste estudo, 156 proteínas foram obtidas de tecidos parafinizados, sendo possível identificar por espectrometria de massas, levando a estudos posteriores para identificação de possíveis biomarcadores relacionados ao CCR. / Colorectal cancer (CRC) is the second cause of cancer-related death, with approximately 500,000 deaths per year in developed countries. Patients with locally advanced or metastatic disease have a poor prognosis and, thus, early detection is needed to reduce the high mortality associated with this disease. Yet, the screening methods used at the time have good accuracy, but are expensive, time consuming and are uncomfortable for patients. Colonoscopy is the most commonly used method for the detection of CRC. However, present two major limitations: i) the need to be clean intestinal lumen without fecal waste, which is achieved only after rigorous preparation with laxatives and diet, in the days before the exam, and ii) it is examiner-dependent, i.e., depends on the professional\'s experience in locating anatomical changes, and even the ability to select the best site for biopsy. Therefore, the development of a rapid and sensitive method for the diagnosis of colorectal cancer is extremely important. The purpose of this dissertation was to develop an effective method for the preparation of tissues and protein extraction experiments for two-dimensional electrophoresis (2DE). Tissues fixed in formaldehyde and embedded in paraffin have the advantage of preserving the morphological quality, abundance and storage facility, which allows for further studies. However, for proteomic analysis, the recovery of biological material is inefficient. From this study, 156 proteins were obtained from paraffinized tissues, and can be identified by mass spectrometry, leading to further studies to identify potential biomarkers related to CCR.

A VDAC é a proteína mais abundante da membrana mitocondrial externa. Possui diversas funções, tais como o controle da troca de metabólitos, através da membrana, e a participação no maquinário apoptótico. Estudamos o interactoma da VDAC com as proteínas mitocondriais neuronais do cérebro bovino e murino, a fim de compreender se a expressão diferenciada da VDAC1 e VDAC2 verificada entre essas células estão associadas às diferenças nas interações da VDAC. Os complexos proteicos foram analisados por 2D Blue Native SDS-PAGE e identificados via MALDI-TOF TOF usando o software Mascot e o banco de dados NCBInr. Foram identificados 27 e 46 spots em murino e bovino, respectivamente. Nós identificamos proteínas solúveis e incorporadas na membrana que não são participantes da fosforilação oxidativa, dentre elas a aldeido deidrogenase e muitas outras constituintes de complexos mitocondriais já conhecidos tão bem como novos, tais como a putative stomatin-like protein 2 complex e a switch-associated protein 70. Nossos resultados mostraram que os neurônios bovinos possuem mais complexos (5) contendo a VDAC do que em ratos (1), os quais indicam uma cinética diferencial de acoplamento e desacoplamento. Interessantemente, a lista contendo as proteínas identificadas inclui algumas proteínas conhecidas ou supostamente localizadas em compartimentos não-mitocondriais, por exemplo, a myc-induced nuclear antigen. O interactoma diferencial da VDAC entre as espécies bovina e murina, evidencia a presença de uma base comum, porém com diferentes ambientes estruturais, as quais podem ser a base da diferença entre os sítios de ligação A e B observados nas diferentes espécies. / The voltage dependent anion channel (VDAC) is the most abundant protein of outer mitochondrial membrane. VDAC controls metabolite exchange through this membrane and the apoptosis machinery. We studied the interactome of VDAC with mitochondrial proteins of neuronal cells from rat and bovine brain. We wished to understand if the differential expression of VDAC1 and VDAC2 verified between these cells was linked to differences in the VDAC interactions. Protein complexes were analyzed by 2D Blue Native SDS-PAGE and were identified by MALDI-TOF TOF using Mascot software against the NCBInr database. Number of 27 e 46 spots were identified from rat and bovine brain, respectively. We identified soluble and membrane-embedded non-OXPHOS proteins, among them aldehyde dehydrogenase, and many as constituents of known mitochondrial complexes as well as novel ones such as putative stomatin-like protein 2 complex and switchassociated protein 70. Our results showed that bovine neurons had more protein complexes (5) containing VDAC than rat cells (1), which indicates a differential kinetics of assembly or disassembly. Interestingly, the identification list included some proteins known or presumed to be localized to nonmitochondrial compartments, for example, myc-induced nuclear antigen. Our results support evidences of differential apoptotic and energetic mechanisms verified in these brains. The differential VDAC interactome between bovine and murine, support evidences of a common base, but whith different structural environment, which may be the basis of the difference between the binding sites A and B observed in these brains.

Bactéria e fungos são as principais fontes de enzimas envolvidas na transformação de compostos chave para o fluxo de carbono em solos de manguezal, caracterizado por alta prevalência de anaerobiose, salinidade e elevado teor de matéria orgânica. A decomposição de plantas ou resíduos de animais nestas condições é muito lenta, devido à pressão seletiva sobre a evolução de enzimas envolvidas nos processos de mineralização de nutrientes. A metagenômica, permiti o acesso a grande maioria da diversidade microbiana no ambiente, por meio da geração de bibliotecas de clones, o que resulta em um cenário promissor para bioprospecção de novas atividades enzimáticas. Neste estudo, foi relatada a descrição e caracterização de uma nova ?-N-acetil-hexosaminidase (EC 3.2.1.52) da família GH3, envolvida na degradação da matéria orgânica em solo de manguezal contaminado por derramamento de óleo localizado no município de Bertioga-SP, por meio de uma triagem de 12.960 clones metagenômicos. O clone positivo para a atividade celulolítica foi sequenciado e um total de 1.175.586 reads foram gerados com tamanho médio de 198 pb. As sequencias foram trimadas com base na qualidade de índice PHRED >= 30.0, e remoção de sequencias do hospedeiro (E. coli) e do vetor (fosmídeo), originando um contig final com 39.586 Kb. Entre as ORF\'s anotadas a partir do contig gerado, uma sequencia de 1.065 nucleotídeos foi identificada como codificante para a enzima ?-N-acetil-hexosaminidase, evidenciando baixa similaridade (32 %) com as demais encontradas no bancos de dados comparativos. A enzima foi expressa e purificada, onde uma banda isolada foi visualizada por SDS-PAGE com massa molecular prevista de 43 kDa. Por fim, as atividades ótimas da enzima (30 °C; pH 5.0; 0.5 M de NaCl; diminuição de atividade após 3hs de incubação) foram caracterizadas por meio do indicador p-nitrophenol (pNP) ligados aos substratos GlNac, GalNac e Glc. A detecção da enzima por meio da metagenômica, evidenciou que os manguezais são reservatórios de novas enzimas com características diferenciadas e altos potenciais de aplicabilidades biotecnológicas / Bacteria and fungi are major sources of enzymes involved in the transformation of key compounds for the carbon fluxes on mangrove soils, characterized by the high prevalence of anaerobiosis salinity and high content of organic matter. The decomposition of plant or animals residues under these conditions is very slow, acting as a selective pressure on the evolution of enzymes involved in the mineralization process of nutrients. Metagenomics has provided access to the vast majority of the microbial diversity in the environment through the generation of fosmid libraries, resulting in a promising scenario for bioprospection enzymatic activities. In this study, we report the description and characterization of a novel ?-N-acetylhexosaminidase (EC 3.2.1.52) of GH3 family, involved in the degradation of organic matter in mangrove soils contaminated by oil spill located in the city of Bertioga-SP through of a screening of 12.960 metagenomic clones. The positive clone for cellulolytic activitie was sequenced and a total of 1.175.586 reads were generated with measuring size 198 bp. The sequences were trimmed based on the index of quality PHRED >= 30.0 and removing the sequences to host (E. coli) and vector (fosmid) resulting in a contig of 39.586 Kb. Between the anoted ORF\'s from generated contig a sequence of 1.065 nucleotides was identified coding for a ?-N-acetylhexosaminidase showing low similatrity (32 %) with the other found in comparatives databases. The enzyme was expressed and purified where an isolated band can be visualized by SDS-PAGE with molecular mass of 43 kDa. Finally, as optimum activity of the enzyme (30 °C; pH 5.0; 0.5M NaCl; decreased activity after 3 h incubation) were characterized by the indicator p-nitrophenol (pNP) linked to the substrates GlNac, GalNac and Glc. The detection of the enzyme through metagenomics indicated that mangroves are reservoirs of novel enzymes with different characteristics and high potential for biotechnological applicability

O objetivo deste trabalho é estudar o nível de organização dos cristalitos de surfactantes aniônico, catiônico e zwiteriônico com o aumento na densidade destas moléculas sobre um cristal de germânio. As análises foram realizadas por espectroscopia de infravermelho com transformada de Fourier acoplada à técnica de reflexão total atenuada (FTIR-ATR). Este estudo apresenta importantes aspectos na organização do dodecilsulfato de sódio (SDS), do N-hexadecil-N,N-dimetil-3-amônio-1-propanosulfato (HPS), do brometo de hexadeciltrimetilamônio (CTAB) e do brometo de dodeciltrimetilamônio (DTAB). No SDS é observado um deslocamento de 1.7 cm-1 para valores de maior frequência na banda de estiramento assimétrico do CH2, vass (CH2), enquanto que é observado um deslocamento de 0.9 cm-1 na banda de estiramento simétrico, vsim (CH2). Este deslocamento para valores de maior frequência nas bandas de estiramento está associado com um aumento na desorganização da cadeia alifática com o aumento na densidade de moléculas sobre o elemento de ATR. A banda de deformação angular do CH2, &delta;(CH2), apresenta um valor em 1468 cm-1 que é também um indicativo de desorganização. No CTAB não é observado variações acentuadas nos valores das frequências vibracionais. Na banda vass (CH2) do DTAB é observado um deslocamento de 4.5 cm-1 para valores de menor frequência. Embora seja observado valores próximos de 2920 cm-1 para a banda vass (CH2), que é um indicativo do estado líquido de surfactantes, o que é observado nesse estudo são cristalitos de DTAB. O deslocamento da banda vsim (CH2) do DTAB é da ordem de 2 cm-1. Estas mudanças nas bandas vass (CH2) e vsim (CH2) são um indicativo da diminuição nas conformações gauche e um aumento nas conformações trans ao longo da cadeia alifática. O valor da freqüência em torno de 1472 cm-1 para a banda &delta;(CH2) é também um indicativo de uma maior organização na cadeia de CH2 do DTAB. Para o HPS é observado um deslocamento de 2.6 e 2.7 cm-1 para valores de maior frequência nas bandas vass (CH2) e vsim (CH2), respectivamente. A banda &delta;(CH2) do HPS apresenta um deslocamento de 4 cm-1 para valores de maior frequência. A variação nas bandas vass (CH2), vsim (CH2), e &delta;(CH2) ressalta o aumento na desorganização da cadeia alifática com o aumento na densidade de moléculas de HPS sobre o germânio. / The objective of this work is study the order level of anionic, cationic and zwitterionic surfactants with the increase of their density packing on the surface of a germanium element. The analyses were performed by a Fourier transform infrared-attenuated total reflection spectroscopy. This study shows important insights on the conformational order of sodium dodecyl sulfate (SDS), N-hexadecyl-N-N -dimethyl-3-ammonio-1-propane-sulfonate (HPS), hexadecyl-trimethylammonium bromide (CTAB) and dodecyl trimethylammonium bromide (DTAB). It is observed a shift of 1.7 and 0.9 cm-1 to higher frequency values of the CH2 asymmetric (vass (CH2)) and symmetric (vsim(CH2)) stretching bands for the SDS molecules, respectively. The latter shift to higher frequency values is associated with the disorder of the aliphatic chain due to the increase of density packing of SDS molecules on the germanium element. The CH2 scissoring band [&delta; (CH2)] shows a value in 1468 cm-1, which is also an indicative of conformational disorder. It is not observed any accentuated change on the vibrational frequency values of the CTAB molecules. The vass (CH2) band of the DTAB molecules is shifted 4.5 cm-1 to lower frequency values. Although it is observed values near 2920 cm-1 for the vass(CH2) band, indicating a surfactant liquid phase, it is shown in this study that there are indeed DTAB crystallites. The shift of DTAB vsim(CH2) band is around 2 cm-1. These changes in vass(CH2) and vsim(CH2) band are an indicative of a decrease in gauche conformations and an increase in all-trans conformations over the aliphatic chain. The frequency value around 1472 cm-1 for the &delta;(CH2) band is also an indicative of the order in CH2 chain of DTAB. It is observed a shift of 2.6 and 2.7 cm-1 to higher frequency values of vass (CH2) and vsim(CH2) bands of the HPS molecule, respectively. The &delta;(CH2) band of the HPS molecule presents a shift of 4 cm-1 to higher frequency values. These variations in vass (CH2), vsim(CH2), and &delta;(CH2) bands stand out the disorder of the aliphatic chain due to the increase of the density packing for the HPS molecules on the germanium surface.

Bactéria e fungos são as principais fontes de enzimas envolvidas na transformação de compostos chave para o fluxo de carbono em solos de manguezal, caracterizado por alta prevalência de anaerobiose, salinidade e elevado teor de matéria orgânica. A decomposição de plantas ou resíduos de animais nestas condições é muito lenta, devido à pressão seletiva sobre a evolução de enzimas envolvidas nos processos de mineralização de nutrientes. A metagenômica, permiti o acesso a grande maioria da diversidade microbiana no ambiente, por meio da geração de bibliotecas de clones, o que resulta em um cenário promissor para bioprospecção de novas atividades enzimáticas. Neste estudo, foi relatada a descrição e caracterização de uma nova ?-N-acetil-hexosaminidase (EC 3.2.1.52) da família GH3, envolvida na degradação da matéria orgânica em solo de manguezal contaminado por derramamento de óleo localizado no município de Bertioga-SP, por meio de uma triagem de 12.960 clones metagenômicos. O clone positivo para a atividade celulolítica foi sequenciado e um total de 1.175.586 reads foram gerados com tamanho médio de 198 pb. As sequencias foram trimadas com base na qualidade de índice PHRED >= 30.0, e remoção de sequencias do hospedeiro (E. coli) e do vetor (fosmídeo), originando um contig final com 39.586 Kb. Entre as ORF\'s anotadas a partir do contig gerado, uma sequencia de 1.065 nucleotídeos foi identificada como codificante para a enzima ?-N-acetil-hexosaminidase, evidenciando baixa similaridade (32 %) com as demais encontradas no bancos de dados comparativos. A enzima foi expressa e purificada, onde uma banda isolada foi visualizada por SDS-PAGE com massa molecular prevista de 43 kDa. Por fim, as atividades ótimas da enzima (30 °C; pH 5.0; 0.5 M de NaCl; diminuição de atividade após 3hs de incubação) foram caracterizadas por meio do indicador p-nitrophenol (pNP) ligados aos substratos GlNac, GalNac e Glc. A detecção da enzima por meio da metagenômica, evidenciou que os manguezais são reservatórios de novas enzimas com características diferenciadas e altos potenciais de aplicabilidades biotecnológicas / Bacteria and fungi are major sources of enzymes involved in the transformation of key compounds for the carbon fluxes on mangrove soils, characterized by the high prevalence of anaerobiosis salinity and high content of organic matter. The decomposition of plant or animals residues under these conditions is very slow, acting as a selective pressure on the evolution of enzymes involved in the mineralization process of nutrients. Metagenomics has provided access to the vast majority of the microbial diversity in the environment through the generation of fosmid libraries, resulting in a promising scenario for bioprospection enzymatic activities. In this study, we report the description and characterization of a novel ?-N-acetylhexosaminidase (EC 3.2.1.52) of GH3 family, involved in the degradation of organic matter in mangrove soils contaminated by oil spill located in the city of Bertioga-SP through of a screening of 12.960 metagenomic clones. The positive clone for cellulolytic activitie was sequenced and a total of 1.175.586 reads were generated with measuring size 198 bp. The sequences were trimmed based on the index of quality PHRED >= 30.0 and removing the sequences to host (E. coli) and vector (fosmid) resulting in a contig of 39.586 Kb. Between the anoted ORF\'s from generated contig a sequence of 1.065 nucleotides was identified coding for a ?-N-acetylhexosaminidase showing low similatrity (32 %) with the other found in comparatives databases. The enzyme was expressed and purified where an isolated band can be visualized by SDS-PAGE with molecular mass of 43 kDa. Finally, as optimum activity of the enzyme (30 °C; pH 5.0; 0.5M NaCl; decreased activity after 3 h incubation) were characterized by the indicator p-nitrophenol (pNP) linked to the substrates GlNac, GalNac and Glc. The detection of the enzyme through metagenomics indicated that mangroves are reservoirs of novel enzymes with different characteristics and high potential for biotechnological applicability

Streptococcus pneumoniae (pneumococo) é um importante patógeno humano, responsável por graves infecções das vias respiratórias. O principal fator de virulência desse microrganismo é a cápsula polissacarídica (PS), antígeno das vacinas atuais, que são elaboradas com os PS purificados de cepas de pneumococo prevalentes na população. O objetivo desse trabalho foi desenvolver um processo de purificação do PS do sorotipo 14, responsável por 39% das doenças em crianças de 0-6 anos no Brasil. A metodologia de purificação envolveu separação celular por microfiltração tangencial e concentração do microfiltrado com membrana de ultrafiltração tangencial de 50 kDa. O produto dessa etapa foi diafiltrado com dodecil sulfato de sódio em membrana de ultrafiltração tangencial de 30 kDa, seguido de precipitação com ácido tricloroacético a 5%, precipitação por etanol (20% e 60%) e cromatografia de troca aniônica. A pureza do PS foi avaliada pelo conteúdo de proteínas e ácidos nucleicos remanescentes e o tamanho por cromatografia de exclusão molecular. O PS foi obtido com pureza e tamanho requeridos pelos órgãos regulatórios e o rendimento final do processo foi de 65%. / Streptococcus pneumoniae (pneumococcus) is an important human pathogen responsible for severe respiratory tract infections. The main virulence factor of this microorganism is the capsular polysaccharide (PS), which is the antigen of all current vaccines, which are prepared with purified PS of pneumococcal strains prevalent in the population. The objective of this work was to develop a new purification process for PS of serotype 14, responsible for 39% of diseases in children of 0-6 years old in Brazil. The purification method involved cell separation by tangential microfiltration and concentration of cell-free culture broth containing PS by tangential ultrafiltration (50 kDa). The product of this step was diafiltrated with sodium dodecyl sulfate by tangential ultrafiltration (30 kDa), following by 5% trichloroacetic acid precipitation, 20% and 60% ethanol precipitation and anion exchange chromatography. The PS purity was evaluated by the content of residual proteins and nucleic acids, and the molecular mass by size exclusion chromatography. The purity and molecular mass requirements were achieved and the process global yield was 65%.

Appropriate solubilization of membrane proteins can be achieved by incorporating detergents such as sodium dodecyl sulfate (SDS). Unfortunately, SDS interferes with liquid chromatography (LC) and mass spectrometry (MS). This thesis presents “MS-friendly’ alternatives to solubilize membrane proteins and provides an evaluation of current protocols for SDS removal. Considering the limitation of SDS in a proteome analysis workflow, fluorinated surfactants have previously been proposed. Our results showed that APFO similar proteome solubilization to that of SDS. Unfortunately, APFO was only marginally more tolerable to LC and MS than SDS. Nonetheless, an important advantage of using APFO is that it can be easily removed from the sample by evaporation. The efficiency of precipitation protocols was explored. Our results demonstrate that high recovery is possible. Quantitative re-solubilization of membrane proteins following precipitation was made possible through addition of 80% formic acid. This solvent system may present a promising pathway for top-down MS analysis.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Recovering deoxyribose nucleic acid (DNA) from items of evidence can provide critical information in criminal cases. Since the development of the polymerase chain reaction (PCR) and use of short tandem repeats (STR) to create unique profiles from an individual’s genome1, sampling items of evidence for the presence of DNA has become routine. Biological evidentiary specimens are commonly collected at crime scenes as well as sampled from collected items of interest by using a cotton swab which can then be easily stored and tested as needed. However, even with modern advances in technology and methods, large amounts of DNA can be either lost throughout processing or remain on the substrate used for collection of the sample, such as a cotton swab2. While many of the downstream processes of evidence evaluation have been vastly improved through the use of automated procedures, engineered buffers, and commercially available extraction kits, the front-end procedures are typically more technician dependent; it is an area in which opportunities to fine-tune techniques remain. The most recent change to generalized stain recovery occurred after Sweet et al. achieved an increased efficiency of recovery by using what they referred to as the “double swab technique”. The classic method of collection before this time used a single, wet cotton swab. Based on a need to increase the effective collection of DNA from saliva samples, the double swab method was developed. The classic method was modified by using a second, dry swab to collect remaining moisture deposited by the first, wet swab3. To continue the effort to maximize cellular and DNA recovery from cotton swabs the use of trehalose in the cotton swab wetting solution was explored. D-(+)-Trehalose dihydrate is a naturally occurring disaccharide composed of two alpha glucose molecules. An alpha, alpha-1, 1 bond connects the two molecules which lends high resistance to acid hydrolysis, giving the molecule unique properties. Specifically, these properties allow the compound to maintain stability even during exposure to high temperatures and in acidic conditions4,5. In nature, trehalose can be found in plants and small organisms where it is thought to act as a protectant against fluctuations in moisture and temperature. Synthesis and release of trehalose by lower life forms during stressed states shows protective properties to cellular integrity by inhibiting protein denaturation6. The objectives of this study are to investigate the use of trehalose as an additive in DNA collection processes. The experiments examine the ability of trehalose to increase efficiency of cellular release from cotton swabs during the elution step and compares trehalose to other common buffer additives, bovine serum albumin (BSA) and sodium dodecyl sulfate (SDS), when utilized as a pre-treatment or moistening agent on the cotton swab. Two procedures were developed to test the ability of trehalose to increase efficiency of cellular and DNA release from cotton swabs. The first procedure tested trehalose at 0.2 molar (M) and 1 M concentrations as the incubating solution over1 hour and 18 hour time periods after which the cotton swab was eluted using a spin-x insert and centrifugation. Both eluate and cotton swab were then processed using ZyGEM direct lysis and quantified. Quantification results of the eluate and swabs incubated in trehalose solution were not significantly different from controls. However, it is apparent that a large portion of deposited DNA remained on the swabs even after elution and ZyGEM direct lysis. The second procedure tested trehalose against BSA and SDS as treatments to cotton swabs before DNA collection. A pre-treated group (solution was applied to the swab and dried overnight; DNA was deposited to the dried swab) and a moist group (solution was applied and DNA deposited immediately) were tested after deposition of a set volume of saliva cell suspension. Quantification and amplification results of SDS treated samples indicated significant differences of DNA recovery and average peak height of profiles compared to water and buffer controls. Trehalose samples did have some significant improvement in DNA yield; however, the addition of trehalose as a moistening agent for cotton swabs does not prove to be of forensic value.

No description available.