Orientador: Denise Vaz de Macedo / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-14T06:07:53Z (GMT). No. of bitstreams: 1 Lazarim_FernandaLorenzi_D.pdf: 1781611 bytes, checksum: 278e3fd2103780698a1b6dc5452c8b5d (MD5) Previous issue date: 2009 / Resumo: A resposta adaptativa decorrente de um programa de treinamento está relacionada a um intenso processo de síntese protéica, cujo efeito cumulativo de várias sessões de exercício leva a alterações fenotípicas do músculo e aumento de rendimento em capacidades biomotoras diversas. Para isso é necessário um tempo adequado de recuperação entre os estímulos. Um processo contínuo de treinamento intensificado sem o tempo de recuperação adequado é denominado overtraining. Este pode culminar em basicamente dois estados diferenciados em relação ao desempenho: overreaching funcional (FOR), com manutenção ou mesmo melhora de desempenho após o descanso, e overreaching não funcional (NFOR), caracterizado pela queda no desempenho por tempo prolongado. A visualização das alterações agudas e crônicas do perfil protéico tanto de células como de fluidos pode auxiliar na compreensão dos mecanismos envolvidos nos estados FOR e NFOR, e possibilitar a identificação de marcadores que auxiliem na detecção desses estados. Nesse contexto a análise proteômica pode ser uma ferramenta bastante útil, pois permite separar, quantificar e identificar o perfil protéico de tecidos e fluidos biológicos. A presente tese está dividida em duas partes: pesquisa (Parte I) e ensino (Parte II), que refletem as experiências vividas desde a iniciação científica, sendo igualmente relevantes para minha formação acadêmica. A Parte I é constituída por três capítulos cujo objetivo principal foi investigar as alterações agudas e crônicas decorrentes do exercício físico no perfil protéico do soro de ratos através da análise proteômica. O capítulo 1 apresenta uma revisão sobre os mecanismos moleculares envolvidos na resposta adaptativa ao treinamento, as proteínas do soro, as técnicas utilizadas na análise proteômica e sua aplicabilidade nas pesquisas com exercício. O capitulo 2 apresenta as alterações agudas no perfil proteico do soro de ratos submetidos a um exercício exaustivo de média duração em esteira, 3 e 24 horas após o estímulo. As proteínas diferencialmente expressas 24 horas após o exercício corresponderam a proteínas de fase aguda sintetizadas em resposta à instalação de um processo inflamatório, indicando que a geração de microtraumas e a inflamação são partes integrantes da resposta aguda ao exercício. O capítulo 3 apresenta as alterações no perfil proteico do soro de ratos submetidos a um protocolo de indução ao continuum treinamento-overtraining, desenvolvido recentemente no nosso laboratório, e que produz animais nos estados FOR e NFOR. As proteínas diferencialmente expressas indicam um quadro antiinflamatório nos animais do grupo FOR e alterações protéicas que favoreceram os processos adaptativos envolvidos na biogênese mitocondrial e regeneração do tecido danificado. Também apresentaram melhora no perfil lipídico. O grupo NFOR apresentou alterações de proteínas de fase aguda indicando um processo inflamatório instalado e alterações de algumas proteínas que podem ter prejudicado o desencadeamento da resposta adaptativa, resultando na queda da performance. A Parte II da tese apresenta uma proposta de atividade prática, aplicada num curso de especialização com enfoque em bioquímica para alunos de Educação Física e Nutrição. Essa atividade consiste na discussão dos conceitos de Índice Glicêmico e Carga Glicêmica a partir de dados obtidos pelos próprios alunos. Utilizamos essa aula para a introdução ao estudo das vias de síntese e integração metabólica no estado alimentado / Abstract: The adaptive response to a training program is related to an intensive process of protein synthesis, which cumulative effect of multiple sessions of exercise leads to muscle phenotypic alterations and increases different physical capacities. For such adaptation an appropriate time for recovery between stimuli is required. A continuous process of intensified training without adequate recovery time is called overtraining. It can result in basically two different states concerning performance: functional overreaching (FOR), with maintenance or even improvement of performance after the recovery period, and non-functional overreaching (NFOR), characterized by performance decrement for a prolonged period. The visualization of acute and chronic changes on the protein profile of both cells and fluids may help one to understand the mechanisms involved on FOR and NFOR states, and it can enable the identification of biomarkers helping to detect these states. Within this context the proteomic analysis can be an interesting tool as it enables to separate, identify and quantify the protein profile of tissues and biological fluids. This work is divided in two parts: research (Part I), and education (Part II), which represent the experiences that I have been living since my scientific initiation and therefore, both are relevant for my education. Part I consists of three chapters in which the main goal is to investigate the acute and chronic changes in response to exercise in serum proteins profile of rats by proteomic analysis. Chapter 1 presents a review of the molecular mechanisms involved in the adaptive response to training, serum proteins, the techniques used in proteomics analysis and its applicability on exercise research. Chapter 2 presents the acute changes in serum protein profile of rats submitted to an exhaustive exercise of average duration on a treadmill, 3 and 24 hours after the stimulus. The proteins differently expressed 24 hours after the exercise were the acute-phase protein synthesized in response to installation of the inflammatory process, indicating that the generation of micro trauma and inflammation are parts of the acute response to the exercise. Chapter 3 reveals the changes in the serum protein profile of rats, submitted to an exercise protocol developed recently in our laboratory, to induce the animals through the continuum training-overtraining, leading the animals to the FOR and NFOR states. The differently expressed proteins indicate an anti-inflammatory process in the animals that were in the FOR group and protein changes which favored the adaptive processes involved in mitochondrial biogenesis and the complete recovery of tissue damage, as well as the improvement on the lipid profile. The NFOR group presented changes of acute phase proteins indicating the instalation of an inflamatory process and alterations in some proteins that may have impaired the development of the adaptive response, which results in performance decrement. Part II of this work shows a proposal for a practical activity implemented in a specialization course with focus on Biochemistry for Physical Education and Nutrition students. This activity consists in the discussion of the Glycemic Index and Glycemic Load concepts through data obtained by the students. This class is used to introduce the study of synthesis pathways and metabolic integration in the fed state / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

O eixo hipotálamo-pituitária-adrenal é o principal sistema neuroendócrino envolvido na regulação e adaptação da resposta ao estresse e, o principal hormônio secretado é o cortisol. O cortisol exerce seus efeitos por meio dos receptores mineralocorticoide (MR) e glicocorticoide (GR). Variações nos genes desses receptores têm sido associadas à sensibilidade aos glicocorticoides e mudanças no perfil metabólico. O objetivo geral desse trabalho foi compreender a variabilidade existente em relação às respostas fisiológicas de bovinos por meio da identificação de polimorfismos genéticos em genes envolvidos na resposta ao estresse e, verificar as consequências dessa variação genética em características de qualidade da carne. Dessa forma, três abordagens foram propostas: (1) avaliar a incidência de carne DFD (dark, firm and dry) e seu impacto no perfil metabólico, endócrino e características de qualidade da carne bovina, uma vez que o estresse é um dos principais fatores que levam a essa condição desfavorável; (2) avaliar a contribuição de fatores genéticos, por meio da identificação de polimorfismos de nucleotídeo único (SNPs), no gene do MR e GR, e suas associações com as características mensuradas; (3) avaliar os efeitos desses polimorfismos sobre o perfil proteico do músculo bovino. Foram utilizados 241 bovinos da raça Nelore. Os resultados evidenciaram implicações direta do pH 24 horas post-mortem nos atributos de cor e perdas por cozimento da carne. A incidência de carnes DFD (pH>=5,8) foi de 18,7%. Os polimorfismos identificados mostraram influenciar em algumas características mensuradas. Os SNPs NR3C2_1 e NR3C2_2 no gene do MR foram associados ao conteúdo de glicogênio muscular e nível plasmático do hormônio adrenocorticotrófico (ACTH) post-mortem, e o SNP NR3C1_1 no gene do GR foi associado aos níveis plasmático de cortisol post-mortem. As análises proteômicas demonstraram que a maioria das proteínas reguladas por esses SNPs estão envolvidas na contração muscular, metabolismo e defesa celular. Portanto, é possível inferir que o pH tem impacto nas características de qualidade da carne e que polimorfismos em MR e o GR levam a mudanças na atividade do eixo HPA, no perfil metabólico do organismo e no perfil proteico do músculo, sugerindo que esses genes estão envolvidos em uma complexidade de funções e podendo ser alvos de estudos em sistemas de produção que visam melhorar a produtividade. / The hypothalamic-pituitary-adrenal axis is the main neuroendocrine system involved in the regulation and adaptation in stress response and the primary hormone secreted is cortisol. Cortisol exerts its effects through the mineralocorticoid (MR) and glucocorticoid (GR) receptors. Variations in the genes of these receptors have been associated with sensitivity to glucocorticoids and changes in the metabolic profile. The general objective of this work was to understand the variability in relation to physiological responses of cattle through identification of genetic polymorphisms in genes involved in stress response and, checking the consequences of this genetic variation in meat quality traits. Thus, three approaches have been proposed: (1) evaluate the incidence of DFD meat (dark, firm and dry) and its impact on metabolics, endocrines profiles and meat quality traits, since stress is the major factor that lead to this unfavorable condition; (2) evaluate the contribution of genetic factors through identification single nucleotide polymorphisms (SNPs) in the MR and GR gene and its association with the measured traits; (3) evaluate the effects of these polymorphisms on the protein profile of bovine muscle. A total of 241 Nellore cattle were used. The results evidenced direct implications of 24 hours pH post-mortem in color attributes and cooking losses. The incidence of DFD meat (pH >= 5.8) was 18.7%. The polymorphisms identified demonstrated to influence some on measured characteristics. The NR3C2_1 and NR3C2_2 SNPs in MR gene were associated with muscle glycogen content and post-mortem adrenocorticotropic hormone (ACTH) plasma levels and, the NR3C1_1 SNP in GR was associated with post-mortem cortisol plasma levels. The proteomic analysis demonstrated that most proteins regulated by these SNPs are involved in muscle contraction, metabolism and cellular defense. Therefore, it is possible to infer that pH has impact on meat quality traits and MR and GR polymorphisms lead to changes in the HPA axis activity, metabolic profile and protein muscle profile, suggesting that these genes are involved in a complexity of functions and may be targets for studies on production systems to improve productivity.

O complexo salivar da sanguessuga H. depressa é composto pelas glândulas salivares e probóscide. Análises bidimensionais (2D) mostraram que a maioria das proteínas apresentam pI entre 3.5 à 9.5 e MM entre 10 à 105 kDa. O mapa 2D do complexo salivar apresentou 352 spots totais, sendo 249 exclusivos da saliva (corado por nitrato de prata) e 219 spots totais (corado por Coomassie Blue). As proteínas foram identificadas após sequenciamento tandem MS (proteoma) pela complementariedade às sequências traduzidas do cDNA (transcriptoma). As proteínas mais abundantes foram: antiagregante plaquetário; miohemeritrina e anidrase carbônica. Estas proteínas devem exercer um papel na digestão ou anticoagulação do sangue durante a alimentação. A zimografia também identificou uma protease gelatinolítica (45 kDa). Porém, lefaxin (inibidor de FXa) e hementerina (fibrinogenolítico e inibidor de agregação plaquetária) não foram identificados por estas técnicas biotecnológicas, o que mostra a necessidade de técnicas adicionais para a completa elucidação da constituição desta amostra. / The salivary complex from H. depressa leech is composed of salivary gland and proboscis. Two-dimensional (2D) analysis showed that majority of proteins have pI from 3.5 to 9.5 and MW from 10 to 105 kDa. The 2D gel of salivary complex showed 352 total spots, 249 of them were from saliva (silver stained) and 219 total spots (Coomassie Blue stained). Proteins were identified after tandem MS sequencing (proteome) and complementar analysis of translated sequences from cDNA (transcriptome). The most abundant proteins were: antiplatelet protein; myohemerytrin; carbonic anhydrase. These proteins may have a role in digestion or antihemostatic system during blood feeding. The zymographic assay identified a gelatinolytic protein (45 kDa). But, lefaxin (inhibitor of Fxa) and hementerin (fibrinogenolytic and antiplatelet function) were not identified by these biotechonolgical techniques, showing that additional techniques are necessary for complete knowledge about the composition of this sample.

A soja geneticamente modificada tolerante ao herbicida glifosato tem sido a cultura derivada da engenharia genética mais cultivada atualmente no mundo. Como todo alimento GM a soja tem sido alvo de investigação em relação a sua Biossegurança. Novas estratégias têm sido desenvolvidas e aplicadas neste campo de pesquisa, sendo que métodos rápidos e eficientes de análise proteômica têm sido utilizados para avaliação e monitoramento da segurança e inocuidade alimentar, indicando mudanças no perfil protéico entre variedades convencionais e GM. O objetivo do presente trabalho foi avaliar os mapas protéicos de amostras de soja convencionais e suas derivadas geneticamente modificadas tolerantes ao herbicida glifosato, utilizando técnicas de análise proteômica com ênfase para inocuidade alimentar. Foram utilizadas seis amostras de soja, sendo três convencionais parentais e três derivadas GM, cultivadas entre 2004-2005, em Goiás. O extrato bruto protéico foi submetido à análise por eletroforese unidimensional e bidimensional. A eletroforese 2D, foi realizada utilizando tiras com gradiente de pH de 3-10 e 4-7. As imagens dos mapas protéicos das seis variedades, produzidas em replicatas, foram analisadas pelo software ImageMaster 2D Platinum. O potencial alergênico do extrato protéico bruto foi avaliado para todas as variedades utilizando soro de pacientes alérgicos à soja através de immunoblotting. Nos resultados obtidos observou-se a presença das principais frações protéicas da soja pela eletroforese unidimensional sem alteração significativa entre as amostras parentais e GM, exceto para uma banda de 115 kDa presente nas amostras parentais, mas ausente nas amostras GM. A partir da análise por eletroforese 2D foram identificadas as formas peptídicas correspondentes às frações de β-conglicinina e glicinina bem como diversas outras proteínas encontradas na soja como o inibidor de tripsina e a lipoxigenase. Através do software foi possível observar que um spot apresentou diferença estatística entre as amostras analisadas, expresso em maior concentração nas amostras GM do que nas parentais. Nos testes de alergenicidade, os extratos protéicos das variedades GM demonstraram reatividade similar em relação as suas respectivas variedades parentais. A proteína de 115 kDa foi sequenciada e identificada como a proteína precursora da cadeia α da β-conglicinina e o spot das amostras GM que apresentou diferença estatística significativa foi identificado como a proteína precursora de G4 glicinina. A diferença observada entre as variedades parentais e GM para as subunidades α de β-conglicinina e G4 glicinina pode ter ocorrido devido a variações normais observadas entre diferentes variedades de soja. Os resultados demonstram a viabilidade de aplicação das ferramentas proteômicas na identificação de alterações de perfis protéicos de amostras de soja parentais e GM. Pelos dados obtidos podemos concluir que as diferenças apresentadas não comprometem a inocuidade alimentar das amostras de soja GM em relação a suas respectivas variedades parentais. / Genetically modified soya-tolerant to the herbicide glyphosate culture has been derived from the more cultivated genetic engineering in the world today. As GM soya beans whole food has been investigated in relation to your biosafety. New strategies have been developed and applied research in this field, and fast and efficient methods of analysis proteomics have been used for assessment and monitoring of food security and safety, indicating changes in own protein profile between conventional and GM varieties. The aim of this work was to assess the maps soy protein samples of conventional and genetically modified their derived to the herbicide glyphosate-tolerant, using Proteomics analysis techniques with emphasis on food safety. Six samples were used for conventional soya, three and three derived from GM parental, grown between 2004-2005. The crude protein extract own was subjected to analysis by electrophoresis one-dimensional and two-dimensional. 2D electrophoresis using Strip was held with pH gradient of 3-10 and 4-7. Protein maps images of six varieties produced in replicates have been analysed by the 2D Platinum software ImageMaster. The potential allergenic in crude protein extracts was evaluated for all varieties using allergic patient serum soya by immunoblotting. In the results obtained noted the presence of the main protein fractions of soya by one-dimensional electrophoresis without significant change between parental and GM samples, except for a band of 115 parental kDa present in the sample, but absent in GM samples. From the analysis by 2D electrophoresis peptides forms were identified corresponding to fractions of β-conglicinina and glicinina as well as several other proteins found in soy as trypsin inhibitor and lipoxygenase. Through the software has been possible to observe that a spot presented statistical difference between the samples tested, expressed in greater concentration in the samples GM in parenting. In tests of allergenicity, GM varieties protein extracts showed similar reactivity in respect of their parental varieties. 115 KDa protein was sequenced and identified as the protein precursor of α subunit of β-conglicinina and the spot that GM samples presented significant statistical difference was identified as the G4 glicinina protein precursor. The difference between parental and GM varieties for subunits α of β-conglicinina and G4 glicinina may have occurred due to normal variation between different varieties of soy. The results demonstrate the viability of applying the tools Proteomics in identification of protein profiles changes of soya samples parental and GM. By data obtained can be concluded that the differences do not compromise the safety of food GM soybean samples with regard to their parental varieties.

No description available.

This PhD thesis showed the applicability of a gel-based proteomic separation tool, 2-D electrophoresis in three independent projects. Supplemented with results obtained using different techniques the proteomic studies enabled a global imaging of proteoms in the studied biological systems. Comparing total proteoms of E. coli 61 protein changes were identified and connected with the development of the bacterial population in the presence of an antibiotic compound, erythromycin. This classic proteomic approach included sample extraction, optimization of its 2D separation followed by 2D gel analysis and protein identification by MS methods. A disadvantage of this work was an enourmously large amount of data to be analyzed by computer analysis. For the study of membrane proteom of B. subtilis during a pH induced stress, on the other hand, a modification of isolation techniques for membrane and membrane associated proteins was required first to improve the subsequent protein separation by 2-D electrophoresis. The optimalization of protein extraction included changes in detergents used for protein solubilization and a prolongation of time periods in the protein solubilization protocol. 5 relevant protein changes were then described that play a role in the bacterial response to pH stress. The proteins were...

Die vorliegende Arbeit befaßt sich mit Nachweismöglichkeiten von Helicobacter pylori und deren Vergleich. Hierfür ist eine genaue Kenntnis der Helicobacter-Proteine notwendig. Zu diesem Zweck wurde die humorale Immunantwort gegenüber Helicobacter pylori unter Anwendung der Methodik des zweidimensionalen Immunoblots analysiert. Zunächst wurden Proteine des autologen Helicobacter pylori-Stammes über zweidimensionale Gelelektrophorese aufgetrennt, auf Nitrocellulose-Membranen geblottet und sodann mit Antikörpern aus autologem Plasma sowie Antikörpern aus dem Überstand von in vitro kultiviertem autologem Biopsiematerial detektiert. Zur Bestimmung einer Helicobacter-Infektion wurden andere invasive und nicht invasive Tests genutzt. In einer prospektiven Untersuchung wurden über 200 konsekutive Patienten mit gastrointestinalen Beschwerden und unbekanntem H. pylori-Status, die für eine Gastroskopie vorgesehen waren, routinemäßig untersucht. Bei jeder Gastroskopie wurden zwei Antrum- und zwei Korpusbiopsien zur Gastritis-Diagnostik und zur Bestimmung des H. pylori-Status entnommen. Es wurden Assoziationen zwischen einer Infektion mit Helicobacter pylori und Erkrankungen wie akute und chronische Gastritis, gastraler und duodenaler Ulkus, Magenkarzinom und Folgeerkrankungen der Gastritis überprüft. Dabei wurden auch die eindeutig Helicobacter pylori-negativen Seren mit den positiven Seren verglichen. Trotz ungleichmäßiger Verteilung der Patientenzahlen über die einzelnen Krankheitsgruppen (Gastritis, Ulkus, Karzinom) wurden bestimmte Proteine nur bei einer der Erkrankungen erkannt. Einige Proteinspots kamen deutlich intensiver bei einer einzigen Krankheitsgruppe vor. Anzustreben sind Studien mit größeren Patientenzahlen innerhalb der einzelnen Krankheitsgruppen, um mögliche weitere Assoziationen bestimmter Helicobacter-Antigene mit Folgeerkrankungen zu analysieren und zu verifizieren. Ferner wurde das Vorliegen einer Assoziation des zweidimensionalen Immunoblots mit anderen invasiven und nicht invasiven Nachweisverfahren der H. pylori-Infektion analysiert. Dabei wurde das Antigenprofil des H. pylori, sowohl qualitativ als auch quantitativ, berücksichtigt. Durch die Charakterisierung und Identifizierung einer bedeutenden Anzahl von Helicobacter-Proteinen erhöht sich die Wahrscheinlichkeit für ein zukünftig beschleunigtes Screening in Richtung protektiver Vakzine-Kandidaten. / The present study is concerned with practicable methods of detecting Helicobacter pylori and comparing them. As a precondition, a precise knowledge of the proteins of Helicobacter is necessary. To this end the humoral immune response of Helicobacter pylori was analysed by using the method of two-dimensional immuno blots. First, the proteins of each autologous Helicobacter pylori strain were separated by two-dimensional electrophoresis, then blotted onto nitrocellulose membranes and eventually detected by using antibodies from autologous plasma and antibodies taken from the overflow of in vitro cultivated autologous biopsy material. For determining a Helicobacter infection various invasive and non-invasive tests were carried out. In a prospective study on more than 200 patients with gastrointestianal disorders but unknown H. pylori status were consecutively tested. At each gastroscopy two bioptic specimen each were taken from the antrum and from the corpus region in order to determine the H. pylori status. Associations were assessed between Helicobacter pylori infections and manifestations such as acute or chronic gastritis, gastric or duodenal ulcers, gastric carcinoma and gastritis induced disorders. In the process, clearly Helicobacter pylori negative sera were also compared with positive sera. Inspite of the unequal distribution of numbers of patients over different groups of disorders (gastritis, ulcers, carcinoma) certain proteins were only detected in connection with one group of disorder. Several of the protein spots only occurred in a single group of disorders. More studies will be necessary using greater numbers of patients within each group of diseases in order to analyse and verify associations between Helicobacter antigenes and other disorders. Further, evidences of an association between two-dimensional immunoblots and other invasive and non-invasive methods of assessing H. pylori were analysed with regard to respective antigene profiles, qualitatively as well as quantitatively. Based upon the presented characterizations and identifications of an unusually great number of Helicobacter proteins the probability is thus increased considerably with regard to improved screening methods towards protective vaccine candidates.

Orientador: Antonio Carlos Cicogna / Resumo: A obesidade é uma doença metabólica complexa considerada uma pandemia global e associada à alta incidência de doença cardiovascular. O excesso de tecido adiposo pode promover mal adaptação que resulta em alterações na estrutura e função do coração; no entanto, os mecanismos não estão totalmente elucidados. A proteômica pode fornecer uma compreensão mais profunda do processo fisiopatológico e contribuir para a identificação de novos potenciais alvos terapêuticos. Portanto, o objetivo deste estudo foi avaliar a expressão proteica miocárdica em ratos saudáveis e obesos por dieta Ocidental, empregando duas abordagens proteômicas, para melhor compreender a rede de mecanismos inerentes à disfunção cardíaca na obesidade. Ratos Wistar foram distribuídos em dois grupos: controle (C, n = 13; dieta controle) e obeso (Ob, n = 13; dieta Ocidental) alimentados por 41 semanas. A obesidade foi determinada pelo índice de adiposidade. A função cardíaca foi avaliada pelo ecocardiograma e análise do músculo papilar isolado. A proteômica foi baseada em eletroforese em gel bidimensional (2-DE) juntamente com espectrometria de massa (LC-MS/MS) e cromatografia-líquida em nanofluxo com espectrometria de massa em tandem (nanoLC-MS/MS) seguida de quantificação label-free. Ratos obesos apresentaram aumento do índice de adiposidade e disfunção cardíaca sistólica e diastólica comparados aos controles. Um total de 82 proteínas miocárdicas foram identificadas como diferencialmente expressas entre os grupos ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Obesity is a complex metabolic disease considered a global pandemic and associated with high incidence of cardiovascular disease. The excess of adipose tissue may promotes maladaptation that result in alterations in structure and function of the heart; however, the mechanisms are not fully elucidated. Proteomics may provide a deeper understanding into the pathophysiological process and contribute to the identification of new potential therapeutic targets. Thus, the aim of this was evaluate the myocardial protein expression in healthy and obese rats, employing two proteomic approaches to better comprehend the network of mechanisms inherent to cardiac dysfunction in obesity. Male Wistar rats were distributed into two groups: control (C, n=13; standard diet) and obese (Ob, n=13; Western diet) fed for 41 weeks. The obesity was determined by adipose index. Cardiac function was evaluated by echocardiogram and isolated papillary muscle analysis. The proteomics was based on two-dimensional gel electrophoresis (2-DE) along with mass spectrometry identification (LC-MS/MS) and nano-liquid chromatography with tandem mass spectrometry (nanoLC-MS/MS) followed by label-free quantification. Obese rats showed increased adiposity index and systolic and diastolic cardiac dysfunction. A total of 82 myocardial proteins was identified as differentially expressed between C and Ob groups using two proteomic strategies, being 43 up- and 39 down-regulated by obesity. These proteins are involved in imp... (Complete abstract click electronic access below) / Doutor

Today, assessment of the health of coastal waters is recognized as being important for both the conservation of nature and well-being of humans. Anthropogenic pollution has been the focus of extensive research for some time and a variety of programs for the monitoring and assessment of environmental pollution have been developed. Determination of the levels of pollution in sensitive ‘sentinels’ such as mussels, allows monitoring of these levels in a given area over a prolonged period of time. Furthermore, the biological effects of pollution are reflected in a series of biomarkers, none of which provides a general picture of the sentinel’s state of health and all of which are individually specific for certain pollutants and influenced by both biotic and abiotic factors. In an attempt to improve biomonitoring of marine pollution, we have developed two proteomic approaches here. In the first portion of the thesis, a proteomic analysis was performed on peroxisomes isolated from mussels exposed either to one of three model anthropogenic pollutants, or two different types of crude oil, or from mussels exposed to the Prestige oil spill. Application of two-dimensional electrophoresis (2-DE) provided protein expression signatures (PES) for exposure to these different pollutants.Furthermore, several individual protein components of these PES could be putatively identified. In the second portion of this work, such analysis of subproteomes was developed further in order to improve the applicability of this approach to biomonitoring. A simple fractionation procedure in combination with liquid chromatography and 2-DE provided samples from mussels residing in different regions of a pollution gradient around the harbor of Gothenburg, as well as from mussels exposed to two types of fuel oil similar to that of the Prestige that were suitable for environmental proteomics. In addition, we constructed a model for this approach that can be cross-validated in the future and applied to assess sources of fuel oil pollution in connection with biomonitoring programs.

L'adenocarcinoma pancreàtic és una neoplàsia amb mal pronòstic per la que no existeixen marcadors específics. En aquesta tesi s'han estudiat possibles alteracions de les estructures glucídiques de la ribonucleasa pancreàtica humana (RNasa 1) de sèrum amb l'objectiu de determinar el seu ús diagnòstic. S'han descrit les estructures glucídiques de la RNasa 1 sèrica i de línies cel·lulars endotelials, i s'ha observat un increment en la proporció d'estructures biantenàries amb Fc en la RNasa 1 sèrica de pacients amb càncer de pàncreas, fet que podria ser d'utilitat diagnòstica. També, donada la gran similitud entre les estructures glucídiques descrites per la RNasa 1 sèrica i per l'endotelial, l'origen de la RNasa 1 sèrica sembla ser principalment endotelial. La RNasa 1 també s'ha avaluat per electroforesi bidimensional i s'ha establert una correlació entre el contingut d'àcid siàlic i el seu pI, fet que pot ajudar a la interpretació dels mapes bidimensionals d'altres glicoproteïnes. / Pancreatic adenocarcinoma has one of the highest mortality rates of all neoplasias and it has no specific markers. In this work, possible alterations in the glycan structures of human pancreatic ribonuclease (RNase 1) present in serum are investigated with the aim of determining its diagnostic utility. Serum and endothelial RNase 1 glycan structures have been described and an increase of biantennary structures with Fc in serum RNasen 1 from pancreatic cancer patients could be observed, what can be of diagnosis utility. Also, due to the high similarity between serum RNase 1 and endothelial RNase 1 glycan structures, it was concluded that endothelium can be the main producer of serum RNase 1. RNase 1 was also evaluated by two-dimensional electrophoresis and a correlation between the sialic acid content and the observed pI decrease could be establish, what may be very significant for understanding and interpreting two-dimensional maps from other glycoproteins.