Studies in animals and humans link both physical and psychological stress with an increased rate and severity of infections and onset of diseases. Stress is a very broad and complex topic. It can be defined as a condition occurring in response to adverse external influences capable of affecting physical health which leads to activation of a stress response in the body. There are two prominent stress responsive systems: the hypothalamic-pituitary-adrenal axis and the sympathetic adrenomedullary axis. These systems are responsible for the majority of the changes in the body, which occur in response to stress. Stress has been linked to many detrimental effects in cattle including immune suppression, increased disease susceptibility and decreased reproduction. These cause huge economic losses to the cattle industry every year. Weaning has been identified as one of the main stressors implicated in these negative effects. For this reason it is important to be able to identify animals stressed by weaning and do so using samples which are easily obtainable and useful for future diagnostic purposes. We hypothesize that weaning will cause sufficient stress in cattle to alter protein profiles in serum, which can be used to identify this type of stress. To do this we employed proteomic methodologies including two-dimensional gel electrophoresis and mass spectrometry to compare an abrupt weaned group of calves to a never weaned group and a previously weaned group (preconditioned). We have included a preconditioned group to examine the differences between this group and animals which have never been weaned. Preconditioned animals are typically used as a control group in weaning studies. A total of 83 distinct protein bands were identified after image analysis. Out of 83 protein bands, we found 9 spots which were significantly different in abundance among the treatment groups. Two out of 9 spots were significantly different between the abrupt weaned and the never weaned groups. Five protein bands were also found to be significantly different between the abrupt weaned group and the preconditioned group. Five protein bands were found to be significantly different between the never weaned group and the preconditioned group. Identification of these proteins, however, had limited success since the bovine protein database is not as extensive as that for humans or mice. Among the proteins identified were alpha-1-acid glycoprotein and collagen precursor. The differences in intensities found between the abrupt weaned group and the never weaned group may be useful as markers of calves going through weaning stress. We have also seen that animals who have undergone weaning and through the stress associated with that event are not exactly the same as animals which have never been weaned. This has implications to research where a preconditioned group is used as a control rather than a never weaned group. Despite the limitations of the methodology used for the current system, the overall results revealed specific changes in serum proteins which were associated with abrupt weaned animals. Future studies can be planned to determine the specificity of these protein changes and possibly identify the molecular basis of stress dependent disease susceptibility.

Studies in animals and humans link both physical and psychological stress with an increased rate and severity of infections and onset of diseases. Stress is a very broad and complex topic. It can be defined as a condition occurring in response to adverse external influences capable of affecting physical health which leads to activation of a stress response in the body. There are two prominent stress responsive systems: the hypothalamic-pituitary-adrenal axis and the sympathetic adrenomedullary axis. These systems are responsible for the majority of the changes in the body, which occur in response to stress. Stress has been linked to many detrimental effects in cattle including immune suppression, increased disease susceptibility and decreased reproduction. These cause huge economic losses to the cattle industry every year. Weaning has been identified as one of the main stressors implicated in these negative effects. For this reason it is important to be able to identify animals stressed by weaning and do so using samples which are easily obtainable and useful for future diagnostic purposes. We hypothesize that weaning will cause sufficient stress in cattle to alter protein profiles in serum, which can be used to identify this type of stress. To do this we employed proteomic methodologies including two-dimensional gel electrophoresis and mass spectrometry to compare an abrupt weaned group of calves to a never weaned group and a previously weaned group (preconditioned). We have included a preconditioned group to examine the differences between this group and animals which have never been weaned. Preconditioned animals are typically used as a control group in weaning studies. A total of 83 distinct protein bands were identified after image analysis. Out of 83 protein bands, we found 9 spots which were significantly different in abundance among the treatment groups. Two out of 9 spots were significantly different between the abrupt weaned and the never weaned groups. Five protein bands were also found to be significantly different between the abrupt weaned group and the preconditioned group. Five protein bands were found to be significantly different between the never weaned group and the preconditioned group. Identification of these proteins, however, had limited success since the bovine protein database is not as extensive as that for humans or mice. Among the proteins identified were alpha-1-acid glycoprotein and collagen precursor. The differences in intensities found between the abrupt weaned group and the never weaned group may be useful as markers of calves going through weaning stress. We have also seen that animals who have undergone weaning and through the stress associated with that event are not exactly the same as animals which have never been weaned. This has implications to research where a preconditioned group is used as a control rather than a never weaned group. Despite the limitations of the methodology used for the current system, the overall results revealed specific changes in serum proteins which were associated with abrupt weaned animals. Future studies can be planned to determine the specificity of these protein changes and possibly identify the molecular basis of stress dependent disease susceptibility.

Discovery of Novel Serum Biomarkers for Diagnosing and Staging Alzheimer’s DiseaseDipti Jigar ShahDepartment of Chemistry and Biochemistry, BYUDoctor of PhilosophyAlzheimer’s disease (AD) is an untreatable neurologic disease affecting more than 5 million Americans, most over 60 years of age. Protein plaques and neurofibrillary tangles typify AD brain pathology and are thought to cause the progressive dementia and brain shrinkage observed in AD. Currently there are no methods to diagnose the disease at a time before damage becomes irreversible.Biochemical tests for AD using cerebrospinal fluid analysis or neuroimaging are not yet sufficiently sensitive and specific, and they are invasive. This points to a need for a more easily applied and more sensitive diagnostic test. Although the gross anatomical changes are localized to the brain, AD is likely to involve changes throughout the body. As a result of this, changes in the abundance of certain biomolecules present in the circulation system are likely to occur. Consequently, a serum proteomics approach able to measure such changes, when applied to AD, would likely find quantitative changes in relevant molecules that can help diagnose the disease correctly, ideally early in the disease process. The goal of this work was to discover and validate novel diagnostic serum biomarkers for AD. For biomarker discovery and validation, we used a novel serum proteomics approach involving reversed phase capillary-liquid chromatography-electrospray ionization-quadrupole-time of flight mass spectrometry. Our samples were protein depleted, which helped us survey low molecular weight species in the serum without ion suppression from larger proteins like albumin. We were able to observe more than 8000 molecular species in a single run. The overall project was comprised of four studies: (i) discovery of novel potential serum AD markers, (ii) blinded validation of diagnostically promising biomarkers found in the initial study, with their further chemical identification, (iii) exploring gender-based serum AD biomarkers, and (v) discovery of biomarkers that distinguish early versus moderate stage AD. In the first study, the approach found 38 significant (p < 0.05) biomarkers and 21 near significant (p = 0.05 to 0.099) biomarkers. On using the forward selection approach, we built multi-marker panels with specificities and sensitivities higher than 80%.The second study reports on a blinded validation study that was performed on a new set of serum samples. We focused on the 13 most promising AD biomarkers found as part of the initial study. We successfully validated 4 of these biomarkers that showed highly significant statistical p-values. As part of this study, research was conducted to identify these 4 biomarkers, which was accomplished using tandem mass spectrometry with fragmentation experiments. The third study used data from the initial study but looked at gender specific biomarkers. We found 31 significant and near significant serum AD biomarkers for women, 16 for men, and 25 that were gender independent. Multi-marker panels of AD biomarkers for women or men had sensitivities of >60% and specificities >85%.In the fourth study, cases with moderate AD were compared to cases with very mild or mild AD to find novel biomarkers that could be used for staging. We found 44 significant and near significant biomarkers that were quantitatively different between mild and severe AD. In conclusion, we were successful in accomplishing the goal of this work of finding, validating and identifying novel serum biomarkers that diagnose AD.

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