A hipertensão arterial é uma condição clínica grave acompanhada por alterações estruturais do aparelho cardiovascular. Os antagonistas dos receptores 1-adrenérgicos são drogas usadas na terapêutica anti-hipertensiva. O nebivolol é um antagonista seletivo dos receptores 1 de terceira geração, que estimula a liberação endotelial de NO e diminui a ativação da -nicotinamida adenosina dinucleotído fosfato (NAD(P)H) oxidase. O metoprolol é um antagonista seletivo dos receptores 1de segunda geração, que não apresenta efeitos vasodilatadores. As metaloproteinases da matriz (MMPs), principalmente a MMP-2, são enzimas que participam ativamente do processo de remodelamento vascular. Elas passam de seu estado latente para seu estado ativo pela ação de proteases e espécies reativas de oxigênio (EROs). Como na hipertensão há aumento de EROs, de MMPs e remodelamento vascular, é possível que o nebivolol impeça o aumento dos níveis de MMPs vasculares e o remodelamento vascular hipertrófico associados à hipertensão, além do seu efeito de antagonismo do receptor 1. Primeiro, realizamos uma avaliação das alterações aórticas da hipertensão 2-rins,1-clipe (2R-1C), pois há poucas informações sobre essas alterações durante o desenvolvimento da hipertensão. Para isso, ratos hipertensos e controles foram estudados com 2, 4, 6 e 10 semanas após a indução da hipertensão. A pressão arterial sistólica foi monitorada semanalmente. As alterações na parede aórtica foram estudadas em hematoxilina/eosina (H&E), picrosirius e orceína. Foram avaliados também a atividade da NAD(P)H oxidase, a produção de ânion superóxido, a atividade gelatinolítica por zimografia in situ; os níveis e localização de MMP-2, MMP-14 e TIMP-2 por imunofluorescência zimografia e imunohistoquímica. No segundo protocolo, após 6 semanas de hipertensão foi iniciado o tratamento com metoprolol (Meto -20/mg/kg/dia) ou nebivolol (Nebi -10mg/kg/dia), realizado durante 4 semanas. Foram avaliados a pressão arterial sistólica, as alterações na parede aórtica por H&E, a atividade da NAD(P)H oxidase, produção de ânion superóxido, a atividade gelatinolítica, os níveis e localização de MMP-2 por imunofluorescência e zimografia em gel, e os níveis de nitrotirosina por imunohistoquímica. Observamos no protocolo temporal que a pressão foi gradualmente aumentada nos animais hipertensos quando comparado aos controles. Houve hiperplasia e hipertrofia da aorta, com aumento da deposição de colágeno e elastina. Observamos aumento nos níveis de estresse oxidativo, MMPs e atividade gelatinolítica em todas as semanas de estudo. Ao final do tratamento com metoprolol e nebivolol, observamos que a pressão diminuiu nos animais hipertensos, e que essa redução da pressão ocorreu de modo semelhante com os diferentes tratamentos. Os animais hipertensos apresentaram hipertrofia da aorta, aumento nos níveis de EROs, de MMP-2 e da atividade gelatinolítica. Essas alterações foram revertidas apenas pelo tratamento com nebivolol, mas não pelo metoprolol. Assim, o remodelamento da hipertensão 2R-1C parece estar estabelecido com duas semanas de hipertensão arterial, com elevados níveis de espécies reativas de oxigênio e MMPs, e o tratamento com nebivolol, mas não o metoprolol, atenuou o estresse oxidativo e o remodelamento vascular associado à hipertensão. / Hypertension is a serious clinical condition with structural changes in the cardiovascular system. Beta-adrenoreceptor antagonists are used in hypertension therapy. Nebivolol is a third-generation selective 1-adrenoreceptor antagonist that stimulates endothelial cell NO production and prevents vascular NAD(P)H oxidase activation. Metoprolol is a second-generation selective 1-adrenoreceptor antagonist, without vasodilatory effect. The matrix metalloproteinases (MMPs), especially MMP-2, participate actively in the vascular remodeling process, and are activated by reactive oxygen species (ROS). There are increases in ROS, of MMPs, and vascular remodeling in hypertension. Because of these, it´s possible that nebivolol prevent MMP increases and vascular remodeling associated with hypertension beyond its 1-receptorblocking properties. First, structural aortic changes in the development of two-kidney, one-clip hypertension (2K-1C) were evaluated. Sham or 2K1C hypertensive rats were studied after 2, 4, 6, and 10 weeks of hypertension. Systolic blood pressure (SBP) was monitored weekly. Morphometry of structural changes in the aortic wall was studied in hematoxylin/eosin, orcein and picrosirius red sections. Aortic NAD(P)H activity and superoxide production was evaluated. Aortic gelatinolytic activity was determined by in situ zymography, and MMP-2, MMP-14, and tissue inhibitor of MMPs (TIMP)-2 levels were determined by gelatin zymography, immunofluorescence and immunohistochemistry. Six weeks after surgery, hypertensive and sham rats were treated with metoprolol (20 mg/kg/ day) or nebivolol (10 mg/kg/day), for four weeks, in the second-protocol. Systolic blood pressure (SBP) was monitored weekly Aortic structural were studied in hematoxylin/eosin sections. NAD(P)H oxidase activity and ROS and nitrotyrosine production were evaluated. MMPs levels and activity were determined by zymography and in situ zymography. In the temporal study, 2K-1C hypertension was associated with increased aortic collagen and elastin content in the early phase of hypertension, which was associated with vascular hypertrophy, increased vascular MMPs levels, and increased gelatinolytic activity, possibly as a result of increased vascular NAD(P)H oxidase activity and oxidative stress. In the second-protocol, similar reductions in SBP were found with both metoprolol and nebivolol treatments. However, only Nebi reversed aortic hypertrophy, the increases in aortic NAD(P)H oxidase activity, in aortic ROS levels, in nitrotyrosine staining, in aortic MMP-2 levels and in aortic MMP activity. These results indicate that vascular remodeling of renovascular hypertension is an early process associated with increases in MMPs activities, enhanced matrix deposition and oxidative stress, and that treatment with Nebi attenuate the oxidative stress and the vascular remodeling associated with 2K-1C hypertension.

DNA damage from both normal metabolic activities and environmental factors such as UV and radiation can cause as many as 1 million individual lesions to the DNA per cell per day (Lodish et al 2004). Cells respond to this continuous damage by employing many, highly efficient DNA repair mechanisms and undergo apoptosis when normal DNA repair fails. Of the many types of DNA damage that can occur, double strand breaks (DSBs) are the most toxic (Featherstone & Jackson 1999). A single unrepaired DSB is enough to induce cellular apoptosis and several mechanisms have developed to repair DSBs. The recognition, signalling and repair of DSBs involve large multi-­‐subunit complexes that bind to both the DNA and modified histone tails, which require modification of the chromatin in order to access their bind sites and function effectively (Allard et al 2004). Consequently several chromatin-­‐remodelling proteins have been implicated in DSB repair (van Attikum et al 2004, Chai et al 2005). LSH (Lymphoid specific helicase) is a putative chromatin-­‐remodelling enzyme that interacts with DNA methyltransferases and has been connected to DNA methylation (Myant & Stancheva, 2008). Knockouts of LSH or its homologues in A. thaliana and M. musculus show a reduction in DNA methylation of 60-­‐70% (Jeddeloh et al 1999, Dennis et al 2001). However in addition to this phenotype, knockout A. thaliana also have an increased sensitivity to DNA damage (Shaked et al 2006). A homologue of LSH has also been identified in S. cerevisiae, which interacts with known repair proteins (Collins et al 2007) and may be involved in DSB repair. Although the majority of Lsh-­‐/-­‐ mice die shortly after birth, 40% of the line produced by Sun et al survive and show unexplained premature aging (Sun et al 2004). As premature aging is a hallmark of increased acquisition of DNA damage there is the possibility of a conserved role for LSH in mammalian DNA damage repair. Here I show that LSH depleted mammalian cells have an increased sensitivity specifically to DSB inducing agents and show increased levels of apoptosis. Further analysis shows that cells lacking LSH repair DSBs slower, indicating a novel role for LSH in mammalian repair of DSB. I performed an in depth analysis of the DSB defects in LSH depleted cells in an attempt to elucidate the function of LSH in DSB repair. I found that LSH depleted cells can correctly recognise DSBs but recruit downstream signalling and repair factors, such as γH2AX, less efficiently. I show that reduced recruitment of downstream DSB repair factors is not accompanied by extended cell cycle checkpoint signalling. This suggests that LSH depleted cells continue through the mitosis with unrepaired DSBs, which most likely leads to apoptosis and the increased sensitivity to DSB inducing agents. These experiments also showed that recruitment of DSB signalling and repair factors is not impaired equally at all breaks, and I present a model system created to quantitatively compare individually breaks between WT and LSH depleted cells to identify DSB that require LSH for efficient repair. I also preformed an analysis of Lsh-­/-­ MEFs containing WT or catalytic null mutant LSH rescue constructs and I show that WT but not catalytic null LSH can restore efficient DSB repair. These studies identify a novel role for LSH in mammalian DSB repair and demonstrate the importance of its catalytic activity.

Orientador: Jorge Kennety Silva Formiga / Coorientadora: Vivian Silveira dos Santos Silva Bardini / Banca : João Maurício Ferraz da Silva / Banca: Denilson Paulo Souza dos Santos / Resumo: Os modelos matemáticos utilizados atualmente para análise variação da densidade óssea quando submetido a estímulos mecânicos, consideram apenas carregamentos estáticos ou a variação dentro de um curto espaço de tempo e permanecendo estático novamente. Esse artigo tem o objetivo de desenvolver dois novos modelos que permitam simular o comportamento do tecido ósseo de remodelagem e determinar as faixas de subcarga e sobrecarga onde ocorrem a reabsorção óssea, quando submetido a estímulos mecânicos variados oscilando no tempo. Tradicionalmente os estudos realizados para determinação da variação da densidade óssea, vem utilizando um modelo adotando uma equação diferencial ordinária (EDO) para analisar essa variação. A partir do modelo dessa EDO, foram desenvolvidas duas novas equações matemáticas para simular o comportamento do tecido ósseo quando submetido à estímulos mecânicos variados no tempo. A primeira equação utiliza uma variação da tensão seguindo o padrão de oscilação de uma onda senoidal e a segunda equação utiliza um padrão de onda resultante da combinação linear entre seno e cosseno. A resolução dessa EDO foi feita utilizando o método de Runge-Kutta de 5ª ordem, um integrador de maior precisão e permitindo uma melhor análise do comportamento do tecido ósseo através dos resultados obtidos com maior precisão para diversos níveis de tensão. A análise dos resultados obtidos através das simulações matemáticas empregando três tipos de carregamentos, estático, variável de ac... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract : The mathematical models currently used for bone density variation analysis when subjected to mechanical stimuli, consider only static loading or variation within a short time and remaining static again. This article aims to develop two new models that allow simulating the behavior of bone remodeling tissue and determine the underload and overload ranges where bone resorption occurs when subjected to varying mechanical stimuli oscillating over time. Traditionally, studies performed to determine bone density variation have been using a model using an ordinary differential equation (ODE) to analyze this variation. From the model of this ODE, two new mathematical equations were developed to simulate the behavior of bone tissue when subjected to varying mechanical stimuli over time. The first equation uses a voltage variation following the sine wave oscillation pattern and the second equation uses a wave pattern resulting from the linear combination of sine and cosine. The resolution of this ODE was performed using the 5th order Runge-Kutta method, a more accurate integrator and allowing a better analysis of bone tissue behavior through the results obtained with greater precision for various stress levels. The analysis of the results obtained through mathematical simulations employing three types of loads, static, variable according to the oscillation of a sine wave and variable through the oscillation of a sine-cosine wave, demonstrated that the behavior of bone tissue in relation to the stimuli Mechanics vary by type of loading. The stress levels applied for the three simulations were the same, but the bone remodeling behavior response was different for each type of loading. The resorption tension range, remodeling tension range and overload range change according to the type of loading applied, demonstrating that bone tissue behavior may ....(Complete abstract click electronic access below) / Mestre

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Osteoporosis affects the human skeleton through the direct effects of the disease on the function and structure of bone. Individuals who are affected by osteoporosis may be subject to serious fractures and it is estimated that annually approximately 1.5 million fractures can be attributed to this disease [1]. The disease is categorized as the direct side effect of increased bone porosity and bone loss and is directly linked to estrogen deprivation [2]. Animal models are often used to make initial conclusions about the effects of the disease or pharmacological treatments. In this study, sheep were chosen as a representative animal model due to their similar metabolic characteristics to that of a human. Like most animals, the ovine does not undergo a natural menopause and an ovariectomy was necessary to replicate the condition. The study objective was to quantify compact bone density present in ovine at three months post ovariectomy. The study included 112 ovine separated into different treatment groups. The treatment groups were separated into 4 groups of 28 based on season of surgery: autumn, winter, spring, and summer. Each seasonal group was further divided into 2 groups of 14; the first group underwent an ovariectomy; and the second group underwent a sham surgery, in which the ovaries were visualized and handled but left in the abdomen. One group was sacrificed 3 months post operatively and the other group was sacrificed at 12 months post operatively. This study specifically looks at ewe sacrificed at 3 months. The radius from each sheep was cut into the anatomical sectors: cranial, caudal, craniolateral, craniomedial, caudomedial, and caudolateral. Each anatomical sector was turned into a microradiograph for analysis. Densitometry was performed to determine the density of each specimen using the estimated thickness of aluminum (ETA) as the key. Statistical analysis assessed the resulting data to understand the effects of treatment, season of sacrifice, season of surgery, and anatomical sector by comparing both mean ETA and standard deviation ETA to understand changes in bone density. The results revealed significant differences between the ovariectomy and sham groups as well as variation within season of surgery and season of sacrifice in both groups. Anatomical sector showed no significant variation. The differences in the thickness of aluminum seen in the sheep that underwent a sham operation can be attributed to the presence of estrogen. The sheep that underwent an ovariectomy showed differences in the estimated thickness of aluminum that can be attributed to other seasonal characteristics including the influence of Vitamin D. The results and conclusions within this study can be used to influence bone material characteristics and bone loss test protocols in future osteoporosis and estrogen depletion studies.

Dauer larva formation in C. elegans is a life-history polyphenism that relies on the function of several pathways, including insulin, TGFβ and nuclear hormone receptor signaling. The downstream effectors of these pathways, DAF-16/FOXO, DAF-3/Co-Smad and DAF-12/VDR, are transcription factors (DAF TFs) with broad or ubiquitous expression patterns, null mutations in which result in the inability to form dauers regardless of environmental conditions. In preparation for the dauer diapause, all tissues of the worm undergo extensive morphological and functional remodeling in a coordinated manner. The broad goal of my thesis is to understand how these transcription factors act in different tissues of the worm to regulate the dauer-specific tissue remodeling and gene expression changes. In addition to characterizing dynamic expression pattern of chemosensory GPCR genes in dauer, which revealed an additional layer of plasticity and provided novel entry points to studying remodeling in distinct neuron classes and non- neuronal tissues, I have developed molecular tools – conditional alleles of the daf TFs – that allowed me to address the question of tissue-specificity and cell-autonomy of the DAF TFs in a previously inapproachable way. I have found that DAF TFs act in both cell-autonomous (DAF-16 in neurons, intestine, pharynx) and non-autonomous manner (DAF-16 in the pharynx) to control dauer tissue remodeling. Unlike DAF-16 and DAF-12, the function of DAF-3 in the dauer decision appears to be largely determined by its action in neurons, and specifically in sensory neurons. The three TFs also differ in their roles in pharynx remodeling: while DAF-16 controls dauer pharyngeal morphology and activity both cell-autonomously and non- autonomously, DAF-12 or DAF-3 depletion from pharyngeal muscle does not affect the dauer pharyngeal phenotypes. Yet, all three TFs are required continuously throughout all tissues to maintain the dauer state, once the decision to enter dauer has been made. This work is a first attempt to characterize tissue-specific roles of all transcriptional effectors of the dauer pathways in a systematic way, and contributes to a fundamental understanding of a polyphenic developmental switch regulated by highly conserved molecular pathways.

PURPOSE: To understand the influences of mechanical loading on bone adaptation, the ground reaction force (GRF) applied to the bone must be quantified. The use of force plates in a lab setting is the accepted method for quantifying GRFs; however, this is not feasible in free-living situations. Recent developments in accelerometer technology may provide the ability to evaluate the effects of mechanical loading on bone outside of laboratory settings. The purpose of this project was to validate an accelerometer for the measurement of mechanical loading by comparing its output against GRFs. METHODS: Male and female participants (n = 20 males, 20 females; 18 to 49 yr) completed 10 repetitions of 9 common everyday movements (stand, walk, jog, run, 15 cm jump, step down from curb, drop down from curb, forward hop, and side hop) on a force plate with an accelerometer worn on their right hip. Then, a subset (n = 5 males, 5 females) wore an accelerometer on their right hip and played basketball, volleyball, and dodgeball as a group. Finally, all 40 participants wore an accelerometer home for 7 days. All activities were organized into derived activity categories labeled as low-, moderate-, and high-mechanical-load-intensity and used with 59 possible accelerometer variables to predict mechanical load. Models were fit using the randomForest package in R. Model performance (coefficient of determination [R2] and median absolute error) was evaluated using cross-validation. RESULTS: The percentage of variation mechanical load intensity explained by the models ranged from 0.27 to 0.78 with median absolute errors ranging from 0.20 to 0.49. The model with R2 = 0.78 contained the known activity categories and the accelerometer variables, but this is not realistic for free-living situations where activity categories will not be known. The two free-living models with the highest R2 values included derived activity categories and accelerometer variables, and estimated, on average, 21.1 and 20.7 hours per day in low-intensity, 1.6 and 1.7 hours per day in moderate-intensity, and 0.0 and 0.5 hours per day in high-intensity osteogenic activity, respectively. CONCLUSION: It is assumed that higher intensity activities (i.e., jumping vs. jogging) result in higher GRF values, but depending on the actual execution of the movement, this is not always the case. This research demonstrated that models containing the accelerometer variables performed better in predicting GRF than those containing only the derived activity categories. This supports the hypothesis that accelerometers provide valuable objective information when evaluating mechanical loading on bone.

Zur Untersuchung des Einflusses des klassischen Komplementaktivierungsweges auf das Remodelling nach Myokardinfarkt wurden C1qKO-Tiere mit Wildtyp-Tieren (WT) vor und nach Myokardinfarkt echokardiographisch und hämodynamisch untersucht. Nach Myokardinfarkt erfolgten außerdem eine Infarktgrößenbestimmung sowie eine fluoreszenzmikroskopische Messung des Kollagengehaltes. Die Anzahl neutrophiler Granulozyten, Makrophagen sowie apoptotischer Zellen wurde drei Tage nach Myokardinfarkt bestimmt. In der C1qKO-Gruppe zeigten sich vor Myokardinfarkt signifikant höhere Ejektionsfraktionen im Vergleich zur WT-Gruppe. Dies ließ sich nach Myokardinfarkt nicht mehr beobachten. Hier zeigten sich keine signifikanten Unterschiede zwischen C1qKO-Gruppe und WT-Gruppe. Ebenso zeigten sich keine Unterschiede in den Parametern Mortalität, Infarktgrößen, Organgewichte, Kollagengehalt des Gewebes, Makrophagenanzahl und Neutrophilenanzahl. Allerdings zeigte sich drei Tage nach Myokardinfarkt eine deutlich geringere Anzahl apoptotischer Zellen in der C1qKO- Gruppe im Vergleich zur WT-Gruppe. Insgesamt lassen sich die beobachteten Effekte aus einer vorherigen Studie bei C3-Defizienz nicht in den C1qKO- Tieren reproduzieren. Die Komplementaktivierung nach Myokardinfarkt ist somit nicht ausschließlich auf eine Aktivität des klassischen Komplementaktivierungsweges zurückzuführen. Vielmehr scheint ein Zusammenspiel aller drei Komplementaktivierungswege sowie zusätzlich eine Komplementaktivierung durch komplementunabhängige Proteasen zum linksventrikulären Remodelling nach Myokardinfarkt beizutragen. / To investigate the influence of the classical complement activation pathway on the remodeling after myocardial infarction, C1qKO animals and wild type animals (WT) were examined echocardiographically and hemodynamically before and after myocardial infarction. Furthermore, infarct size determination and fluorescence microscopic measurements of the collagen content were performed after myocardial infarction. The number of neutrophilic granulocytes, macrophages and apoptotic cells was determined three days after myocardial infarction. Significantly higher ejection fractions were found in the C1qKO group before myocardial infarction compared to the WT group. This was no longer observed after myocardial infarction. There were no significant differences between C1qKO and WT animals. Likewise, there were no differences in the parameters mortality, infarct size, organ weight, collagen content of the tissue, number of macrophages and number of neutrophils. However, three days after myocardial infarction, there was a significantly lower number of apoptotic cells in the C1qKO group compared to the WT group. Overall, the observed effects from a previous study with C3 deficiency cannot be reproduced in the C1qKO animals. Complement activation following myocardial infarction is thus not exclusively due to activity of the classical complement activation pathway. Rather, an interaction of all three complement activation pathways as well as additional complement activation by complement-independent proteases seems to contribute to left ventricular remodeling after myocardial infarction.

Over the past five years the role of epigenetic modifiers in brain development has become increasingly evident. In this regard, Snf2l, a homolog of the chromatin remodeling protein ISWI, was shown to have enriched expression in the brain and be important for neuronal differentiation. Mice lacking functional Snf2l have hypercellularity of the cerebral cortex due to increased cell cycle re-entry. In this thesis I demonstrate the effects of Snf2l-ablation on cortical progenitor cells including increased proliferation and cell cycle deregulation, the consequence of which is a delay in neuronal migration and altered numbers of mature cortical neurons. This phenotype arises from increased expression of Foxg1, a winged-helix repressor expressed in the forebrain and anterior optic vesicle. Moreover, genetically reducing its overexpression rescues the Snf2l-ablated phenotype. Snf2l is bound directly to a promoter region of Foxg1 suggesting that it acts as a repressive regulator in vivo and is an important factor in forebrain differentiation.

In vertebrates, the embryonic hindbrain is transiently subdivided along its anterior-posterior (A-P) axis into 8 well defined segments termed rhombomeres (r1-8). Each rhombomere represents a true cellular compartment in transcriptional profile, lineage restriction and neuronal organization. Thus, the vertebrate hindbrain provides a beautiful model for studying mechanisms of anterior-posterior patterning, signal transduction and interpretation, initiation and maintenance of transcriptional profiles, cell sorting and border formation. The Kreisler/MafB gene, which encodes a basic leucine zipper (bZIP) transcription factor that regulates some Hox genes, is one of the first genes to be expressed segmentally in the hindbrain, and is subject to a dynamic and complex regulatory process. However, unlike the Hox genes, Kreisler/MafB is not located within a large cluster of genes and therefore provides a simple system for dissecting the molecular mechanisms involved in hindbrain compartmentalization. In dissecting the mechanisms that govern Kreisler/MafB regulation, we have identified the S5 regulatory element that directs early MafB expression in the future r5-r6 domain. We have found a binding site within S5 that is specific for the Variant Hepatocyte Nuclear Factor 1 (vHNF1) to be essential, but not sufficient for early induction of r5-r6-specific expression. Thus, early inductive events that initiate MafB expression are clearly distinct from later acting ones that modulate its expression levels. Using mouse mutants, we have shown that MafB is dependent on the M33 polycomb protein and other mechanisms of chromatin remodeling. We then utilized transgenic flies and mice as well as binding assays to identify and validate a PcG/trxG response element (PRE), PRE1 which acts to reorganize the surrounding chromatin, regulating S5-dependent expression. To our knowledge, PRE1 is the first validated vertebrate PcG/trxG response element. Thus, PRE1 provides a springboard for further exploration of the mechanisms governing chromatin remodeling.