The pathophysiology of atherosclerotic cardiovascular disease (CVD) is highlighted by vascular dysfunction and low-grade vascular inflammation. Furthermore, the site-specific distribution of atherosclerosis throughout the arterial vasculature is primarily determined by local hemodynamic force. Therefore, this dissertation outlines three experiments designed to investigate the role of acute mental and physical (i.e., aerobic exercise), and vascular wall shear stress (SS) on the inflammatory aspects of atherosclerosis. Chapter 2 examines the effect of acute laboratory-induced mental stress on intracellular pro-inflammatory signaling pathways in peripheral blood mononuclear cells. Chapter 3 investigates the impact of acute laboratory-induced mental stress and maximal aerobic exercise on the concentration of soluble VCAM-1 (sVCAM-1) and CX3CL1/fractalkine (sCX3CL1) in human serum. Lastly, Chapter 4 examines the role of short- (30 min) and long-term (24 hr) low-to-negative oscillating SS (LOSS) and high laminar SS (HLSS) on the expression and secretion (i.e., cleavage) of cell-membrane VCAM-1 and CX3CL1 by human umbilical vein endothelial cell cultures in vitro. Together, these experiments provide evidence that acute psychological stress, maximal aerobic exercise, and HLSS influence vascular inflammation and adhesive properties of the vessel wall. More specifically, the results from Chapter 2 provide evidence that acute mental stress promotes the immune-cell mediated synthesis of pro-inflammatory cytokines in circulation. In addition, Chapter 3 and Chapter 4 demonstrate that the elevations in blood flow and hemodynamic force associated with maximal aerobic exercise, and unidirectional high SS may have the capacity to alter the expression of endothelial-bound cellular adhesion molecules, in part by eliciting their release from the vessel wall.

The unicellular cyanobacterium Prochlorococcus is the numerically dominant photosynthetic organism throughout the temperate and tropical open oceans, but it is difficult to grow in pure cultures. We developed a system for rendering spontaneous streptomycin-resistant mutants of Prochlorococcus axenic by diluting them to extinction in the presence of “helper” heterotrophic bacteria, allowing them to grow to high cell concentrations, and then killing the helpers with streptomycin. Using axenic strains obtained in this fashion, we demonstrated that Prochlorococcus experiences a number of growth defects in dilute axenic culture, including reduced growth rate, inability to form colonies on solid media, and higher incidence of mortality (i.e., catastrophic failure of liquid cultures). These defects were eliminated when Prochlorococcus was grown in co-culture with a phylogenetically diverse array of helper bacteria. The primary mechanism of helping was enzymatic removal of hydrogen peroxide (HOOH) from the culture medium. Axenic Prochlorococcus cultures were profoundly sensitive to HOOH additions in comparison with reported tolerance levels for all other wild-type aerobic bacteria, but in co-culture their resistance was similar to that of the helpers. Neither is dependence on helpers limited to the laboratory. Sterile-filtered seawater exposed to sunlight accumulated enough HOOH in 24h to kill ecologically relevant cell concentrations of Prochlorococcus. We also refined a method for delivering HOOH at a defined, steady rate using the buffer HEPES to more accurately simulate the steady accumulation of HOOH in natural waters. Even at the lowest production rates that could sustain the in situ HOOH concentration in the ocean, HEPES-generated HOOH was lethal to Prochlorococcus; again, co-culture with helpers prevented this effect. We speculate on the ecological consequences of Prochlorococcus’ dependency on other organisms for survival, as well as the evolutionary forces that have led to this lack of self-sufficiency.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease leading to decreased lung volume and eventual respiratory failure. At present, the median post-diagnosis lifespan is between three and six years. Myofibroblasts are collagen-secreting cells essential for wound healing, but also implicated in the fibroproliferation and extra cellular matrix deposition commonly seen in IPF. The nitric oxide (NO) signaling pathway is implicated in protomyofibroblast to myofibroblast transition and regulation. Previous work has shown that in pulmonary myofibroblasts, endothelial nitric oxide synthase (eNOS) is the primary NOS isoform expressed. The current study used cultured rat pulmonary myofibroblasts between passages two and five as a cell model. The cells were grown in normal growth media (DMEM + 10% FBS) or serum starved (DMEM + 0% FBS) to induce cellular differentiation. In this study, immunocytochemistry was used to show localization of eNOS is dependent on cellular differentiation, with protomyofibroblasts expressing eNOS primarily in the nucleus and protomyofibroblasts expressing eNOS in the perinuclear region. We also show catalytic activity and localization of eNOS are correlated by visualizing nitric oxide production in the cells using a permeable fluorescein chromophore. By using western blot analysis on fractionated cell lysates we found eNOS expressed in the nucleus under normal growth conditions. eNOS is at least partially regulated by intracellular calcium (Ca2+) and calmodulin (CaM). Western blot analysis using native eNOS and phospho-specific eNOS antibodies on fractionated cells treated with the protein kinase C (PKC) activator phorbal 12-myristate 13-acetate (PMA) with and without addition of its antagonist ethylene glycol tetraacetic acid (EGTA) was conducted to investigate PKC’s role in eNOS regulation by phosphorylation. Indeed, PKC activation was found to mitigate expression in the nucleus, while inhibition of the activator restored the activity expression above basal levels. This finding correlates with previous data from our lab showing a decrease in activity in myofibroblasts treated with PMA and assayed amperometrically with an NO electrode.

Growth hormone (GH) was upregulated in the zebrafish inner ear following sound exposure in a previous study. To identify the specific role of GH in hair cell regeneration and the possible cellular mechanisms of this regeneration, groups of zebrafish were divided into baseline (no sound exposure, no injection), buffer-injected and GH-injected groups. Buffer- and GH-injected fish were exposed to a 150 Hz tone at a source level of 179 dB re 1 μPa root mean squared (RMS) for 36 h. Phalloidin-staining was used to assess the effects of GH on hair cell bundle density; BrdU-labeling was used to assess the effects of GH on cellular proliferation; TUNEL-labeling was used to assess the effects of GH on apoptosis in the zebrafish inner ear following acoustic trauma. The time-course of hair cell bundle density, cell proliferation, and apoptosis was established by combining data for baseline fishes and sound-exposed fishes at post-sound exposure day 1 (psed1), psed2, and psed3. GH-injected fish exhibited greater densities of hair cells than bufferinjected controls. In addition, GH-injected fish had higher levels of cell proliferation and lower levels of apoptosis than buffer-injected controls. This suggests that GH may play an important role in zebrafish inner ear hair cell regeneration by stimulating cellular proliferation and inhibiting cellular apoptosis.

TRPA1 channels are sensors for noxious stimuli in a subset of nociceptive neurons. TRPA1 channels are also expressed in cells of the mammalian inner ear, but their function in this tissue remains unknown given that Trpa1–/– mice exhibit normal hearing, balance and sensory mechanotransduction. Here we show that non-sensory (supporting) cells of the hearing organ in the cochlea detect tissue damage via the activation of TRPA1 channels and subsequently modulate cochlear amplification through active cellshape changes. We found that cochlear supporting cells of wild type but not Trpa1–/– mice generate inward currents and robust long-lasting Ca2+ responses after stimulation with TRPA1 agonists. These Ca2+ responses often propagated between different types of supporting cells and were accompanied by prominent tissue displacements. The most prominent shape changes were observed in pillar cells which here we show possess Ca2+-dependent contractile machinery. Increased oxidative stress following acoustic overstimulation leads to the generation of lipid peroxidation byproducts such as 4-hydroxynonenal (4-HNE) that could directly activate TRPA1. Therefore, we exposed mice to mild noise and found a longer-lasting inhibition of cochlear amplification in wild type than in Trpa1–/– mice. Our results suggest that TRPA1-dependent changes in pillar cell shape can alter the tissue geometry and affect cochlear amplification. We believe this novel mechanism of cochlear regulation may protect or fine-tune the organ of Corti after noise exposure or other cochlear injuries.

The left ventricle of the heart relaxes when it fills with blood and contracts to eject blood into circulation to meet the body’s metabolic demands. Dysfunction in either relaxation or contraction of the left ventricle can lead to heart failure. Transmural heterogeneity is thought to contribute to normal ventricular wall motion but it is not well understood how transmural modifications affect the failing left ventricle. The overall hypothesis of this dissertation is that normal left ventricles exhibit transmural heterogeneity in cellular level contractile properties and with aging and heart failure there are region-specific changes in cellular level contractile mechanisms. Age is the biggest risk factor associated with heart failure and therefore we investigated transmural changes in Ca2+ handling and contractile proteins in aging F344 rats before the onset of heart failure. We found that in 22-month old F344 rats there is a region-specific decrease in cardiac troponin I phosphorylation in the sub-epicardium that may contribute to slowed myocyte relaxation in the sub-epicardial cells of the same age. We then investigated the transmural patterns of contractile properties in myocardial tissue samples from patients with heart failure. Force and power output reduced most significantly in the samples from the mid-myocardial region when compared to sub-epicardium and sub-endocardium of the failing hearts. There was a region-specific increase in fibrosis is the mid-myocardium of the failing hearts. Myocardial power output was correlated with key sarcomeric proteins including cardiac troponin I, desmin and myosin light chain-1. The results in this dissertation reveal novel region-specific modifications in contractile properties in aging and heart failure. These transmural effects can potentially contribute to disruption in normal wall motion and lead to ventricular dysfunction.

Congenital heart disease (CHD) is the most common birth defect in infants. Identifying factors that are critical to embryonic heart development or CHDs in general could further our understanding of the disease and may lead to new strategies of its prevention and treatment. Endothelial nitric oxide synthase (NOS3/eNOS) is known for many important biological functions including vasodilation, vascular homeostasis and angiogenesis. Previous studies have shown that deficiency in NOS3 results in congenital septal defects, cardiac hypertrophy and postnatal heart failure. In addition, NOS3 is pivotal to morphogenesis of aortic valve and myocardial capillary development. The aim of my thesis was to investigate the role of NOS3 in the embryonic and adult heart. I discovered that NOS3 deficiency resulted in coronary artery hypoplasia in fetal mice and spontaneous myocardial infarction in postnatal hearts. Coronary artery diameters, vessel density and volume were significantly decreased in NOS3-/- mice at postnatal day 0. Lack of NOS3 also down-regulated the expression of Gata4, Wilms tumor-1, vascular endothelial growth factor, basic fibroblast growth factor and erythropoietin in the embryonic heart at E12.5, and inhibited migration of epicardial cells into the myocardium. In addition, my data show that the overall size and length of mitral and tricuspid valves were decreased in NOS3-/- compared with WT mice. Echocardiographic assessment showed significant regurgitation of mitral and tricuspid valves during systole in NOS3-/- mice. Immunostaining of Snail1 was performed in the embryonic heart. Snail1 positive and total mesenchymal cells in the AV cushion were decreased in NOS3-/- compared with WT mice at E10.5 and E12.5. Finally, in the adult aortic valves, NOS3 is important in inhibition of thrombosis formation. Deficiency in NOS3 leads to aortic valve thrombosis and calcification. At 12 months old, 72% (13/18) of NOS3-/- mice showed severe spontaneous aortic valve thrombosis compared with WT mice (0/12). Ex vivo culture of aortic valves showed that platelet aggregation and adhesion were significantly increased in NOS3-/- aortic valves compared with WT aortic valves. There was also a significant regurgitation of the aortic valve during systole in the NOS3-/- compared with WT mice. In addition, NOS3 deficiency resulted in significant aortic valve stenosis, calcification and fibrosis. In summary, these data suggest NOS3 plays a critical role in embryonic heart development and morphogenesis of coronary arteries and inhibits thrombosis formation in the adult aortic valves.

The purpose of this study was to investigate the mechanism of action of lipolysis by growth hormone in rat adipocytes. GH-induced lipolysis, in contrast to that of isoproterenol (ISO), is slow in onset (lag time >1h), small in magnitude (~2X basal). and requires corticosteroid. Evidence for direct coupling between GH receptors and adenylyl cyclase or G-proteins is lacking, and although we could detect no measurable change in cAMP content after treatment with GH + dexamethasone (Dex), it is likely that cAMP activation of protein kinase A is a central event in GH-induced lipolysis. Rp-cAMPS, a competitive antagonist of cAMP was equally effective in decreasing lipolysis in tissues treated with GH/Dex or a comparably lipolytic dose of ISO. Incorporation of 32P from γ-32P-ATP into kemptide, a synthetic oligopeptide substrate for protein kinase A, was increased in homogenates of GH/Dex-treated tissue. This increase was correlated with increased lipolysis. Earlier estimates based upon 32P-ribosylation of Gi catalysed by pertussis toxin (PTx) suggested that the abundance of Gi in adipocyte membranes was decreased 4h after treatment of hypophysectomized rats with GH. We therefore examined the possibility that changes in amount or distribution of G-proteins in adipocyte membranes might account for the lipolytic action of GH. Homogenates of GH/Dex-treated and control adipocytes were subjected to differential centrifugation and the abundance of G-proteins in low speed, l6k x g (16k), pellets and high speed, 100k x g (100k), pellets were determined by quantitative Western analysis with densitometry. A 35% loss of Giα2 from the l6k pellet compared from tissues treated with GH/Dex was associated with a 70% increase of Giα2 in the 100k pellet. No change in Gsα was observed in the l6k pellet but a 35% loss of Gsα was seen in the 100k pellet. The G proteins in the l6k pellet were fractionated on a continuous sucrose gradient followed by quantitation with Western analysis or autoradiography after 32P-NAD ribosylation. Giα2 was consistently shifted from heavier to lighter fractions of the l6k pellet after treatment with GH/Dex. Similar shifts of Gsα were not seen. The distribution of 32P-labelled proteins was comparably altered after incubation of homogenates of control and GH/Dex treated adipocytes with PTx and 32P-NAD. These shifts were blocked by treatment of adipocytes with 100μM colchicine which also blocked the lipolytic action of GH/Dex. We propose that an action of GH/Dex on the cytoskeleton of fat cells may change the cellular distribution of G-proteins in a manner that produces a relative decrease in the tonic inhibitory influence of Gi on adenylyl cyclase.

Na gravidez, o aumento da resistência à insulina é compensado por alterações morfo-funcionais na ilhota pancreática, como o aumento da proliferação e da secreção de insulina. Após o parto ocorre retorno do pâncreas às condições basais e aumento simultâneo da sensibilidade periférica à insulina. Neste trabalho estudamos os mecanismos moleculares responsáveis pelo remodelamento da ilhota no período perinatal. Os resultados mostram que a fosforilação em serina do STAT3 regula a expressão de SERCA2 e assim, modula a primeira fase da secreção de insulina. A fosforilação do STAT3 é dependente da ativação da ERK1/2. No início da lactação, ocorre redução da fosforilação das ERK1/2 devido ao aumento da expressão da fosfatase MKP1. A ação dos glicocorticóides é essencial para este fenômeno, e depende da expressão diferencial de 11<font face=\"symbol\">b-HSD1 e 11<font face=\"symbol\">b-HSD2. O aumento da sensibilidade periférica à insulina ocorre especificamente em músculos oxidativos no início da lactação, e depende do aumento da expressão de Glut4, IR, da via IRS2-PI3K-AKT e da diminuição da PTP1B. / During pregnancy, the increase in insulin resistance is compensated by morfofunctional adaptations of the pancreatic islets, as seen by an increase in cell proliferation and insulin secretion. After delivery, the endocrine pancreas returns to basal conditions, simultaneously to an increase in peripheral insulin sensitivity. In this work we studied the molecular mechanisms involved in islets remodeling during the peripartum period. Our results show that STAT3 serine phosphorylation regulates SERCA2 expression, thus modulating first phase of insulin secretion. STAT3 serine phosphorylation is dependent on ERK1/2 activation. At the beginning of lactation, ERK1/2 phosphorylation is downregulated by the overexpression of the phosphatase MKP1. The action of glucocorticoids is essential in this mechanism, which depends on the differential expression of 11<font face=\"symbol\">b-HSD1 and 11<font face=\"symbol\">b-HSD2. The increase in peripheral insulin sensitivity occurs specifically on oxidative skeletal muscles, which involves an increase in Glut4 and IR expression, IRS2-PI3K-AKT pathway, and a decrease in PTP1B.

Foi realizado o pirossequenciamento de duas bibliotecas de cDNA do intestino médio de Tenebrio molitor e as sequências foram submetidos à montagem através do programa Newbler. Visando sanar alguns questionamentos a respeito de muitos tipos de transportadores que pudessem estar envolvidos com funções presumíveis em tamponamento luminal, absorção de nutrientes, envolvimento em mecanismos de secreção de enzimas como a &#945;-manosidases e secreção e absorção de água, foram analisadas sequências de interesse que pudessem esclarecer os fenômenos fisiológicos em questão. O pirossequenciamento revelou 19 sequencias de &#945;-manosidases. Após alinhamentos múltiplos, desconfiou-se que o contig 12 era a continuação da sequência original da &#945;-manosidase. Utilizando-se de iniciadores apropriados, a suspeita foi confirmada e uma sequência completa foi obtida e denominada de TmMan1. Através de cladogramas gerados com as sequências de todos os contigs obtidos, assim como de sequências representativas das famílias 38 e 47 das glicosídeos hidrolases, mostrou que todas a nossas sequências, exceto o contig 6 e 7, pertencem à família 38. Todas as sequências com mais de 100 reads (exceto o contig 9) tiveram a sua expressão tecidual avaliada por RT-PCR. Os resultados mostraram que só são expressos no intestino ou intestino e túbulo de Malpighi, implicando na possibilidade de serem digestivas. Dessas sequências, as únicas com peptídeo sinal são a TmMan1 (contig 12) e o contig 14 e, portanto, devem corresponder às atividades Man1 e Man2. Levando em conta o número de reads, TmMan1 deveria corresponder a Man2 e o contig 14 à Man1. É possível, embora necessite de confirmação, que os contigs 8 e 15 sejam de expressão lisossômica. Um peptídeo sintetizado que correspondia a sequencia única da TmMan1 foi usado para gerar anticorpos, que reconheceram a Man2, mas não a Man1, confirmando a identificação de TmMan1 com a Man2. Esse anticorpo foi também utilizado para imunolocalizar a TmMan1 nas células intestinais de T. molitor. Os resultados mostraram que a TmMan1 é secretada de forma apócrina pela região anterior de intestino de T. molitor. Esse trabalho é o primeiro que mostra a ocorrência de &#945;-manosidases com especificidade similar àquelas lissossômicas, mas que são secretadas apócrinamente para fora da célula, devendo agir no lúmen intestinal, removendo resíduos de manoses de oligossacarídeos manosilados. Foram identificados 10 tipos diferentes de transportadores e na elaboração dos modelos fisiológicos só foram levados em conta aqueles expressos exclusivamente no intestino médio ou no intestino médio e túbulos de Malpighi. A V-ATPase em T.molitor parece ser uma bomba usada para energizar muitos dos transportes ao longo do intestino médio como, por exemplo, o de oligopeptídeos. Já as bombas de Na+ e K+ são responsáveis pelo equilíbrio de cargas e, portanto estão presentes na maioria dos tipos celulares. Duas sequências de cotransportadores de oligopeptídeos/H+ foram encontradas no pirossequenciamento e sua expressão é maior na região posterior, uma vez que ali é a última possibilidade de absorção dos oligopeptídeos que ainda estiverem no lúmen, remanescentes da digestão final de proteínas. Foi demonstrado que T.Molitor absorve aminoácidos e açúcares ao longo de todo o intestino médio, pois estes tipos de transportadores possuem uma expressão uniforme ao longo do intestino médio. Já a expressão dos transportadores de NH3/NH4+ em T.molitor, encontra-se confinada ás regiões onde o pH do intestino médio do inseto é mais ácida. Também há uma expressão de transportadores de cloreto que se manifesta mais intensamente na região anterior. Podemos visualizar que a distribuição dos contigs dos transportadores de bicarbonato encontra-se mais expressiva na região posterior do intestino médio. Os resultados sugerem que a acidificação na região anterior do intestino de T.molitor pode resultar da secreção de NH4+ acompanhado do íon cloreto e a alcalinização na região posterior do lançamento no lúmen de bicarbonato. A importância dos canais de cloreto é que o mesmo balanceia as cargas e desta forma pode ser útil juntamente com o transporte de NH4+, que gera uma carga no lado onde é transportado. Há absorvição de água (junto com glicose) ao longo de todo o intestino médio, enquanto que a secreção de água ocorreria apenas nos dois terços finais do intestino médio com o auxílio de aquaporinas complementado por transportadores de íons, teria como consequência a abosorção líquida de água na região anterior e uma secreção líquida no final do intestino médio. Isso esclarece qual a base molecular para a ocorrência do contrafluxo intestinal evidenciado por experimentos fisiológicos. / Pyrosequencing was performed with two cDNA libraries in the midgut of Tenebrio molitor and the sequences were subjected to assembly with the Newbler program. In order to tackle questions concerning proteins which may be involved in midgut buffering, nutrient absorption, in the secretion of enzymes such as &#945;-mannosidases , and water absorption and secretion, sequences of interest were analyzed in order to clarify those physiological phenomena. The pyrosequencing revealed 19 sequences of &#945;- mannosidases . After multiple alignments, it was suspected that the contig 12 was the continuation of the original sequence of the &#945;-mannosidase. Using appropriate primers, the hypothesis was confirmed and a complete sequence was obtained and named TmMan . Through cladograms generated from the sequences of the contigs obtained, as well as of sequences representing families 38 and 47 of glycoside hydrolases, it was showed that all sequences except the contig 6 and 7 belong to family 38. All sequences with over 100 reads (except contig 9) had their tissue expression assessed by RT-PCR. The results showed that they are expressed only in the midgut or midgut and Malpighian tubules, implying the possibility of having a digestive function. Among these sequences, the only ones with a signal peptide are TmMan1 (contig 12) and contig 14 and therefore they should correspond to the activities Man1 and Man2. Taking into account the number of reads, TmMan1 should correspond to Man2 and contig 14 to Man1. It is possible, though it requires confirmation, that the contigs 8 and 15 are lysosomal. A peptide corresponding to the unique sequence TmMan1 was synthesized and used to generate antibodies that recognized Man2 , but not Man1, confirming the identification of TmMan1 with Man2. This antibody was also used to immunolocalyze TmMan1 in the midgut cells of T. molitor. The results showed that TmMan1 is secreted in an apocrine way by the anterior region of T. molitor midgut. This is the first study that shows the occurrence of &#945;-mannosidases with similar specificity to those lysosomal, but that are secreted in an apocrine way, acting in the midgut lumen, removing mannoses from mannosylated oligosaccharides. We identified 10 different types of carriers and in the development of physiological models it was only taken into account those expressed exclusively in the midgut or midgut and Malpighian tubules . The V- ATPase in T.molitor appears to be a pump used for powering many transports along the midgut, as of oligopeptides. The Na+ and K+ pumps are responsible for charge load balancing and therefore are present in most cell types. Two sequences of oligopeptide / H+ cotransporters were found in the transcriptome and their expression is higher in the posterior region. This agrees with the fact that there is the last possibility of oligopeptides remaining in the lumen to be absorbed. It is highly probable that T.molitor absorbs amino acids and sugars throughout the midgut, once these types of carriers have a uniform expression throughout the midgut . The expression of NH3/NH4+ transporters in T.molitor is confined to the regions where the pH of the insect midgut is more acidic. There is also chloride transporter expression there. The expression of bicarbonate transporters is more significant in the posterior midgut. The results suggest that acidification in the anterior T.molitor midgut may result from the secretion of NH4+ and chloride ions together, whereas the alkalization in the posterior midgut results from bicarbonate release. There is water absortion (along with glucose) throughout the midgut , while the water secretion occurs only in the final two-thirds of the midgut with the aid of aquaporins, complemented by ion transporters. It would result in the net absorption and net secretion of water in the anterior and postertior midgut, respectively. This clarifies the molecular basis of the midgut countercurrent fluxes evidenced by physiological experiments.