Evidence shows the activation of neutrophils in the systemic vasculature of obese and preeclamptic women. In this study, I evaluated whether expression of neutrophil products, myeloperoxidase (MPO) and matrix metalloproteinase 8 (MMPS), was associated with neutrophil infiltration in systemic vascular tissue of obese and preeclamptic women. I tested my hypotheses by using immunohistochemical studies to look at the expression of MPO and MMP8 in the vasculature of obese and preeclamptic women. There was a significantly greater expression of MPO and MMP8 in the vasculature of preeclamptic women as compared to normal pregnant and normalnonpregnant women. The vasculature of obese women also had a significantly greater expression of MPO and MMP8 as compared to overweight and normal weight patients.These studies are the first to report that activated neutrophils in systemic vasculature are releasing MPO and MMP8. These findings also indicate that the vascular phenotype of obese and preeclamptic women is similar in that they both show an increased presence of MPO and MMP8 in the systemic vasculature as a result of neutrophil infiltration. This suggests that obese women are at increased risk for preeclampsia because their vasculature is already exposed to increased levels of MPO and MMP8, so when they become pregnant and experience further oxidative stress imposed by the placenta along with an increase in neutrophil number, they develop the clinical symptoms of preeclampsia.

It is well known that the overlap between the thick and thin filaments in striated muscle is responsible for the single active length-tension (L-T) curve. With the lack of visible striations, a sarcomeric unit has not been identified in smooth muscle. Though once thought to function like striated muscle via a sliding filament mechanism of contraction, recent studies on length-adaptation (L-adaptation) in airway smooth muscle (ASM), in which increased tension is generated with repeated contraction, have led to the hypothesis of a dynamic L-T curve in smooth muscle. Although more established in ASM, two studies have shown L-adaptation in vascular smooth muscle (VSM). In this project, the L-T curve over a 3-fold length range in rabbit femoral artery was investigated and the presence of more than one active and passive L-T curve was identified. The third of three repeated KCL-induced contractions at a single, shortened length resulted in L-adaptation in which the phasic and tonic phases of contraction demonstrated a 10-15% increase in active tension (Ta) relative to the first contraction. Experiments investigating possible mechanism(s) responsible for this phenomenon demonstrated that neither an increase in [Ca2+]i nor an increase in MLC20 phosphorylation was responsible for the increased tension. However, actin polymerization did appear to play a role in the L-adaptation of both phases of contraction. Thus directions for future research could include further study of actin polymerization in VSM that contributes to L-adaptation and may ultimately result in artery remodeling.

Overactive bladder (OAB), involuntary contractions during bladder filling, is a common condition affecting 17% of the adult population worldwide, and in the U.S. ranks ahead of diabetes in a list of the 10 most common chronic disorders (Mullins 2009). Mechanical mechanisms contributing to OAB are not completely understood and because of the unique function and broad volume range of the bladder, there may be mechanical characteristics that distinguish detrusor smooth muscle (DSM) in bladder from other smooth muscles. Recent studies have shown that the length-passive tension curve in DSM exhibits adjustable passive stiffness (APS) characterized by a passive curve that can be shifted along the length axis as a function of strain history and activation history; however, the mechanical mechanisms responsible for APS remain to be determined. Also, whether DSM exhibits a dynamic length-active tension relationship, as has been identified in airway and vascular smooth muscles, has not been investigated. This dissertation focused on both the passive and active length-tension relationships in DSM and the mechanical mechanisms responsible for these relationships. The first objective was to study the impact of APS on the length-total tension relationship and identify the mechanical mechanisms responsible for generating APS. The second objective was to determine whether the length-active tension relationship is adaptive and identify specific mechanical mechanisms contributing to any adaptive behavior. The results showed that a shift in the length-passive tension curve due to APS corresponded with a shift in the length-total tension curve in DSM, and that APS was 27.0±8.4% of active tension at the optimum length for active tension generation. Most importantly, low-grade rhythmic contraction (RC), which can occur spontaneously in rabbit and human bladders, regenerated APS. Results also showed that the length-active tension curve shifted due to stretch to and then activation at long lengths, as well as either multiple KCl-induced maximal contractions or RC. Thus, DSM exhibits length adaptation, and RC may contribute to both APS and length adaptation. Because increased RC has been correlated with OAB, understanding RC, APS and length-adaptation in bladder may enable the identification of specific targets for new treatments for OAB.

Novel Ca²⁺ signalling pathways in both endothelial cells and smooth muscle cells of rat small resistance arteries were investigated using a combination of confocal imaging, isometric tension recordings, and electrophysiology to study freshly isolated arteries and cells. We first examined the hypothesis that hyperpolarization could alter endothelial cell Ca²⁺ events. Hyperpolarization evoked by direct opening of K_ATP channels in the smooth muscle with levcromakalim triggered an increase in the frequency of Ca²⁺ events in the endothelium of rat cremaster arterioles. These Ca²⁺ events were discrete in nature, requiring subcellular regions of interest to reliably identify them. Opening of K_ATP channels indirectly through β-adrenoceptor stimulation with isoprenaline, caused a similar increase in the frequency of endothelial cell Ca²⁺ events in rat mesenteric third order arteries. These events also had a similar, focal profile. Pharmacological investigation suggested that the response to isoprenaline was receptor-mediated, and dependent on Ca²⁺ influx and opening of K_ATP channels. The presence of β-adrenoceptors on endothelial cells was confirmed using fluorescently-tagged β-adrenoceptor ligands, which showed punctate labelling in smooth muscle and endothelial cells of rat mesenteric arteries. Freshly isolated endothelial cells also showed Ca²⁺ increases to isoprenaline, although this was not consistently observed. Following on from the observed endothelial cell Ca²⁺ response to hyperpolarization, we tested the hypothesized involvement of hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels using the channel inhibitor, ZD7288. Pre-treatment with ZD7288 (1 μM) reduced both the endothelial cell Ca²⁺ response to isoprenaline (in mesenteric arteries) and levcromakalim (in cremaster arterioles). HCN channel subtypes were identified in cremaster arterioles through immunolabelling. We also observed an interesting effect of higher concentrations of ZD7288 to potentially inhibit K⁺ channels, including endothelial cell KCa channels, since hyperpolarization to isoprenaline, levcromakalim or acetylcholine (ACh) was reduced by 10 μM ZD7288, and relaxation to ACh was partially inhibited. ACh-mediated relaxation was also partially inhibited by the clinically used HCN channel blocker, ivabradine (0.3-30 μM). Finally, we identified an interaction of the Ca²⁺-releasing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) with BKCa channels in the smooth muscle. NAADP-mobilised Ca²⁺ has been reported to interact with ryanodine receptors hence we hypothesized an interaction with BK_Ca channels via Ca²⁺ sparks. We found that NAADP-AM relaxed and hyperpolarized rat mesenteric arteries, which was blocked by iberiotoxin (BK_Ca channel inhibitor) and high extracellular [K⁺] (45 mM). Furthermore, NAADP increased paxilline-sensitive K⁺ currents and the frequency and amplitude of spontaneous transient outward currents (STOCs) in freshly isolated vascular smooth muscle cells patched in the whole-cell configuration, further supporting an action at BK_Ca channels. All together these data identify novel Ca²⁺ signalling pathways in resistance arteries that are both activated by and promote hyperpolarization, which is a key determinant of vascular tone.

The presented thesis refers to the administrative transgressions against the safety and smooth traffic flow on highways. This is very actual topic currently, because everybody meets with these administrative transgressions in his life. The thesis is composed of seven chapters. The first chapter introduce us with the history of legislation in the period from the formation of the Czechoslovak republic in 1918 to the presence. The following chapter describes relevant legislation. This chapter contains general regulation of liability for transgressions, as well as discusses the constituent elements of transgressions and ends with characteristic of the constituent elements defined by the Law of highway traffic. In the third chapter, I focused on the comparison of transgressions and crimes. First I compared the general arrangement for liability for crimes and transgressions. Furthermore, I have listed which crimes could be committed in connection with the highway traffic, and then I looked on, in my opinion, mostly committed crimes. The fourth chapter deals with the sanctions that may be imposed for transgressions. The rules on penalties are prescribed by Law of transgressions; the specific amount of sanctions is set by the Law of highway traffic. The administrative transgressions against the safety and smooth...

Bundles and morphisms between bundles are defined in the category of Fr¨olicher spaces (earlier known as the category of smooth spaces, see [2], [5], [9], [6] and [7]). We show that the sections of Fr¨olicher bundles are Fr¨olicher smooth maps and the fibers of Fr¨olicher bundles have a Fr¨olicher structure. We prove in detail that the tangent and cotangent bundles of a n-dimensional pseudomanifold are locally diffeomorphic to the even-dimensional Euclidian canonical F-space R2n. We define a bilinear form on a finite-dimensional pseudomanifold. We show that the symplectic structure on a cotangent bundle in the category of Fr¨olicher spaces exists and is (locally) obtained by the pullback of the canonical symplectic structure of R2n. We define the notion of symplectomorphism between two symplectic pseudomanifolds. We prove that two cotangent bundles of two diffeomorphic finite-dimensional pseudomanifolds are symplectomorphic in the category of Frölicher spaces.

[pt] Neste trabalho um novo modelo de mistura de distribuições é proposto, onde a estrutura da mistura é determinada por uma árvore de decisão com transição suave. Modelos baseados em mistura de distribuições são úteis para aproximar distribuições condicionais desconhecidas de dados multivariados. A estrutura em árvore leva a um modelo que é mais simples, e em alguns casos mais interpretável, do que os propostos anteriormente na literatura. Baseando-se no algoritmo de Esperança- Maximização (EM), foi derivado um estimador de quasi- máxima verossimilhança. Além disso, suas propriedades assintóticas são derivadas sob condições de regularidades. Uma estratégia de crescimento da árvore, do especifico para o geral, é também proposta para evitar possíveis problemas de identificação. Tanto a estimação quanto a estratégia de crescimento são avaliados em um experimento Monte Carlo, mostrando que a teoria ainda funciona para pequenas amostras. A habilidade de aproximação universal é ainda analisada em experimentos de simulação. Para concluir, duas aplicações com bases de dados reais são apresentadas. / [en] In this work a new model of mixture of distributions is proposed, where the mixing structure is determined by a smooth transition tree architecture. Models based on mixture of distributions are useful in order to approximate unknown conditional distributions of multivariate data. The tree structure yields a model that is simpler, and in some cases more interpretable, than previous proposals in the literature. Based on the Expectation-Maximization (EM) algorithm a quasi-maximum likelihood estimator is derived and its asymptotic properties are derived under mild regularity conditions. In addition, a specific-to-general model building strategy is proposed in order to avoid possible identification problems. Both the estimation procedure and the model building strategy are evaluated in a Monte Carlo experiment, which give strong support for the theorydeveloped in small samples. The approximation capabilities of the model is also analyzed in a simulation experiment. Finally, two applications with real datasets are considered.

A obesidade pode mudar as características das células endoteliais e musculares lisas (CMLVs). Reatividade e genes inflamatórios e de marcadores de fenótipo de CMLVs foram avaliados em artérias mesentéricas de resistência (AMRs) e de aorta de camundongos alimentados com dieta hiperlipídica (OB). RNAm para citocinas pró-inflamatórias e IL-10 foi aumentado em AMRs e aorta de OB. O relaxamento não foi alterado, mas a contração foi reduzida em AMRs e aorta de OB. AMRs apresentaram redução global da contração e a aorta apresentou redução específica para agonista adrenérgico. Maior modulação negativa por NO e prostanoides vasodilatadores foi observada em aorta, mas não em AMRs. RNAm para marcador do fenótipo sintético foi aumentado em AMRs de OB. Esses resultados mostram que as células endoteliais e as CMLVs de AMRs e aorta respondem diferentemente à obesidade. Inflamação e mecanismo de contrarregulação são induzidos em AMRs e aorta, que impediria a disfunção endotelial, mas não a mudança fenotípica das CMLVs em AMRs, que então, comprometeria sua capacidade contrátil. / Obesity may change the vascular endothelial cells and smooth muscle cells (VSMC) characteristics. Vascular reactivity and inflammatory and phenotypic markers for VSMC gene expression in resistance mesenteric arteries (RMA) and aorta from mice fed with high fat diet (OB). Pro-inflammatory cytokines mRNA and IL-10 were elevated in OB\'s RMA and aorta. The relaxation wasnt altered, however a reduction in contractility was observed in OB\'s RMA and aorta. A global reduction in contractility was observed in RMA and aorta demonstrated a specific reduction to adrenergic agonist. Higher negative modulation by NO and vasodilator prostanoids were seen only in aorta. Phenotypic markers mRNA were elevated in OB\'s RMA. The results shows that endothelial cells and VSMC from RMA and aorta respond differently to obesity. Inflammation and counter regulatory mechanisms are induced in RMA and aorta of which would prevent endothelial dysfunction but not VSMC phenotypic changing from RMA, compromising the contractile ability.

As fosfolipases A2 secretadas (sFLA2) de veneno de serpente apresentam homologia estrutural e funcional com as sFLA2s do GIIA de mamíferos, cujos níveis estão elevados em doenças inflamatórias, como a aterosclerose. Nesta doença, as células de músculo liso vascular (CMLVs) acumulam corpúsculos lipídicos (CLs) e se diferenciam em células espumosas. Porém, o papel das sFLA2s neste fenômeno não é conhecido. Neste estudo foram avaliados os efeitos das FLA2 MT-III, cataliticamente ativa, e da BthTx-II, sem atividade catalítica, em CMLVs, com ênfase na formação de CLs e a participação de fatores da homeostasia lipídica. Os resultados obtidos demonstraram que a MT-III e a BthTx-II induziram a formação de CMLVs espumosas. Para tanto, estas enzimas recrutaram diferentes fatores envolvidos na síntese e acúmulo de lipídios. Nesta condição, os CLs constituem um local de síntese de prostaglandinas. Ainda, a MT-III induziu a diferenciação de CMLVs para fenótipo e função de macrófagos. A atividade catalítica não é relevante para a formação de CLs induzida por FLA2s. / Bothrops snake venom secreted phospholipases A2 (sPLA2s) share structural and functional features with mammalian GIIA sPLA2s, which are highly expressed during inflammatory diseases, such as atherosclerosis. In this disease, vascular smooth muscle cells (VSMCs) are loaded with lipid droplets (LDs) differentiating into foam cells. However, the role of these enzymes in this process is still unknown. In this study the effects of snake venom PLA2s MT-III with catalytic activity and BthTx-II, devoid of catalytic activity in VSMCs, with focus on LDs formation and mechanisms involved were investigated. Results here obtained show that both MT-III and BthTx-II induce formation of foam VSMCs and recruit distinct factors of synthesis and storage of lipids in these cells. In this condition, LDs constitute sites for synthesis of prostaglandins. Moreover, MT-III showed the ability to modulate VSMCs functions, leading them to a phenotipic switch to macrophage-like cells. In addition, the catalytic activity is not relevant to sPLA2-induced LDs formation.

Avaliou-se prospectivamente 19 pacientes com obstrução infravesical por hiperplasia prostática benigna selecionados pra prostatectomia transvesical. Obteve-se um fragmento da parede da bexiga e quantificou-se o colágeno no epimísio, perimísio e endomísio do detrusor. Amostra vesicais de oito doadores cadavéricos de órgãos serviram como controles. Observou-se padrão focal de aumento do colágeno na camada muscular da bexiga dos pacientes com obstrução. Demonstrou-se que a idade correlaciona-se positivamente com o aumento da quantidade de colágeno com a diminuição da complacência vesical, o aumento da prevalência de hiperatividade detrusora e a maior chance de retenção urinária / We prospectively evaluated 19 patients with bladder outlet obstruction by benign prostatic hyperplasia selected for transvesical prostatectomy. A sample of the bladder wall was obtained and the collagen was measured at the level of epimysium, premysium and endomysium. Bladder samples from eight cadaveric organ donors were used as controls. A focal pattern of collagen increase was observed at the bladder smooth muscle layer of obstructed patients. Age was shown to correlate with increased collagen quantity. Increased collagen quantity also correlated with decreased bladder compliance, higher prevalence of detrusor overactivity and of urinary retention