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Die Anwendung von körperfremden Materialien zur Behandlung verschiedenster Krankheitsbilder, wie zum Beispiel als Zahnersatz oder Knochenstabilisierung, ist seit Jahrtausenden fester Bestandteil in der Medizin. Während damals hauptsächlich stabile Materialien genutzt wurden, die möglichst wenig mit dem menschlichen Körper interagieren, wird heutzutage ein anderer Ansatz verfolgt. Intelligente Materialien können nicht nur passiv die Heilung unterstützen, sondern aktiv zu ihr beitragen. Ein berühmtes Beispiel hierfür ist das Formgedächtnismaterial Nitinol, das in Stents zur Behandlung verengter Arterien eingesetzt wird. Diese Arbeit beschäftigt sich mit Eisen-Palladium, einem neuen Formgedächtnismaterial, bei dem der Effekt nicht wie bei Nitinol über eine Temperaturänderung sondern durch ein äußeres Magnetfeld induziert wird. Da man somit körpertemperaturbedingte Restriktionen in biomedizinischen Anwendungen umgehen kann, birgt Eisen-Palladium ein hohes Potential für Drug-Delivery Systeme oder mikromechanische Pumpen. Da eine optimale Verträglichkeit des Materials mit seiner biologischen Umgebung absolut unabdingbar ist, untersucht diese Arbeit verschiedene Möglichkeiten, die Oberfläche zu modifizieren und somit die Adhäsion biologischer Zellen zu unterstützen. Zu diesem Zweck wurden das Peptid RGD als spezifische Zelladhäsionssequenz, ein Plasmapolymer auf L-Lysin Basis als unspezifische Beschichtung und die Nanostrukturierung der Eisen-Palladium Oberfläche durch Glanzwinkeldeposition untersucht. Die verwendeten Methoden beinhalten Immunofluoreszenztests zur Quantifizierung der fokalen Kontakte zwischen Zellen und Material, theoretische Dichtefunktionaltheorie Rechnungen, sowie Kontraktilitätsmessungen mittels eines selbst entwickelten Biegebalkenaufbaus. Somit gelingt es in dieser Arbeit, die gegenseitigen Beziehungen des Materials mit der jeweiligen Oberflächenmodifikation mit den lebenden Zellen aus verschiedenen Blickwinkeln zu analysieren. Durch eine Kombination aus experimentellen und theoretischen Methoden werden die Stärken und Schwächen der einzelnen Funktionalisierungsmethoden beleuchtet und die Bildung fokaler Kontakte für eine verbesserte Zelladhäsion wird maßgeblich verbessert.

Although prostaglandins (PG) and oxytocin are crucial mediators of uterine contractility, their receptor-mediated effects during the menstrual cycle, pregnancy and labour are not fully understood. The aim of this thesis was to elucidate the functional expression of EP, FP, TP and oxytocin receptors in isolated human myometrium relative to myocyte mRNA and signal transduction pathways. Myometrial samples were obtained from consenting non-pregnant and pregnant donors. Functional techniques were used to determine isometric muscle contractions. Primary uterine myocytes and fibroblasts were cultured at term to identify stimulated changes in calcium (Ca2+), cyclic adenosine monophosphate (cAMP) and mRNA. Myometrial strips exhibited spontaneous contractions, which were most active midcycle under oestrogenic conditions. At this time intrinsic contractility and responsiveness to uterotonins decreased towards the fundus. PGE2 produced bellshaped responses with predominant utero-relaxant effects mediated via the EP2 subtype. Although activity was partially restored by PGE2 through EP3/1 receptors, tissue excitation was more pronounced at FP, TP and oxytocin receptors. Despite high FP mRNA expression, the lower segment uterus was particularly responsive to U46619 and oxytocin at term pregnancy. Even so, Ca2+ mobilisation by oxytocin was greater via principal release from intracellular stores. Incubations with atosiban, progesterone and a rho-kinase inhibitor reduced oxytocin-stimulated Ca2+ transients. EP2 also attenuated oxytocic effects but this appeared to be mediated through cAMP rather than Ca2+ signalling pathways. With advancing labour, intrinsic myogenic activity declined in parallel with oxytocin desensitisation. However, TP-induced contractions were continued in the lower parturient uterus. These findings demonstrate that PG and oxytocin receptor expression are regulated in a hormone-dependent temporal and spatial manner. EP2-mediated cAMP formation appears to promote uterine quiescence, whilst TP receptors may control muscle tonus during parturition. These receptors and their messenger systems represent effective tocolytic targets for uterine hypercontractile disorders, such as dysmenorrhoea and preterm labour.

Modulation of cardiac rate and contraction through calcium-dependent and independent means are of central import to the ability of an organism to adapt to its environment. Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent calcium-releasing second messenger across a broad range of tissues and organisms. In cardiac myocytes, NAADP is thought to stimulate calcium release from acidic stores which then bolsters filling and release during CICR. Questions remain: as to the potential need for amplification to generate the size of responses observed and the physiological role of the NAADP pathway. In contractile myocytes, photorelease of NAADP caused significant increase in calcium transient amplitude and velocity of transient upstroke and decay. Effects were absent during NAADP photorelease in the presence of Ned-19 or CaMKII inhibitors. Cellular calcium transient responses to β-adrenergic stimulation were significantly reduced in the presence of inhibitors of the NAADP pathway. These data support the hypothesis that NAADP-induced calcium release is relevant during adrenergic stimulation and requires amplification through CaMKII. Rate modulation at the sino-atrial node can occur through the hyperpolarisation-activated current I_(f). Basal cardiac rate is a major determinant in cardiac mortality and compounds which specifically affect rate have clinical utility. A compound currently used to treat inflammatory conditions was found to have a significant rate-reducing effect in sino-atrial node preparations mediated by inhibition of I_(f). Apelin, an endogenous peptide, has been reported to potently generate improved contractility without development of hypertrophy. Study of its effects in single cells have provided conflicting information, at least in part because of the difficulty in working with the compound. A method for the consistent observation of apelin-mediated contractile responses is presented, focusing on the timecourse of cell contraction. These observations suggest a role for apelin in both inotropy and lusitropy and will enable further research.

Opioid/adrenergic interactions were studied to evaluate two hypotheses: (1) naloxone potentiates the effect of epinephrine on cardiac contractility by increasing circulating epinephrine concentrations; and (2) endogenous and exogenous opioids alter left cardiac nerve stimulationinduced norepinephrine release and cardiac function. A canine isolated heart-lung preparation was used for the first study. Plasma epinephrine was determined and myocardial epinephrine uptake was calculated during intravenous epinephrine infusion. Naloxone (4 mg) was given and the epinephrine infusion was repeated. Naloxone increased cardiac contractility, coronary blood flow, and the coronary sinus epinephrine concentration. When coronary blood flow was subsequently held constant (100% above resting), naloxone increased only contractility. This result indicated that the previously observed increase in coronary sinus epinephrine was flow dependent. Corticosterone (an uptake II blocker) was employed as a positive control. Corticosterone increased the contractile response to epinephrine, but unlike naloxone, corticosterone was accompanied by a clear decrease in myocardial epinephrine uptake. The stereospecificity of the response to naloxone was investigated and (+) naloxone equaled or exceeded (-) naloxone in potentiating the inotropic effect of epinephrine. In the second study, the left cardiac nerve was isolated and electrically stimulated in intact dogs. Norepinephrine overflow gradually declined during successive control stimulations. Pretreatment with naloxone (100 Mg/kg) prevented or delayed the decline. An intracoronary dynorphin 1-9 infusion (2 nmol/min/kg for 20 minutes) reduced both norepinephrine overflow and cardiac performance, and both effects were prevented by pretreatment with naloxone (100 /xg/kg) . To summarize, naloxone potentiated the inotropic effect of infused epinephrine without altering circulating epinephrine concentrations or myocardial epinephrine uptake. This effect of naloxone was not stereospecific and probably not mediated through a traditional opiate receptor. Endogenous and exogenous opioids inhibited the left cardiac nerve stimulation-induced norepinephrine overflow, suggesting that opiate receptors may regulate cardiac excitability by modulating norepinephrine release.

O infarto do miocárdio (IM) é uma patologia causada pela obstrução parcial ou total das artérias coronárias responsáveis pela irrigação do miocárdio. Sabe-se que logo após o IM, o treinamento físico (TF) promove melhora e adaptações benéficas ao sistema cardiovascular. O objetivo do presente estudo foi traçar o perfil de microRNAs na área remanescente ao IM de ratos submetidos ou não ao TF através da técnica de microarray, selecionar e confirmar a expressão de um microRNA específico, por programas de bioinformática que tenha alvos que possam estar envolvidos com a melhora da função cardíaca com o TF. Também foi objetivo deste estudo, verificar a expressão do microRNA-1, do microRNA-29 e do microRNA-214 e dos respectivos alvos destes microRNAs, como o colágeno, NCX e SERCA2a. Para isso, ratos Wistar foram divididos em quatro grupos: SEDENTÁRIO SHAM (SED-SHAM), SEDENTÁRIO INFARTADO (SED-IM), TREINADO SHAM (TR-SHAM) e TREINADO INFARTADO (TR-IM). A função ventricular foi acompanhada com ecocardiografia antes e após oito semanas do protocolo de TF de natação. Os animais foram sacrificados, o RNA foi extraído e posteriormente foi traçado o perfil de microRNAs. Para seleção dos microRNAs foram estipulados dois padrões de expressão entre os grupos. De acordo com os critérios aplicados foram observados, apenas 6 microRNAs para o primeiro padrão de expressão e 3 microRNAs para o segundo padrão de expressão que satisfizeram todos os critérios. O microRNA-339-5p foi escolhido por apresentar expressão diferencial pronunciada como efeito do TF por ter dois genes (alvos selecionados por programas de bioinformática TargetScan, Miranda e PicTar), o Myo1c e o PGC-1 ?, que estão envolvidos com a regulação da função cardíaca. Neste estudo, foi possível verificar que nos grupos TR-SHAM e TR-IM ocorreu aumento da expressão da Myo1c e redução da expressão do microRNA-339-5p. Nossos resultados também mostraram que o TF induziu aumento na expressão dos microRNAs-29a/c, o que está relacionado com significativa diminuição na expressão gênica do COLIAI, COLIIIAI, diminuição na concentração de hidroxiprolina e fração volumétrica de colágeno de ratos com IM. Outro resultado deste estudo é que o TF induziu diminuição na expressão dos microRNAs-1 e 214, o que está relacionado com aumento na expressão protéica dos alvos SERCA-2a e NCX de ratos com IM. Esses efeitos estão associados com a melhora da função ventricular avaliada pela FEAT% (função sistólica) e pela relação E/A (função diastólica), realizado por avaliação ecocardiográfica após o período de TF. / Myocardial infarction (MI) is a disease caused by partial or total blockage of the myocardial coronary arteries. It is known that after MI, physical training (PT) improves and promotes beneficial adaptations to the cardiovascular system. The aim of this study was to analyse the microRNAs profile in the remaining area to MI in rats submitted to PT by microarray and select a specific microRNA by bioinformatics software that has targets involved with the improvement of cardiac function by PT. Also, the aim of this study was to verify the expression of the microRNA-1, microRNA-29 and microRNA-214 and its targets, such as collagen, NCX and SERCA2a. For this, rats were divided into four groups: SEDENTARY SHAM (SHAM - SED), INFARCTED SEDENTARY (SED-MI), TRAINED SHAM (TR-SHAM) and INFARCTED TRAINED (TR-IM). Ventricular function was monitored with echocardiography before and after eight weeks of the PT. The animals were sacrificed, the RNA was extracted and traced the profile of microRNAs. For selection of microRNAs were stipulated two patterns of expression between the groups. The first standard, 6 microRNAs were selected and in the second standard, 3 microRNAs were selected to satisfy all criteria. The microRNA- 339-5p was chosen because of pronounced differential expression with PT and have two target genes selected by bioinformatics programs (TargetScan, Miranda and PicTar) Myo1c and the PGC-1 ? are involved in the regulation of cardiac function. In this study, we found that the SHAM group and the group with MI undergoing PT increased expression of Myo1c in contrast to a decreased expression of microRNA-339- 5p. Our results also showed that PT induced increased expression of microRNA-29a/c, which is associated with a significant decrease in gene expression of COLIAI, COLIIIAI, the hydroxyproline xvii concentration and collagen volume fraction of rats with MI. Another result of this study is that the PT induced decrease in the expression of microRNA-1 and microRNA-214, which is related to the increase in protein expression of the targets SERCA-2a and NCX of rats with MI. These effects are associated with improved ventricular function assessed by FEAT % (systolic function) and E/A ratio (diastolic function), performed by echocardiography after the period of PT

Atualmente, temos a disposição modernas técnicas de mapeamento eletroanatômico gerando imagens tridimensionais da propagação do impulso nas câmaras cardíacas através de catéteres endocavitários. Apesar disso, poucos estudos estão disponíveis a respeito do mapeamento eletrofisiológico epicárdico. A avaliação intraoperatória de contratilidade miocárdica imediatamente após a revascularização miocárdica é visual e Ecocardiográfica, porém este é muito pouco utilizado para este fim. Há, portanto, a necessidade de desenvolvimento de novos métodos capazes de avaliar o impacto funcional da revascularização miocárdica. Objetivo: Avaliar a resposta intra-operatória de contratilidade miocárdica regional mediante o mapeamento epicárdico com eletrodo decapolar. Métodos: 20 pacientes serão submetidos a revascularização miocárdica e será realizado o mapeamento epicárdico com eletrodo decapolar após confecção da anastomose distal com perfusão seletiva para área revascularizada com a aorta pinçada e portanto sem perfusão para as demais artérias coronárias. Nesta análise será avaliado a duração do impulso do eletrograma. Concomitante será realizada fluxometria dos enxertos e comparada com o resultado obtido no mapeamento epicárdico. Resultados: A técnica é uma forma eficaz de avaliar a contratilidade miocárdica regional após a revascularização miocárdica seletiva. O fluxo intravascular dos enxertos estudados não interfere na duração do impulso durante a perfusão seletiva das áreas estudadas / At present, we have available modern tecnics of electroanatomic mapping generating tridimensional images of impulse propagation on heart chambers through intracavitary catheters. Despite this, very few scientific studies are available about epicardial electrophysiologic mapping. Intraoperative evaluation of myocardial contractility right after coronary artery bypass is visual and Echocardiographic, even though this is rarely used to this end. Therefore, there is a need for development of new methods capable of evaluate the functional impact of myocardial revascularization. Objective: Evaluate the intra-operative response of regional myocardial contractility with epicardial mapping with a decapolar catheter. Methods: 20 patients will be submitted to coronary artery bypass graft surgery and epicardial mapping with decapolar catheter was performed after distal anastomosis is made with selective perfusion on the revascularized area with cross clamped aorta, therefore without blood perfusion to the other coronary arteries. On this basis it will be evaluated the duration of the electrogram impulse. It was held the grafts flowmetry and compared to the results obtained on epicardial mapping. Results: The presented tecnic is an effective way to evaluate the regional myocardial contractility after the selective myocardial revascularization. The graft flowmetry doesn\'t interfere with the duration of the impulse obtained during the selective perfusion for the studied areas.

INTRODUÇÃO: O carcinoma espinocelular de origem bucal (CEC) apresenta uma alta taxa de mortalidade devido à invasividade das células tumorais. A migração celular, principal evento da invasão e metástase, pode ser regulada tanto por fatores intrínsecos, como adesão e contratilidade celular, quanto extrínsecos, como composição, densidade e remodelagem da matriz extracelular (MEC). OBJETIVO: Avaliar o papel de elementos intrínsecos e extrínsecos sobre o processo invasivo do carcinoma espinocelular de origem bucal. MÉTODOS: Foi realizada imuno-histoquímica para as proteínas: Miosina II (isoformas A, B e C), metaloproteinases de matriz (1, 2, 9 e 14); imunofluorescência as proteínas: e-caderina, n-caderina, FAK, paxilina, vinculina e fibronectina em amostras de CEC oral. Foi realizado ensaio de migração nas seguintes condições: 1 – matriz 2D com o substrato de fibronectina, ou laminina ou matrigel; 2 – matriz 3D com colágeno na presença ou não de fibronectina ou laminina; 3 – matriz 3D com diferentes concentrações de colágeno (0,6; 1,2 e 1,8 mg/ml) + fibronectina na presença ou não de um inibidor de MMP. Foi realizado análise de adesão celular utilizando-se o microscópio TIRF e o microscópio confocal, tanto em matrizes 2D quanto 3D. Foram realizados esferoides celulares para avaliar a contratilidade celular, através do plaqueamento das células em gel de agarose e a utilização de drogas que inibem ou que induzem a contratilidade, bem como a partir de células transfectadas com versões fosfomiméticas para a cadeia leve de miosina. Foi realizado ainda western blotting para proteínas: e-caderina, FAK, vinculina, paxilina, N-caderina, integrinas e as isoformas de miosina II, bem como foi avaliado os níveis de ativação das proteínas da família RhoGTPase, as quais estão envolvidas no controle da migração celular. RESULTADOS: A expressão das MMPs analisadas e das isoformas de miosinas foi maior nas zonas de invasão tumoral, sendo que o CEC oral também apresenta uma maior expressão de proteínas associadas à adesão com a MEC. A migração celular foi afetada pela densidade e a composição da MEC, bem como pela atividade das MMPs. Adicionalmente, a modulação das proteínas de adesão célula-matriz altera a velocidade de migração, a direcionalidade dessa migração e também a forma de migração, mudando de uma migração coletiva para uma migração individual. O aumento na contratilidade células resulta numa dispersão celular enquanto que a diminuição da contratilidade resulta numa melhor adesão célula – célula. CONCLUSÕES: O comportamento das células tumorais pode ser modulado através de fatores extrínsecos como, por exemplo, a alteração no microambiente tumoral, seja ela por mudança no substrato ou na densidade da matriz, e também dos fatores intrínsecos como a alteração nos níveis de miosina. / INTRODUCTION: Oral squamous cell carcinoma (OSCC) presents high mortality index due to the invasive phenotype of tumor cells. Cell migration is the main event in cell invasion and metastasis and it can be regulated by intrinsic factor, such as adhesion and cell contractility, and extrinsic factors, such as density and extracellular matrix (EMC) remodeling. OBJECTIVE: Analyze the role of intrinsic and extrinsic factor during the invasive process of oral squamous cell carcinoma. METHODS: We performed immunostaining in OSCC samples for the following proteins: myosin II (isoforms A, B and C), matrix metalloproteinase (1, 2, 9 and 14) e-cadherin, n-cadherin, FAK, paxillin, vinculin and fibronectin. We also performed migration assays with OSCC cell line in the following conditions 1 – 2D matrix with fibronectin or laminin or matrigel; 2 – 3D matrix with collagen in the presence or not of fibronectin or laminin; 3 – 3D matrix with different collagen concentration (0,6; 1,2 e 1,8 mg/ml) with fibronectin in the presence or not of the MMP inhibitor. In order to analyze cell adhesion, it was performed Total Internal Reflectance Fluorescence and Confocal microscopy, in 2D and 3D matrix. To analyze cell contractility, cells were plated in agarose gel in order to produce spheroids, which were treated with drugs that inhibit or induce cell contractility or cells were previously transfected with Myosin Light Chain phosphomimetics mutants. It was also performed western blotting to: e-cadherin, n-cadherin, FAK, paxillin, vinculin and myosin II isoforms, as well as it was analyze the levels in RhoGTPase family, which are involved in cell migration control. RESULTS: The expression to MMPs and myosin II isoforms were higher at invasion zone of the tumor, and the OSCC presented higher expression of proteins associated to adhesion to ECM. Cell migration was affected by the EMC composition and density and by MMP activity. Also, the modulation of cell-matrix adhesion proteins altered migration speed, cell directionality as well as influenced the switch between collective and single cell migration. The increase in cell contractility resulted in cell dispersion while the decrease in cell contractility resulted in a better cell-cell adhesion. CONCLUSIONS: The behavior of cell tumor can be modulate by extrinsic factors, for example, the change in tumor microenvironment, by the change in the EMC substrate or density and by intrinsic factors such as the alteration in myosin levels.

Orientador: Madileine Francely Américo / Resumo: A contratilidade do músculo liso e o trânsito gastrintestinal (GI) são importantes parâmetros da motilidade, cujas alterações podem comprometer a absorção de nutrientes e a biodisponibilidade de fármacos. Diversos fármacos apresentam efeitos adversos associados ao trato GI e, mesmo aqueles utilizados para controlar esses efeitos, podem provocar alterações importantes na motilidade ainda pouco conhecidas. Camundongos das linhagens C57BL/6 e BALB/c são amplamente utilizados nas pesquisas biomédicas, entretanto poucos estudos consideram os parâmetros motores GI. Diante de métodos escassos para avaliar a motilidade in vivo e da importância de conhecer a fisiologia básica dos camundongos, bem como o efeito de fármacos sobre o trato GI, objetivou-se: 1) desenvolver protocolo para avaliação da contratilidade duodenal e trânsito gastrintestinal por meio da Biosusceptometria de Corrente Alternada (BAC), e avaliar comparativamente a morfologia e função gastrintestinal de camundongos das linhagens BALB/c e C57BL/6J e, 2) avaliar o efeito GI do tratamento com cisplatina e a influência da dexametasona como antiemético. A partir deste trabalho foi possível implementar um protocolo experimental inédito, por meio da BAC, para avaliar a contratilidade duodenal e o trânsito gastrintestinal em camundongos. Após prévia implantação de marcador magnético no duodeno de camundongos, observou-se uma alta e uma baixa frequência de contração, expressas em ciclos por minuto (cpm). BALB/c e C57BL/6J apre... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The smooth muscle contractility and gastrointestinal (GI) transit are important parameters of motility, whose alterations may compromise nutrient absorption and bioavailability of drugs. Several drugs have side effects associated with the GI tract and, even those used to control these effects may cause significant changes in motility that are still poorly understood. Mice C57BL/6 and BALB/c strains are widely used in biomedical research, but a few studies consider the GI motor parameters. In front of scarce methods to evaluate the in vivo motility and the importance of knowing the basic physiology of the mice, as well as the effect of drugs on the GI tract. Then, the objectives of this study were 1) to develop protocol for the evaluation of duodenal contractility and gastrointestinal transit through Alternating Current Biosusceptometry (ACB), and comparing the morphology and gastrointestinal function of BALB/c and C57BL/6J mice, and 2) to evaluate the GI effect of cisplatin treatment and the influence of dexamethasone as an antiemetic. From this work, it was possible to implement an original experimental protocol, through the BAC, to evaluate the duodenal contractility and the gastrointestinal transit in mice. After the previous implantation of a magnetic marker in the duodenum of mice, a high and low frequency of contraction, expressed in cycles per minute (cpm), was observed. BALB/c and C57BL/6J presented a high frequency of 40 cpm, while the low frequency in BALB/c was 29 ... (Complete abstract click electronic access below) / Doutor