Recently, we demonstrated the beneficial effects of engineered heart tissues for the treatment of dilated cardiomyopathy in rats. For further development of this technique we started to produce engineered tissue (ET) from mesenchymal stem cells. Interestingly, we observed a malignant tumor invading the heart with an inverse relationship between proliferation markers and connexin expression.

Die vorliegende Dissertation befasst sich mit Entstehungsmechanismen kardialer Arrhythmien auf der Grundlage pathologisch veränderten Myokards. Es wurde eine systematische Analyse der elektrophysiologischen Veränderungen, die als Folge von Gap-Junction- Lateralisierung und zellulärer Hypertrophie auftreten, durchgeführt. Die Analyse beruht auf einem mathematischen Computermodell, das zur Simulation der Aktionspotentialausbreitung innerhalb einer Einzelzellschicht humaner ventrikulärer Kardiomyozyten entwickelt wurde. Ausgehend von bestehenden Einzelzellmodellen wurde ein räumlich und zeitlich hoch aufgelöstes Multizellmodell generiert und in der Programmiersprache Object Pascal implementiert. Nach Validierung des Modells wurde es zur gezielten, an experimentellen Daten orientierten Manipulation geometrischer Eigenschaften der Zellen (Länge, Durchmesser) und des Zellverbandes (Anordnung der Zellen untereinander) sowie der Gap-Junction-Verteilung genutzt. Die Analyse der elektrophysiologischen Effekte im Vergleich zur Kontrolle fand sowohl unter Normalbedingungen als auch unter Bedingungen, die pathologischen Veränderungen entsprechen (Entkopplung der Gap-Junctions, verringerte Aktivität des schnellen Natriumkanals, erhöhte Inhomogenität), statt. Es zeigte sich, dass ein größerer Zelldurchmesser bzw. erhöhte laterale Gap-Junction-Leitfähigkeit (Simulation von kardialer Hypertrophie bzw. Connexin- Lateralisierung) die Entstehungswahrscheinlichkeit eines unidirektionalen Leitungsblocks erhöhte. Die Erregungsausbreitungsgeschwindigkeit in hypertrophierten Zellen war zudem weniger stabil als in normalen Zellen. Beide Effekte gehören zu den Hauptursachen der Entstehung und Aufrechterhaltung ventrikulärer Arrhythmien. Die Ergebnisse der Arbeit erklären somit Ursachen des erhöhten Arrhythmierisikos in pathologisch veränderten und hypertrophierten Herzen und liefern eine theoretische Grundlage für zukünftige Studien.

Recentes estudos sugerem a existência de sinapses elétricas mediadas por junções gap entre fotorreceptores na retina de vertebrados. Neste trabalho, descrevemos um modelo computacional dos circuitos primário e secundário mediados pelos bastonetes da retina de vertebrados. O modelo é composto pelas seguintes populações de células: bastonetes, cones, células bipolares dos bastonetes, células bipolares dos cones, células amácrinas do tipo AII e células ganglionares. As células do modelo estão acopladas entre si por sinapses químicas e elétricas segundo padrões realísticos de convergência e divergência. As sinapses elétricas ocorrem entre os bastonetes, entre os bastonetes e os cones, entre as células amácrinas AII e entre as células bipolares dos cones e a células amácrinas AII. O modelo assume que um estímulo luminoso de baixa intensidade, simulando condições escotópicas, atinge todos os bastonetes da camada receptora, porém menos da metade deles é excitada. A resposta dos bastonetes excitados é controlada por uma fotocorrente cuja amplitude pode ser alterada para simular estímulos de diferentes intensidades dentro da faixa escotópica. O modelo é utilizado para investigar os efeitos dos diferentes graus de acoplamento elétrico entre as células receptoras e entre as células amácrinas AII, além do efeito de diferentes valores de condutância do canal Ih ativado pela hiperpolarização nos bastonetes, sobre a faixa dinâmica da retina. Os resultados das simulações mostram que, para valores realísticos da condutância do canal Ih, a faixa dinâmica medida na camada receptora é maximizada para o índice de conectividade crítico para que haja percolação de ligação. No entanto, quando a faixa dinâmica é medida para as células bipolares ou ganglionares o valor máximo é obtido para um índice de conectividade subcrítico. Este resultado é conseqüência da alta convergência de sinapses químicas entre os bastonetes e células bipolares. / Recent studies suggest the existence of electrical synapses (gap junctions) connecting photoreceptors in the vertebrate retina. In this work we describe a computer model of the primary and secondary rod pathways in the vertebrate retina. The model is composed of the following cell populations: rods, cones, rod bipolar cells, cone bipolar cells, AII amacrine cells and ganglion cells. Cells of the model are connected via chemical as well as electrical synapses according to realistic convergence and divergence factors. There are electrical synapses between rods, rods and cones, AII amacrine cells, and cone bipolar cells and AII amacrine cells. The model assumes that low intensity stimuli simulating scotopic conditions reach all rods in the receptor array but less than half of them are excited. The excited rods response is controlled by a photocurrent waveform whose amplitude can be manipulated to simulate stimuli of different intensities within the scotopic range. The model is used to investigate the effects of different degrees of coupling among photoreceptors and among AII amacrine cells, as well as values of rod hyperpolarization activated current Ih on the dynamic range of the retina. Results show that for realistic values of Ih conductance the dynamic range of the rod array is maximized at the critical connectivity degree for bond percolation. However, the dynamic range of the rod bipolar and ganglion cells is maximized for a photoreceptor connectivity degree below the critical value. The latter result is a consequence of the high convergence of chemical synapses from rods to rod bipolar cells.

Durante o desenvolvimento pós-natal do hipocampo, as junções comunicantes (JC) formadas por conexinas (Cxs) neuronais participam na maturação da circuitaria hipocampal promovendo a regulação da atividade espontânea sincronizada neuronal. Neste estudo investigamos as duas Cxs neuronais mais abundantes no hipocampo, a Cx36 e Cx45, durante o desenvolvimento pós-natal. Identificamos mudanças nos níveis de transcritos e proteicos da Cx36 e Cx45 ao longo deste período. Nossos resultados revelaram que ambas as Cxs neuronais estão presentes nas sub-regiões do hipocampo e que sua distribuição é modulada em função da progressão do desenvolvimento. Atráves da avaliação dos níveis de atividade neuronal identificamos diferenças exercidas pelo bloqueio das JCs em hipocampo de neonatos e na segunda semana de vida. Nossos resultados mostram que as Cxs neuronais são reguladas durante o desenvolvimento pós-natal do hipocampo, assim como sua ação sobre sua excitabilidade, mostrando que as Cxs podem contribuir de forma distinta em periodo específicos do desenvolvimento hipocampal. / Gap junctions (GJ) composed of neuronal connexins (Cx) play a significant role in the activity-dependent circuitry maturation promoting modulation of coherent spontaneous neuronal activity. Herein, we evaluate two major hippocampal neuronal Cxs, Cx36 and Cx45 during postnatal development. We identified changes in Cx36 and Cx45 transcript and protein levels during these developmental periods. Interestingly, immunofluorescence analyses showed that Cx36 and Cx45 are located in all hippocampal subregions. Also, neuronal Cx distribution in these sub-regions is modulated throughout postnatal development. Using electrophysiological recording, we identified changes imposed by GJ blocker over the hippocampal activity in neonates and two-week-old rats. Our finds demonstrate a regulation of neuronal Cxs and distinct GJ role in activity modulation during postnatal hippocampal development, which might be essential to physiological processes that govern proper hippocampal development.

Teoricamente, conexões por sinapses elétricas entre neurônios poderiam levar ao aumento da faixa de resposta dinâmica da rede neural. A faixa de resposta dinâmica de uma rede de neurônios pode ser definida como a faixa de valores de intensidade dos estímulos de entrada para a qual o conjunto de neurônios produz resposta antes de atingir a saturação. Em um cenário biológico, propôs-se que junções gap entre células ganglionares da retina aumentariam a faixa dinâmica da retina. O teste experimental dessa proposta apresenta várias dificuldades, o que torna a modelagem computacional uma alternativa metodológica para o estudo do papel das sinapses elétricas na faixa dinâmica da camada ganglionar da retina. O objetivo deste trabalho foi a construção de um modelo biologicamente plausível da camada ganglionar da retina da salamandra, submetida a sinais de entrada realísticos conforme evidências experimentais e com a inclusão de sinapses elétricas conectando suas células, para estudar in silico os possíveis efeitos dessas sinapses elétricas sobre a faixa dinâmica da camada ganglionar. A camada ganglionar foi modelada como uma rede bidimensional cujos neurônios foram modelados pelo formalismo de Hodgkin-Huxley. Cada neurônio recebeu um de dois tipos de entrada sináptica, transiente ou sustentada. Avaliou-se o efeito da inibição pré-sináptica das células ganglionares e o efeito de diferentes padrões de conectividade mediados pelas sinapses elétricas. Os resultados sugerem que o acoplamento elétrico aumenta a sensibilidade do sistema e altera o ponto de saturação, mas não necessariamente aumenta a faixa dinâmica. / Theoretically, connections by electrical synapses between neurons could lead to an increase in their dynamic range. The dynamic range of a network of neurons can be defined as the range of input stimuli values for which the network responds before saturation. In a biological scenario, it is hypothesized that gap junctions between retinal ganglion cells may increase the dynamic range of the retina. However, the experimental testing of this hypothesis presents several difficulties, which makes computational modeling a methodological alternative to study the role of electrical synapses on the dynamic range of the ganglion cell layer of the retina. In this work we constructed a biologically plausible computational model of the ganglion cell layer of the salamander retina. A bidimensional network was built with cells modeled by the Hodgkin-Huxley formalism connected via gap junctions and subject to realistic inputs constrained by experimental evidence, to study in silico the effects of gap junctions on the dynamic range of the model. We studied the effect of different gap junction-mediated connectivity patterns, input type combinations (transient, sustained and mixed between the two) and presynaptic inhibition on the dynamic range. Our results suggest that gap junction coupling increases the network\'s sensitivity and alters the saturation point but not necessarily increases the dynamic range.

Made available in DSpace on 2014-06-11T19:31:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-27Bitstream added on 2014-06-13T19:19:53Z : No. of bitstreams: 1 duarte_dr_dr_botfm.pdf: 365312 bytes, checksum: 5b54817ed6d69d3fe0e8673385f863f0 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O hábito de fumar apresenta importante impacto na saúde da população. A associação entre fumo e doença cardiovascular tem sido amplamente explorada em relação à aterosclerose. Recentemente, evidências clínicas e experimentais sugerem que a exposição ao cigarro pode modular o processo de remodelação ventricular. O objetivo desse estudo foi avaliar o papel do inibidor da enzima conversora da angiotensina no processo de remodelação induzido pela exposição à fumaça do cigarro. Ratos Wistar foram distribuídos em três grupos: 1) grupo controle (C, n=8); 2) grupo exposto à fumaça do cigarro (EFC, n=8); 3) grupo exposto à fumaça do cigarro e recebendo 20 mg/kg/dia de lisinopril (EFC-LIS, n=8). Após dois meses os animais foram submetidos ao estudo funcional, morfométrico, e bioquímico. Para a análise estatística foi utilizado o teste de variância ANOVA complementado por Holm-Sidak, o teste Kruskal-Wallis complementado por Tukey e o teste Mann-Whitney. O nível de significância foi 5%. A pressão sistólica caudal foi menor no grupo EFC-LIS (C = 116 ± 27, EFC = 126 ± 16, EFC-LIS = 89 ± 12 mmHg; p = 0,003) em relação aos grupos C e EFC; o grupo EFC apresentou maior valor do diâmetro sistólico do ventrículo esquerdo, corrigido pelo peso (C = 8,25 ± 2,16, EFC = 11,76 ± 1,20, EFC-LIS = 9,27 ± 2,00 mm/kg; p = 0,003), em comparação aos grupos C e EFC-LIS; o diâmetro diastólico do ventrículo esquerdo ajustado pelo peso foi maior nos grupos EFC e EFC-LIS... / The habit of smoking has important impact on population health. The association between tobacco and cardiovascular disease has been widely explored in relation to atherosclerosis. Recently, clinical and experimental evidences suggest that the exposure to tobacco smoke can modulate the process of ventricular remodeling. The objective of that study was to evaluate the role of the angiotensin-converting enzyme inhibitor on cardiac remodeling induced by tobacco smoke exposure. Wistar rats were allocated in three groups: 1) group control (C, n=8); 2) group exposed to tobacco smoke (EFC, n=8); 3) group exposed to tobacco smoke and treated with lisinopril 20mg/kg/day (EFC-LIS, n=8). After two months the animals were submitted to the functional study, morphometric, and biochemical. For the statistical analysis was used the ANOVA test of variance complemented by Holm-Sidak, the test Kruskal-Wallis complemented by Tukey and the test the Mann-Whitney. The significance level was 5%. Results: the caudal systolic pressure was smaller in the group EFC-LIS (C = 116 ± 27, EFC = 126 ± 16, EFC-LIS = 89 ± 12 mmHg; p = 0,003) in relation to the groups C and EFC; the group EFC presented higher value of the systolic diameter of the left ventricle, corrected by body weight (BW) (C = 8,25 ± 2,16, EFC = 11,76 ± 1,20, EFC-LIS = 9,27 ± 2,00 mm/kg; p = 0,003), in comparison with the groups C and EFC-LIS; the diastolic diameter of the left ventricle, adjusted by BW was higher in the groups EFC e EFC-LIS (C = 18,42 ± 3,57, EFC = 22,94 ± 1,98, EFC-LIS = 22,05 ± 1,30 mm/kg; p = 0,003); the relationship EPP/DDVE was smaller in the group EFC-LIS in relation to the control (C = 0,20 (0,18-0,23), EFC = 0,15 (0,14-0,18), EFC-LIS = 0,14 (0,14-0,18); p = 0,026). The group EFC presented smaller ejection fraction... (Complete abstract click electronic access below)

Recentes estudos sugerem a existência de sinapses elétricas mediadas por junções gap entre fotorreceptores na retina de vertebrados. Neste trabalho, descrevemos um modelo computacional dos circuitos primário e secundário mediados pelos bastonetes da retina de vertebrados. O modelo é composto pelas seguintes populações de células: bastonetes, cones, células bipolares dos bastonetes, células bipolares dos cones, células amácrinas do tipo AII e células ganglionares. As células do modelo estão acopladas entre si por sinapses químicas e elétricas segundo padrões realísticos de convergência e divergência. As sinapses elétricas ocorrem entre os bastonetes, entre os bastonetes e os cones, entre as células amácrinas AII e entre as células bipolares dos cones e a células amácrinas AII. O modelo assume que um estímulo luminoso de baixa intensidade, simulando condições escotópicas, atinge todos os bastonetes da camada receptora, porém menos da metade deles é excitada. A resposta dos bastonetes excitados é controlada por uma fotocorrente cuja amplitude pode ser alterada para simular estímulos de diferentes intensidades dentro da faixa escotópica. O modelo é utilizado para investigar os efeitos dos diferentes graus de acoplamento elétrico entre as células receptoras e entre as células amácrinas AII, além do efeito de diferentes valores de condutância do canal Ih ativado pela hiperpolarização nos bastonetes, sobre a faixa dinâmica da retina. Os resultados das simulações mostram que, para valores realísticos da condutância do canal Ih, a faixa dinâmica medida na camada receptora é maximizada para o índice de conectividade crítico para que haja percolação de ligação. No entanto, quando a faixa dinâmica é medida para as células bipolares ou ganglionares o valor máximo é obtido para um índice de conectividade subcrítico. Este resultado é conseqüência da alta convergência de sinapses químicas entre os bastonetes e células bipolares. / Recent studies suggest the existence of electrical synapses (gap junctions) connecting photoreceptors in the vertebrate retina. In this work we describe a computer model of the primary and secondary rod pathways in the vertebrate retina. The model is composed of the following cell populations: rods, cones, rod bipolar cells, cone bipolar cells, AII amacrine cells and ganglion cells. Cells of the model are connected via chemical as well as electrical synapses according to realistic convergence and divergence factors. There are electrical synapses between rods, rods and cones, AII amacrine cells, and cone bipolar cells and AII amacrine cells. The model assumes that low intensity stimuli simulating scotopic conditions reach all rods in the receptor array but less than half of them are excited. The excited rods response is controlled by a photocurrent waveform whose amplitude can be manipulated to simulate stimuli of different intensities within the scotopic range. The model is used to investigate the effects of different degrees of coupling among photoreceptors and among AII amacrine cells, as well as values of rod hyperpolarization activated current Ih on the dynamic range of the retina. Results show that for realistic values of Ih conductance the dynamic range of the rod array is maximized at the critical connectivity degree for bond percolation. However, the dynamic range of the rod bipolar and ganglion cells is maximized for a photoreceptor connectivity degree below the critical value. The latter result is a consequence of the high convergence of chemical synapses from rods to rod bipolar cells.

The first part of my thesis involves an investigation into mechanisms underlying the presynaptic regulation of transmitter release from myelinated hindlimb sensory afferents in rodents. The central hypothesis is that in addition to chemical transmission in spinal neuronal networks, electrical synapses formed by connexins are critically involved in presynaptic inhibition of large diameter sensory afferents. Subsequent sections of the thesis present a detailed examination of the distribution of connexins in the rodent spinal cord with a particular emphasis on the neuronal connexin, Cx36. Connexin36 (Cx36) is widely believed to be the protein forming the neuronal gap junctions that create electrical synapses between mammalian neurons in many areas of the central nervous system (Condorelli et al 1998). The first part of thesis concerns a previously unknown role of neuronal connexins in interneurone pathways involved in presynaptic control of synaptic transmission in the lumbar spinal cord of rodents. As far as we are aware, the idea that electrical contacts between spinal neurons contribute to spinal presynaptic inhibition is a novel hypothesis. Evidence will be presented: 1) that Cx36 is present in regions of the spinal cord containing interneurons involved in presynaptic inhibition, 2) that the lack of Cx36 in Cx36-/- knockouts mice results in a severe impairment of presynaptic inhibition, and 3) that blocking gap junctions pharmacologically in wild type mice impairs presynaptic inhibition. The exploration of this hypothesis will involve a combination of electrophysiological and immunohistochemical approaches in juvenile wild-type and knockout mice lacking Cx36, as well as immunohistochemical observations in adult rodents. This first section of the thesis begins with the development of a preparation in which several measures of presynaptic inhibition described in the in vivo adult cat preparation can be examined in vitro in young mice. The following sections of the thesis describe the distribution and features of Cx36 on neurons in mice and rats of different ages in four parts. The first will show that Cx36 is the only connexin associated with spinal neurons and refutes claims in the literature about the existence of a variety of connexions on spinal neurons. The second part will show that while gap junctions between some spinal neurons are only a transient developmental phenomenon, they persist in abundance in adult animals. The third part will present evidence of a previously unsuspected III association of Cx36 gap junctions at the chemical synapse between muscle afferent fibres and motoneurons. Specifically, an association between Cx36 and the glutamate transporter used in primary afferents, Vglut1 will be described. To our knowledge these results are the first to suggest the existence of mixed (electrical and chemical) synapses between primary afferents and motoneurons in the mature mammalian spinal cord. The final part of the thesis will describe the presence of Cx36 gap junctions on adult sacral motoneurons involved in control of sexual, urinary and defecation functions in the rodent.

The first part of my thesis involves an investigation into mechanisms underlying the presynaptic regulation of transmitter release from myelinated hindlimb sensory afferents in rodents. The central hypothesis is that in addition to chemical transmission in spinal neuronal networks, electrical synapses formed by connexins are critically involved in presynaptic inhibition of large diameter sensory afferents. Subsequent sections of the thesis present a detailed examination of the distribution of connexins in the rodent spinal cord with a particular emphasis on the neuronal connexin, Cx36. Connexin36 (Cx36) is widely believed to be the protein forming the neuronal gap junctions that create electrical synapses between mammalian neurons in many areas of the central nervous system (Condorelli et al 1998). The first part of thesis concerns a previously unknown role of neuronal connexins in interneurone pathways involved in presynaptic control of synaptic transmission in the lumbar spinal cord of rodents. As far as we are aware, the idea that electrical contacts between spinal neurones contribute to spinal presynaptic inhibition is a novel hypothesis. Evidence will be presented: 1) that Cx36 is present in regions of the spinal cord containing interneurones involved in presynaptic inhibition, 2) that the lack of Cx36 in Cx36-/- knockouts mice results in a severe impairment of presynaptic inhibition, and 3) that blocking gap junctions pharmacologically in wild type mice impairs presynaptic inhibition. The exploration of this hypothesis will involve a combination of electrophysiological and immunohistochemical approaches in juvenile wild-type and knockout mice lacking Cx36, as well as immunohistochemical observations in adult rodents. This first section of the thesis begins with the development of a preparation in which several measures of presynaptic inhibition described in the in vivo adult cat preparation can be examined in vitro in young mice. The following sections of the thesis describe the distribution and features of Cx36 on neurones in mice and rats of different ages in four parts. The first will show that Cx36 is the only connexin associated with spinal neurons and refutes claims in the literature about the existence of a variety of connexions on spinal neurons. The second part will show that while gap junctions between some spinal neurons are only a transient developmental phenomenon, they persist in abundance in adult animals. The third part will present evidence of a previously unsuspected III association of Cx36 gap junctions at the chemical synapse between muscle afferent fibres and motoneurones. Specifically, an association between Cx36 and the glutamate transporter used in primary afferents, Vglut1 will be described. To our knowledge these results are the first to suggest the existence of mixed (electrical and chemical) synapses between primary afferents and motoneurones in the mature mammalian spinal cord. The final part of the thesis will describe the presence of Cx36 gap junctions on adult sacral motoneurones involved in control of sexual, urinary and defecation functions in the rodent.

Plyšinės jungtys (PJ) yra tarpląstelinę sąveiką užtikrinantys kanalai, svarbūs ląstelių dauginimuisi, diferenciacijai, migracijai bei koordinacijai. Skirtinguose žmogaus organizmo audiniuose atrastas 21 koneksino (Cx) tipas. Tam tikros ligos yra susijusios su Cx mutacijomis. PJ kanalų savybes įtakoja jungties įtampa, viduląstelinis pH (pHi), viduląstelinė dvivalenčių katijonų koncentracija ([Ca2+]i, [Mg2+]i), fosforinimas, įvairios cheminės medžiagos. Disertacinio darbo metu pirmą kartą parodėme, kad plačiai naudojamų skirtingų grupių PJ slopiklių, tokių kaip alkanolių, forano, meflokvino, veikimas priklauso nuo pHi ir Cx tipo. Iškėlėme ir eksperimentiškai patikrinome hipotezę, kad tam tikri PJ slopikliai veikia, sudarydami vandenilinius ryšius su Cx45 baltymo histidinais. Ištyrėme Cx36, Cx40, Cx43, Cx45 ir Cx47 PJ kanalų laidumo priklausomybę nuo pHi. Nustatėme [Mg2+]i įtaką Cx43 ir Cx45 PJ kanalų laidumui bei Cx36 PJ kanalų laidumo priklausomybei nuo pHi, kas iki šiol nebuvo tirta. Cx45 ir Cx36 mutantinių baltymų tyrimai suteikė naujų žinių apie šių baltymų amino rūgštis, kurios gali būti reikšmingos PJ kanalų laidumo reguliavimui viduląsteliniu pH. / Gap junctions (GJ) are intercellular channels, which provide a direct pathway for electrical and metabolic cell-to-cell communication. GJ play an important role in cell proliferation, differentiation, migration, and coordination. The family of connexin (Cx) genes consists of 21 members in the human genome, and a variety of diseases is associated with Cx mutations. Electrical conductance (gj) of gap junctions is regulated by transjunctional voltage, intracellular pH (pHi), intracellular divalent cation concentration ([Ca2+]i and [Mg2+]i), phosphorylation and different chemical compounds. In this work we demonstrate for the first time that the potency of widely used GJ inhibitors such as alkanols, forane and mefloquine in GJ channel uncoupling is pHi- and Cx type-dependent. This type of modulation may occur through the formation of hydrogen bonds between uncouplers and histidines of Cx45 protein. We determined gj-pHi dependence of Cx36, Cx40, Cx43, Cx45 and Cx47 GJ channels, and evaluated the influence of [Mg2+]i on the conductance of Cx43 and Cx45 GJs and on the sensitivity of Cx36 GJs to pHi. The investigation of the Cx45 and Cx36 mutants provided with new knowledge on the structural elements, which may be responsible for the sensitivity of Cxs to pHi.