CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Com o objetivo de verificar a interferÃncia da broncoprovocaÃÃo antigÃnica sobre a contratilidade traqueal, ratos machos (250 -350 g) foram sensibilizados à ovalbumina (OVA) e, 13 a 14 dias depois, desafiados em intervalos de 15 minutos atravÃs da inalaÃÃo do antÃgeno sensibilizante (OVA, 1 mg/ml, seguida de 5 mg/ml). O sacrifÃcio dos animais foi feito imediatamente (SD0) ou 24 horas (SD24) apÃs o desafio antigÃnico atravÃs de anestesia com hidrato de cloral (0,4 g/Kg). Alguns animais foram tratados com metilsergida (1 mg/Kg) 40 minutos antes do desafio. Os animais controle (CONT) inalaram apenas o veÃculo (NaCl 0,9 %). A traquÃia foi removida e montada em cuba para orgÃo isolado contendo 10 ml de soluÃÃo de Krebs-Henseleit modificada (mantida a 37Â0,5 oC) e aerada com mistura carbogÃnica (O2/CO2â95:5). Foram confeccionadas curvas concentraÃÃo-efeito (CCE) para cloreto de potÃssio (KCl), acetilcolina (ACh) ou serotonina (5-HT). Em outros experimentos foram realizadas CCE ao Ca2+ em preparaÃÃes mantidas previamente em soluÃÃo sem Ca2+ (contendo 10-5 M de EDTA, nifedipina e indometacina, 10-6 M para ambas) e estimuladas por KCl, ACh ou 5-HT. A sensibilizaÃÃo e o posterior desafio antigÃnico (SD) promoveram um significativo aumento da resposta mÃxima (RM) das CCE ao KCl (forÃa em grama, mÃdia  E.P.M.: CONT = 0,52  0,01; SD24 = 1,22  0,03; n = 06; p < 0,01), à ACh (CONT = 2,11  0,10; SD24 = 3,53  0,03; n = 06; p < 0,01) ou à 5-HT (CONT = 0,60  0,03; SD24 = 1,48  0,09; n = 06; p < 0,01). As traquÃias de animais sensibilizados e desafiados apresentaram aumento da RM ao Ca2+ apenas quando prÃ-contraÃdas com KCl (CONT = 0,84  0,08; SD24 = 1,98  0,05; n = 6; p < 0,01) ou ACh (CONT = 0,98  0,09; SD24 = 1,54  0,15; n = 6; p < 0,05). Quando o agonista utilizado foi a 5-HT esta diferenÃa nÃo ocorreu, sendo observada uma maior RM somente na ausÃncia de nifedipina (CONT = 0,81  0,06; SD24 sem nifedipina = 1,23  0,08; n = 05; p < 0,01). Foram comparados os animais apenas sensibilizados e nÃo desafiados (SENS) com os SD na presenÃa de Ãcido niflÃmico (AN). O AN inibiu a hiperreatividade para a 5-HT induzida pelo desafio antigÃnico (SENS = 1,28  0,09; SD24 + AN = 1,01  0,08; n = 05), enquanto que foi ineficaz em inibir a hiperreatividade para a ACh (SENS = 1,44  0,01; SD24 + NA = 1,67  0,03; n = 05; p < 0,01). Portanto, os resultados mostram que hà envolvimento de canais de Ca2+, tanto dependentes de voltagem (VOC) como operados por receptor (ROC), na hiperreatividade do mÃsculo liso respiratÃrio, induzida pela reapresentaÃÃo do antÃgeno a animais previamente sensibilizados e que o desenvolvimento da hiperreatividade para a 5-HT està relacionado à abertura de canais de Cl- ativados por Ca2+ / In order to verify the interference of the antigenic challenge on traqueal contractility in vitro, male rats (250 -350 g) were ovalbumine (OVA)-sensitized and, 13 to 14 days later, they were challenged through the sensitizing antigen inhalation (OVA, 1 mg/ml, followed by 5 mg/ml). Animals were sacrificed immediately (SD0) or 24 hours (SD24) after the antigenic challenge through chloral hydrate anaesthesia (0,4 g/Kg). Some animals were treated with metilsergide (1 mg/Kg) 40 minutes before the challenge. Control animals (CONT) received only the vehicle (NaCl 0,9 %) by inhalation. Tracheal rings were carefully removed and mounted in a 10 ml isolated bath chamber with modified Krebs-Henseleit solution (at 37Â0,5 oC) bubbled with a mixture of 5% of CO2 in 95% of O2. Concentration-effect curves (CCE) were constructed for potassium chloride (KCl), acetycholine (ACh) or serotonin (5-HT). In another experiments, CCE for Ca2+ addition were constructed under Ca2+-free conditions (with 10-5 M EDTA, nifedipine and indomethacin, 10-6 M each) in the presence of KCl, ACh or 5-HT. Sensitization and subsequent antigenic challenge (SD) promoted a significant increase of the maximal response (RM) of the CCE to KCl (force in grams, mean  S.E.M.: CONT = 0,52  0,01; SD24 = 1,22  0,03; n = 06; p < 0.01), ACh (CONT = 2.11  0,10; SD24 = 3,53  0,03; n = 06; p < 0.01) or to 5-HT (CONT = 0,60  0,03; SD24 = 1,48  0,09; n = 06; p < 0.01). Tracheal rings of sensitized and challenged animals showed a rise in RM for Ca2+ only when they were pre-contracted with KCl (CONT = 0,84  0,08; SD24 = 1,98  0,05; n = 6; p < 0.01) or ACh (CONT = 0,98  0,09; SD24 = 1,54  0,15; n = 6; p < 0,05). When 5-HT was used as agonist this hyperresponsiveness did not occur, being observed only in the nifedipine absence (CONT = 0.81  0,06; SD24 without nifedipine = 1,23  0,08; n = 05; p < 0.01). The sensitized but not challenged animals (SENS) were compared to SD animals in the presence of niflumic acid (AN). AN inhibited the antigenic shock-induced hyperresponsiveness for 5-HT (SENS = 1,28  0,09; SD24 + AN = 1.01  0,08; n = 05), while it was ineffective in inhibit that for the ACh (SENS = 1,44  0,01; SD24 + AN = 1,67  0,03; n = 05; p < 0.01). Therefore, the results show that there is a participation of Ca2+ channels, both voltage- and receptor-operated channels, in the hyperresponsiveness of the respiratory smooth muscle, induced by the representation of the antigen to previously sensitized animals and, in addition, the Cl- channels Ca2+-activated have an important role on 5-HT hyperresponsiveness development

Orientador: Iscia Teresinha Lopes Cendes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T06:36:18Z (GMT). No. of bitstreams: 1 Gonsales_MarinaCoelho_D.pdf: 5961380 bytes, checksum: 501b9df1659eb15a6a8545ae0943d967 (MD5) Previous issue date: 2013 / Resumo: As epilepsias são distúrbios cerebrais caracterizados por uma predisposição persistente para a geração de crises epilépticas, que são interrupções transitórias no funcionamento normal do sistema nervoso. Acredita-se que a maioria das epilepsias relacionadas com idade de inicio precoce possui etiologia presumivelmente genética. Sendo assim, elas representam um grupo para o qual o uso de testes genéticos seria potencialmente benéfico. Os objetivos principais deste trabalho foram: a caracterização das bases moleculares de diferentes formas de epilepsia da infância e da adolescência e a avaliação do potencial dos genes candidatos estudados para a utilização em testes genéticos para fins clínicos. A estratégia empregada foi à triagem de mutações nos seguintes genes: SCN1A, em pacientes com síndromes de Dravet e de Doose; SCL2A1 em pacientes com síndrome de Doose e epilepsias idiopáticas generalizadas (EIGs), especialmente a epilepsia mioclonica juvenil (EMJ); e EFHC1 e GABRA1, em pacientes com EMJ e outras formas comuns de EIGs. A triagem de mutações foi realizada por sequenciamento automático pela técnica de Sanger. As alterações potencialmente deletérias foram investigadas em um grupo controle composto por 100 indivíduos sem epilepsia. O potencial deletério das substituições que resultam em troca do resíduo de aminoácido na proteína codificada foi estimado utilizando-se diferentes algoritmos de predição. As mutações previamente descritas na literatura foram compiladas e analisadas quanto a sua provável localização na proteína e predição de efeito deletério. Analises por Multiplex Ligation-dependent Probe Amplification (MLPA) foram realizadas para a detecção de variações no numero de copias de SCN1A. A analise de mutações no gene SCN1A revelou alterações potencialmente deletérias em 81% dos pacientes com síndrome de Dravet, e em apenas um paciente com síndrome de Doose. Esses dados, juntamente com os resultados das analises de compilação das mutações descritas na literatura, sugerem que o teste genético em SCN1A para fins clínicos seria altamente recomendável em indivíduos com síndrome de Dravet, mas não para os com síndrome de Doose típica. O gene SLC21A não parece estar envolvido na etiologia da síndrome de Doose e das EIGs em nossa casuística. A frequencia de alterações potencialmente deletérias no gene EFHC1 em indivíduos com EMJ foi relativamente baixa, sugerindo que esse gene não seja o principal causador dessa epilepsia, embora possa ser um fator de predisposição. Por fim, o gene GABRA1 não parece conferir predisposição para as EIGs comuns em nossa casuística / Abstract: Epilepsy is a brain disorder characterized by a long lasting predisposition to generate epileptic seizures, which are transient interruptions of normal brain function. Most epilepsies with early onset presumably have a genetic etiology. Thus, they represent a group for which the use of genetic testing would be potentially beneficial. The main goals of this study were to characterize the molecular basis of different forms of epilepsy in childhood and adolescence and to evaluate the potential clinical use of genetic testing in the context of these disorders. To achieve these goals we searched for mutations in the following genes: SCN1A in patients with Dravet and Doose syndromes; SLC2A1 in patients with Doose syndrome and idiopathic generalized epilepsies (IGEs), particularly juvenile myoclonic epilepsy (JME); and EFHC1 and GABRA1 in patients with JME and other common forms of IGEs. Mutation screening was performed by automated Sanger sequencing using capillary electrophoresis. Potentially deleterious nucleotide changes found were subsequently investigated in a control group of 100 individuals without epilepsy. In addition, the deleterious potential of amino acid changes identified was estimated using different prediction algorithms. Mutations previously described in the literature were compiled and analyzed regarding their putative location on the protein and predicted deleterious effect. Furthermore, Multiplex Ligation-dependent Probe Amplification (MLPA) analyzes were performed to detect the presence of copy number variations in SCN1A. Our results showed potentially deleterious variants in SCN1A in 81% of patients with Dravet syndrome, but only in one patient with Doose syndrome. These data, along with the results of the compilation of mutations reported in the literature suggest that genetic testing for SCN1A is clinically relevant in Dravet syndrome, but not in typical Doose syndrome. SLC21A does not seem to be involved in the etiology of Doose syndrome and EIGs in our cohort. The frequency of potentially deleterious changes in EFHC1 in individuals with JME was relatively low, suggesting that this gene is not the main cause of this form of epilepsy, although it may be a predisposing factor. Lastly, GABRA1 does not seem to confer predisposition to common EIGs in our cohort / Doutorado / Fisiopatologia Médica / Doutora em Ciências

Orientadores: Carlos Amilcar Parada, Helder José Ceragioli / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-19T15:58:34Z (GMT). No. of bitstreams: 1 Ferreira_PriscilaAmaral_M.pdf: 1496649 bytes, checksum: a3c769ea7d0b4a3fe45913f836d3615c (MD5) Previous issue date: 2011 / Resumo: Nanopartículas são estruturas com dimensões nanométricas que vem demonstrando um potencial para várias aplicações, inclusive na área biológica. De acordo com o seu processo de síntese e crescimento estas partículas podem possuir propriedades magnéticas com maior ou menor magnitude. O objetivo deste trabalho foi avaliar se a administração no espaço sub-aracnóide da medula espinal, de nanopartículas magnéticas altera o limiar de resposta a estímulos mecânicos aplicados em patas de ratos. As nanopartículas de carbono (nanotubos ou nanofibras) foram caracterizadas através do espectro de Raman e o seu comportamento magnético caracterizado pelo ferromagnetismo foi avaliado através da curva de histerese. As nanopartículas foram administradas via intratecal em ratos Wistar machos levemente anestesiados com Isoflorano. A resposta comportamental ao estímulo mecânico foi avaliada por um analgesímetro eletrônico (Von Frey eletrônico) que mede o limiar mecânico de retirada da pata (nocicepção mecânica). As nanopartículas foram classificadas de acordo com o comportamento magnético em: alto magnetismo e baixo magnetismo. A administração de nanopartículas (5, 20 ou 80 ?g) com alto magnetismo, mas não com baixo magnetismo, diminuiu o limiar mecânico de retirada da pata, quando comparada com a administração do seu solvente propilenoglicol (20?L). A diminuição do limiar mecânico foi observada nos tempos de 15, 30 e 60 minutos após a administração de 20?g de nanopartículas com alto magnetismo, sendo que após este período as respostas retornaram aos seus limiares basais. Por outro lado, a administração intraplantar das nanopartículas não alterou o limiar mecânico quando comparados com as respostas basais. Portanto, a administração na medula espinal, mas não no tecido periférico, de nanopartículas com alto magnetismo diminuiu o limiar mecânico. Em conclusão, os dados deste trabalho sugerem que a transmissão neuronal na medula espinal envolve uma atividade eletromagnética / Abstract: Nanoparticles are structures with nanometer dimensions that are emerging to various applications, including that one involved in biological area. According to their synthesis and growth techniques these particles can have magnetic properties with greater or lesser magnitude. The objective of this study was to evaluate whether the magnetic nanoparticles administration in the subarachnoid space of the spinal cord induces changes in the mechanical nociceptive threshold in rats hind-paw. The carbon nanoparticles (nanotubes or nanofibers) were characterized by Raman spectrum and their magnetic characterized by the ferromagnetism behavior was evaluated by the hysteresis curve. The nanoparticles were intrathecally administered in Wistar rats lightly anesthetized with Isoflorano. The behavioral response to mechanical stimulation was assessed by an electronic analgesimeter (electronic Von Frey) that measures the withdrawal mechanical threshold (mechanical nociception). The nanoparticles were classified according to their magnetic behavior in: high magnetism and low magnetism. The administration (5, 20 or 80 ?g) of high magnetism nanoparticles, but not of low magnetism nanoparticles, decreased the mechanical withdrawal threshold of the hind-paw, when compared with the administration of propylene glycol (20 ?L). Decreasing in mechanical withdrawal threshold was observed at 15, 30 and 60 minutes after administration of 20?g of high magnetism nanoparticles. After this period, the mechanical withdrawal thresholds returned to their normal baseline. In contrast, intraplantar administration of the nanoparticles did not alter the mechanical withdrawal threshold. Therefore, administration of high magnetic nanoparticles in the spinal cord, but not in peripheral tissue, decreases the mechanical withdrawal threshold. In conclusion, our data suggest that neuronal transmission in the spinal cord involves an electromagnetic activity / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

Neuron types (e.g., pyramidal cells) within one area of the brain are often considered homogeneous, despite variability in their biophysical properties. Here we review literature demonstrating variability in the electrical activity of CA1 hippocampal pyramidal cells (PCs), including responses to somatic current injection, synaptic stimulation, and spontaneous network-related activity. In addition, we describe how responses of CA1 PCs vary with development, experience, and aging, and some of the underlying ionic currents responsible. Finally, we suggest directions that may be the most impactful in expanding this knowledge, including the use of text and data mining to systematically study cellular heterogeneity in more depth; dynamical systems theory to understand and potentially classify neuron firing patterns; and mathematical modeling to study the interaction between cellular properties and network output. Our goals are to provide a synthesis of the literature for experimentalists studying CA1 PCs, to give theorists an idea of the rich diversity of behaviors models may need to reproduce to accurately represent these cells, and to provide suggestions for future research.

The auditory temporal processing deficits associated with age-dependent hearing decline have been increasingly attributed to issues beyond peripheral hearing loss. Age-related hearing loss (ARHL), also known as presbycusis, is linked with changes in the expression of both excitatory and inhibitory neurotransmitters in the central auditory system. There are also age-related changes in the expression and function of the ion channels which mediate action potential firing. The slow, Ca2+ activated, K+ channels of the BK-type are essential in controlling both neurotransmitter release and neural communication via alteration of action potential durations, firing frequency, and neural adaptation. There are many subsets of this type of ion channel located throughout the body, and though it is evident that these channels are involved in cellular activation within the peripheral auditory system, little is known about their contribution to auditory processing in the brain. There is a need for further understanding of the functional involvement and mechanisms of neurotransmitter loss and how this relates to the BK channel and auditory disorders such as presbycusis and tinnitus (the perception of a phantom sound). My research focused on investigating how the downregulation of neurotransmitter production and the reductions in BK channel expression affect ARHL. I also evaluated a custom BK-channel modulating peptide as a path towards a possible therapeutic intervention for age-related hearing loss. This custom peptide is especially useful because it reduces the potential for serious side effects, due to mechanisms which best mimic natural occurring peptide systems. The initial investigation described in this dissertation measured auditory system changes in aged mice that occurred following a drug-induced increase in the availability of the inhibitory neurotransmitter GABA. This increase in GABA decreased minimum response thresholds in the auditory midbrain of aged mice, bringing them to levels seen in young adult animals. The other changes that occurred following increased GABA availability were increased acoustically driven neuronal firing rates, frequency-dependent decreases in spontaneous rates, and increases in the symmetry of the receptive fields. The return of clear and fine-tuned acoustically-evoked responses in aged mice was a major finding of this experiment. The second phase of the dissertation built on this demonstration that modulation of the aged auditory system was possible by changing neurotransmitter levels. This second portion of the study focused on how a novel potent neuropeptide (LS3), which increases the probability of the BK channel remaining in the closed conformational state, might invoke alterations in auditory-evoked responses. First, the LS3 neuropeptide was used to modify addictive behavior in the C. Elegans; followed by evaluation of in vitro changes to a human cell line. This study then confirmed that LS3 is a potent BK channel modulator with a greater affinity than those known toxins classified as high-affinity toxins. In vivo testing demonstrated that LS3 could rapidly cross the blood-brain barrier (BBB) following systemic injections, where it altered auditory evoked activity in a manner similar to that of the direct application to the dura over the midbrain. This work demonstrates that the BK channel is highly responsible for the control of auditory-evoked neurological processes, and that a potent BK channel modulator may be useful for the treatment of certain neurological disorders. The third study was designed to confirm that the BK channel plays an important role in sound-evoked activity generated in the auditory midbrain, by testing the effects of a general BK channel pore blocker, PAX. The results established that the BK channel is vital for sound processing in the midbrain of young adult mice, and is responsible for the maintenance of receptive field properties. I also evaluated the role it plays in temporal processing, which is an underlying mechanism for the processing of neurologically-relevant complex acoustic signals such as speech. Here, blocking of the channel increased (worsened) the threshold for the detection of a silent gap-in-noise and the neural recovery functions that occurred following the stimuli. The fourth study significantly expanded the in vivo testing of the custom peptide channel blocker, LS3, and added a behavioral measure of changes to auditory perception in addition to the electrophysiology recordings. The auditory-evoked receptive fields from midbrain neurons were modulated in a dose-dependent manner following the application of LS3. The neural recordings took place in the inferior colliculus, where the dorsal region responds to low-frequency sounds and ventral areas to high frequencies. The LS3-induced suppression or enhancement of evoked responses was different for the various tonotopic regions of the auditory midbrain. The improvements shown in receptive fields and improvement in auditory perception indicates a plausible route for direct translational treatment of auditory disorders through small custom peptide therapeutics. These studies provide supportive information about how auditory evoked responses in the midbrain, including the coding of different sound features, are affected by the down-regulation of a key inhibitory neurotransmitter (GABA), and how GABA-dependent neural evoked responses are altered in older mice through the modulation of BK channel activity.

Ion channels play a critical role in maintaining homeostasis by moving various ions in and out of cells. The Na+-K+-2Cl- or NKCC1 ion channel is involved in the regulation of Na+, K+, and Cl- across cell membranes, and plays a key role in many forms of cellular physiology. In the cochlea, NKCC1 is involved in endolymph production and maintenance of the endocochlear potential. Our hypothesis is that blocking NKCC1 channels should directly impact auditory sensitivity causing hearing loss. Our lab has also shown that the hormone aldosterone (ALD) can upregulate NKCC1 protein expression in vitro and in vivo. In the present investigation, we use electrophysiology and molecular biology techniques to study the biophysical mechanisms underlying the action of ALD in vitro on NKCC1 in the SH-SY5Y cell line. Our initial protein expression studies using RT-PCR found that proteins specific to NKCC1channels were present in SH-SY5Y neuronal cells. Whole cell currents measured using patch clamp methodology, were used to analyze the effects of various compounds on NKCC1 in the SH-SY5Y cell line. Control data were collected under perfusion of extracellular solution (ECS), then ECS containing 10µM bumetanide was applied, and, finally a washout condition completed the experiment. Similar experiments were conducted using ALD, and we observed an increase in K+ currents when bumetanide as well as when ALD was applied. This is the first report that indicates that ALD can directly regulate K+ channels in SH-SY5Y cells.

Hyperkalemic periodic paralysis is characterized by myotonic discharges followed by paralysis. Caused by a mutation in the gene encoding for NaV1.4 channel, patients do not experience symptoms during infancy, but the onset starts between 1-10 years of age. The symptoms severity then increases with age until adolescence. A large increase in gene expression marked by an increase in oxidative capacity of muscles has also been reported in HyperKPP. It is possible that the onset of symptoms is related solely to NaV1.4 channel content/activity reaching a critical level. It is also possible that the onset of some symptoms are due to defective NaV1.4, while other symptoms and the increase in severity with age are related to changes in membrane components as a result of changes in gene expression. To test these possibilities, the progression of paralysis and changes in fiber types were followed with age in HyperKPP mice in relation to changes in NaV1.4 content and activity. Changes in fiber types (index of changes in gene expression), started after the onset of paralysis was observed, which coincided with NaV1.4 channels reaching maximum expression. Therefore, the onset of symptoms was related to defective NaV1.4 channels.

In lipid membranes lacking activating lipids, the nicotinic acetylcholine receptor adopts an uncoupled conformation that binds ligand, but does not transition into an open conformation. Understanding the mechanisms of lipid-dependent uncoupling is essential to understanding lipid-nAChR interactions, which may be implicated in pathological conditions such as nicotine addition. Here, I tested two structural features of a proposed uncoupling method to elucidate the mechanism of lipid-dependent uncoupling. First, infrared measurements and electrophysiological characterization performed in prokaryotic homologues indicate that lipid sensitivity is largely controlled by the most peripheral α-helix in the transmembrane domain, M4. My data show that tighter association of M4 with the adjacent M1 and M3 transmembrane α-helices decreases a receptor’s propensity to adopt a lipid-dependent uncoupled conformation. Second, I indirectly tested the hypothesis that uncoupling results from a conformational change at the extracellular/transmembrane domain interface that leads to an increased separation between the two domains and ultimately to a constriction of the channel pore. Finally, biophysical studies presented in this dissertation shed light on the complex binding of a number of non-competitive channel blockers to the nicotinic acetylcholine receptor channel pore in both the resting and desensitized states. The data provide further insight into the structural rearrangements that occur upon uncoupling of ligand binding and gating in the nicotinic acetylcholine receptor.

The atrioventricular junction (AVJ) is a complex anatomical structure. It has an important role in maintaining synchronised atrioventricular conduction and protects from ventricular tachycardia, as well as bradycardia. Its embryological development and function is under tight transcription factor control. Heart failure is a chronic systemic condition, affecting one million people in the UK alone. Slowing of atrioventricular conduction in heart failure is associated with increased morbidity and mortality. The molecular and anatomical basis of abnormal atrioventricular conduction was studied in a rabbit model of heart failure due to aortic insufficiency and abdominal aortic constriction. The PR interval was significantly prolonged in heart failure animals. Using laser-assisted microdissection, the tiny tissues of the AVJ were collected for RT-PCR analysis. HCN1, Cav1.3, Cx40 and Cx43 transcripts were significantly downregulated by heart failure, with a compensatory increase in CLCN2, Nav1.1, Navβ1, SUR2A and PAK1. Immunolabelling for Cx43 showed reduction in protein level and longitudinal dissociation not only in the inferior nodal extension but also in the His bundle in heart failure animals. Anatomical studies of the AVJ have previously been limited by its small size and inaccessible location. Contrast-enhanced micro-CT scanning allowed non-destructive imaging of the AVJ anatomy. AVJ length and volume were increased in the rabbit model of heart failure, which is expected to contribute to atrioventricular conduction abnormalities. Micro-CT additionally resolved the anatomy of the canine AVJ and atria, including fibre orientation in the pulmonary vein sleeves and Bachmann’s bundle. The physiological effects of loss of T-box transcription factor 5 (Tbx5) in the AVJ were studied in a transgenic inducible Tbx5 knockout mouse model using optical mapping. Tbx5-deficient mice had a prolonged PR interval in vivo and a higher incidence of atrioventricular block and ventricular conduction abnormalities in Langendorff-perfused hearts.

Les dérivés de platine (cisplatine, oxaliplatine et carboplatine) et les taxanes (paclitaxel et docétaxel) font partie des grandes familles d’agents anticancéreux couramment utilisés en chimiothérapie. Ils permettent de traiter les tumeurs solides telles que les cancers de l’ovaire, du poumon, de la prostate, du sein, tête et cou. Cependant, ils sont à l’origine d’importantes neuropathies périphériques qui peuvent devenir irréversibles et laisser les patients avec des séquelles importantes. Il s'agit de vertiges, acouphènes, engourdissements, pertes de sensibilité, allodynies au toucher ou aux variations de température. Ces effets secondaires sont fortement contraignants et réduisent considérablement la qualité de vie de 30 à 50% des patients. L'importance clinique est considérable, puisque cela conduit environ 1/3 des patients atteints à l’arrêt et/ou au changement des traitements, bien que ceux-ci soient efficaces sur les tumeurs.Les dérivés de platine sont des agents pontants : leurs interactions avec les bases purines de l’ADN forment des adduits, qui vont être reconnus par des protéines de dommage à l’ADN, conduisant à la mort cellulaire. Les taxanes bloquent la dépolymérisation des microtubules, ce qui induit la mort des cellules en divisions. Le fait que deux mécanismes d’action antitumorale très différents provoquent, à court ou à moyen terme, les mêmes neuropathies périphériques, est paradoxal et n’a pas d’explication.Le but de mon projet de thèse était d’étudier le ou les mécanismes à l’origine de ces neuropathies et de développer une formulation visant à les réduire. Pour cela, j’ai étudié les effets des taxanes et dérivés de platine sur les canaux ioniques, impliqués dans la nociception. Ces canaux ioniques, appelés nocicepteurs, sont transcrits dans les corps cellulaires des ganglions spinaux (DRG), avant d’être principalement adressés à l’extrémité des nerfs périphériques. Ils détectent les stimuli mécaniques, thermiques et chimiques et génèrent ou transmettent les potentiels d’action correspondants. Le fait de perturber l’expression génique et/ou l’activité de ces nocicepteurs, aura donc pour conséquence de modifier les seuils de sensibilité et la transmission de différents stimuli.Mes résultats ont permis de quantifier dans des cultures primaires de DRG et in vivo chez la souris, le remodelage génique de nocicepteurs induit par les traitements de chimiothérapie, en corrélation avec l’apparition de neuropathies périphériques que j'ai mesurées par des tests comportementaux. Cela nous a permis d'identifier une famille de molécules candidates, qui pourraient potentiellement contrer le mécanisme identifié dans ces travaux. L'une de ces molécules permet de rétablir l’expression génique de nocicepteurs et aussi de supprimer les neuropathies périphériques chez la souris. Ce travail devrait se poursuivre dans le cadre d'un processus de valorisation, ayant pour objectif d'aboutir à un traitement préventif et/ou curatif, de ces neuropathies chez les patients. / Platinum-based drugs (cisplatin, oxaliplatin and carboplatin) and taxanes (paclitaxel and docetaxel) are among the most common drugs families used in chemotherapy. They are used for treatment of numerous human cancers including bladder, breast, head and neck, lung, ovarian, prostate and testicular cancers. However, these anticancer drugs cause significant peripheral neuropathies, that can become irreversible and leave serious clinical sequelae in patients. These include tinnitus, dizziness, tingling, numbness, loss of sensitivity, allodynia to touch or temperature changes and hyperalgesia. These side effects are highly restrictive and significantly reduce the quality of life of 30-50% of patients. The clinical significance is considerable, since it leads to about one-third of patients with stopping and/or changing treatments, although these are effective on tumors.Platinum-based drugs are DNA binding agents: they generate DNA lesions on the purine bases of DNA, that will be recognized by DNA damage response proteins, leading to cell death. Taxanes block the cell cycle in mitosis, by stabilizing the microtubule cytoskeleton against depolymerization. The fact that these two completely different antitumor mechanisms of action, induce the same peripheral neuropathies in the short to medium term, is paradoxical and has no explanation.The aim of my PhD research project was to study the mechanism(s) behind these neuropathies and to develop a new formulation to prevent and/or reduce them. Therefore, I studied the effects of taxanes and platinum-based drugs on several ion channels, involved in nociception. These ion channels, called nociceptors, are transcribed into the cell bodies of dorsal root ganglia (DRGs) and located to the peripheral nerve endings. They detect mechanical, thermal and chemical stimuli and generate or transmit the corresponding action potentials. Changes in gene expression and/or activity of nociceptors, will therefore modify the nociceptive thresholds and the transmission of different stimuli.My results allowed me to quantify, in primary cell culture of mouse DRGs and in vivo, the remodeling of ion channel expression induced by chemotherapy, in correlation with the development of peripheral neuropathies, that I measured by behavioral assessment. These results allowed us to identify a family of candidate molecules, that could potentially counteract the mechanism identified in this work. I showed that one of these molecules, can restore the gene expression of nociceptors and suppress peripheral neuropathies in mice. This work should continue as part of a valorization process, aiming to lead to a preventive and/or curative treatment, of these neuropathies in patients.