A quantitative evaluation of single photon emission computed tomography (SPECT) and planar scintigraphic imaging may be valuable for both diagnostic and therapeutic purposes. For an accurate quantification it is usually necessary to correct for attenuation and scatter and in some cases also for septal penetration. For planar imaging a background correction for the contribution from over- and underlying tissues is needed. In this work a few correction methods have been evaluated and further developed. Much of the work relies on the Monte Carlo method as a tool for evaluation and optimisation. A method for quantifying the activity of I-125 labelled antibodies in a tumour inoculated in the flank of a mouse, based on planar scintigraphic imaging with a pin-hole collimator, has been developed and two different methods for background subtraction have been compared. The activity estimates of the tumours were compared with measurements in vitro. The major part of this work is attributed to SPECT. A method for attenuation and scatter correction of brain SPECT based on computed tomography (CT) images of the same patient has been developed, using an attenuation map calculated from the CT image volume. The attenuation map is utilised not only for attenuation correction, but also for scatter correction with transmission dependent convolution subtraction (TDCS). A registration method based on fiducial markers, placed on three chosen points during the SPECT examination, was evaluated. The scatter correction method, TDCS, was then optimised for regional cerebral blood flow (rCBF) SPECT with Tc-99m, and was also compared with a related method, convolution scatter subtraction (CSS). TDCS has been claimed to be an iterative technique. This requires however some modifications of the method, which have been demonstrated and evaluated for a simulation with a point source. When the Monte Carlo method is used for evaluation of corrections for septal penetration, it is important that interactions in the collimator are taken into account. A new version of the Monte Carlo program SIMIND with this capability has been evaluated by comparing measured and simulated images and energy spectra. This code was later used for the evaluation of a few different methods for correction of scatter and septal penetration of I-123 brain SPECT. The methods were CSS, TDCS and a method where correction for scatter and septal penetration are included in the iterative reconstruction. This study shows that quantitative accuracy in I-123 brain SPECT benefits from separate modelling of scatter and septal penetration.

To assess the impact of congenital malformations in Down syndrome (DS) on morbidity, mortality and outcome at birth, information on all children with DS born in the northern part of Sweden in 1973-80 (n=211) and 1995-98 (n=88) was collected. Most common were congenital heart defects (CHD), dominated by atrioventricular septal defects (AVSD). Up to age 10 years, morbidity and mortality were more than 10 times higher in DS children with CHD than in healthy DS children. The DS children seemed more vulnerable at birth than Swedish children in general: they had increased frequencies of Cesarean sections, premature birth, asphyxia, and low birthweight, and higher proportions of children small for gestational age, regardless of the presence of CHD. Infant mortality decreased from 14.2% to 2.3% between the two periods. All children with AVSD with and without DS born in Sweden 1973-1997 (n=801) were followed up retrospectively to 2001. Children with isolated AVSD without complex additional CHDs were studied more closely (n=502). A reduction in age at operation and postoperative mortality (from 28 to 1%) was observed. No significant difference in 5-year postoperative mortality between genders or between DS and non-DS children was found. The 5-year postoperative mortality in DS decreased from 35% in 1973-77 to about 10% in 1993-97. CHD had a major influence on morbidity, infectionrate and mortality in DS, but not on DS birth variables. The formerly high mortality in CHD is now reduced. In isolated AVSD measures seem equally successful in DS and non-DS children. Mortality is still 3 times higher in DS children with isolated AVSD than in healthy DS children.

No description available.

Introdução: As comunicações interatriais do tipo ostium secundum (CIA-OS) são tratadas por fechamento percutâneo (FP) ou intervenção cirúrgica (IC). Estudos comparando ambos métodos são escassos e avaliações de custo-efetividade inexistem na literatura nacional. Objetivos: Realizar uma avaliação da segurança e eficácia (ASE) clínica seguida de uma análise de custo-efetividade (ACE) incremental comparando o FP e IC sob a perspectiva do Sistema Único de Saúde (SUS). Materiais e métodos: ASE - Estudo observacional, não randomizado de 2 coortes de crianças e adolescentes < 14 anos com CIA-OS tratadas por FP ou IC. A coleta dos dados foi prospectiva no FP e retrospectiva no IC. ACE - Realizada revisão sistemática de estudos clínicos disponíveis no MEDLINE e a Cochrane Central. Estudos com mais de 50 pacientes e com idade média abaixo de 14 anos foram incluídos. A análise foi baseada em uma árvore de decisão computando os custos e consequências no longo prazo de ambas as opções. Foi utilizado valor da prótese de R$ 16.000 e estipulado valor de honorário médico de R$ 2.355. A efetividade foi estimada em anos de vida. A avaliação utilizou uma taxa de desconto de 5% ao ano e uma disposição a pagar de 3 vezes o PIB per capita no Brasil (63.000). Análises de limiares também foram conduzidas. Resultados: ASE - De Abr/09 a Out/11 foram alocados 75 pacientes (pts) no FP e entre Jan/06 e Jan/11 105 pts na IC. A idade e o peso foram maiores no FP e o diâmetro da CIA foi semelhante entre os grupos. Sucesso técnico foi observado em todos os procedimentos e não houve óbitos. Complicações (a maioria menores) foram encontradas em 68% na IC e em 4% do FP (p<0,001). As taxas de fluxo residual não significativo ou de oclusão total do defeito foram semelhantes nos 2 grupos. A mediana de internação foi de 1,2 dias após o FP e 8,4 dias após a IC (p< 0,001). ACE - A estratégia de busca retornou 2.957 referências, das quais 34 foram incluídas. A taxa de mortalidade foi semelhante nos 2 grupos. O FP teve discreta maior efetividade, menor taxa de complicações moderadas/graves e menor tempo de internação, mas com maior probabilidade de um segundo procedimento. A relação de custo-efetividade incremental (RCEI) do tratamento percutâneo foi de R$ 230.641 por ano de vida salvo. Considerando-se este cenário, seria necessário que o custo indireto associado à cirurgia fosse de R$ 4.960, ou que o valor pago pelo dispositivo percutâneo sofresse redução de R$ 4.960 para que o FP se tornasse aceitável do ponto de vista econômico. Conclusões: Ambos tratamentos são seguros e eficazes com ótimos desfechos, porém o FP apresenta menor morbidade e tempo de internação. Usando os valores diretos estipulados neste estudo, a RCEI foi elevada limitando a incorporação do FP pelo SUS neste momento. Estudos considerando também os custos indiretos são necessários para a adequada ACE de ambas estratégias. / Introduction: Atrial septal defects of the secundum type (ASD-OS) are treated by percutaneous closure (PC) or surgical intervention (SI). Comparative studies are scarce and there is no cost-effectiveness assessment in the Brazilian literature. Objectives: To perform a clinical safety and efficacy (ASE) assessment followed by an incremental cost-effectiveness (CEE) analysis comparing PC and SI under the Brazilian Unified Health System perspective. Materials and methods: ASE - Observational, non-randomized study of two cohorts of children and adolescents under 14 years with ASD-OS treated by PC or SI. Data was collected prospectively in PC and retrospectively in SI. CEE - A systematic review of clinical studies available in MEDLINE and Cochrane Central was performed. Studies with more than 50 patients and mean age under 14 years were included. Analysis was based on a decision tree that took into account costs and consequences during long-term follow-up for both options. The cost of the device and medical honorarium were estimated at R$ 16.000 and R$ 2.355, respectively. Effectiveness was estimated in years of life. Assessment was performed using a discount tax of 5% and a willingness to pay of 3 times the GID in Brazil (63.000). Threshold analyses were also conducted. Results: ASE - From Apr/09 to Oct/11 75 patients (pts) were enrolled in PC and from Jan/06 to Jan/11 105 pts in SI. Age and weight were greater in PC and the ASD diameter was similar. Technical success was achieved in all procedures and there were no deaths. Complications (most minor) occurred in 68% of SI and 4% of PC (p<0,001). The rate of total occlusion or non-significant residual shunts was similar in both groups. Median hospitalization time was 1.2 days in PC and 8.4 days in SI (p<0,001). CEE - Search strategy returned 2957 references and 35 were included. Mortality was similar in both groups. PC was associated with slightly better effectiveness, lower rates of moderate/severe complications and reduced hospital stay despite a higher probability of a second procedure. Incremental cost-effectiveness ratio (ICER) was R$ 230.641 for life-year gained. In this scenario, PC would be acceptable from the economic point of view if the indirect costs of the SI was R$ 4.960 or the cost of the device was reduced by R$ 4.960. Conclusions: Both methods are safe and effective with excellent outcomes, however PC is associated with less morbidity and in-hospital time. Using the direct costs stipulated in this study, the ICER was high limiting the incorporation of PC by the Brazilian Unified Health System (SUS) at this moment. Studies also considering the indirect costs should be performed for better CEE assessment of both strategies.

Introdução: A comunicação interatrial tipo ostium secundum(CIA) é uma cardiopatia congênita frequente, sendo a mais comumente encontrada na população adulta. Seu tratamento está indicado quando há repercussão hemodinâmica caracterizada pelo aumento das dimensões das câmaras direitas à ecocardiografia, independente da presença de sintomas. Nas últimas 2 décadas o fechamento percutâneo da CIA emergiu como a modalidade terapêutica preferencial devido sua alta eficácia e menor morbidade que a correção cirúrgica.Tanto o tratamento cirúrgico como o percutâneo da CIA resultam em remodelamento cardíaco com redução progressiva do tamanho das câmaras direitas e aumento das esquerdas. Recentemente, novas técnicas ecocardiográficas vem sendo empregadas para avaliação das dimensões, geometria e função das câmaras cardíacas incluindo o ecocardiograma tridimensional e o rastreamento de marcadores acústicos. Hipótese e objetivos: Partiu-se da hipótese que o fechamento percutâneo da CIA, por ser um método não invasivo, levaria a rápido remodelamento cardíaco mesmo em adultos com sobrecarga volumétrica crônica das câmaras direitas. Tivemos como objetivo avaliar o comportamento temporal do remodelamento cardíaco e analisar possíveis diferenças existentes entre pacientes de diferentes faixas etárias e com tamanhos diversos de CIA. Material e métodos: Estudo observacional, prospectivo, não randomizado de um braço único de uma coorte de adultos submetidos ao fechamento percutâneo da CIA com a prótese Cera (Lifetech, Shenzheng, China) e acompanhados por um ano. Foram selecionados 29 adultos com CIA com repercussão hemodinâmica com anatomia favorável para a oclusão percutânea e sem contra-indicações para tal. O remodelamento cardíaco foi estudado por meio de várias técnicas ecocardiográficas incluindo as clássicas e outras de introdução recente. A ecocardiografia bidimensional foi usada para a medição das dimensões do átrio direito (AD), ventrículo direito (VD) e ventrículo esquerdo (VE), para determinação da área fracionada do VD (FAC) e da excursão anterior da valva tricúspide (TAPSE) e para análise volumétrica das câmaras cardíacas. A ecocardiografia tridimensional foi empregada para análise volumétrica e funcional do VD. O rastreamento de marcadores acústico foi utilizado para avaliação da função do AD, VD e VE. O teste ANOVA foi usado para avaliação das mudanças observadas nas variáveis repetidas ao longo do tempo com comparações múltiplas de Bonferroni quando aplicável. Uma análise intra e interobservador das medidas foi realizada utilizando coeficientes de concordância. Resultados: A média de idade e peso dos pacientes foi de 45,2 ± 17,0 anos e 68,8 ± 14,0 kgs, respectivamente. Nenhum paciente apresentada hipertensão pulmonar significativa. A média do tamanho da CIA foi de 20,2 ± 5,0 mm e a média do tamanho da prótese implantada foi de 22,9 ± 6,2 mm. Em todos os pacientes houve sucesso no implante. Não houve complicações relacionadas ao procedimento e em todos os pacientes foi observada oclusão do defeito. Após o procedimento, houve redução precoce (< 3 meses) das dimensões do AD (p<0,001) e do VD (p<0,001) e aumento das dimensões do VE (p<0,014). Não houve mudanças significativas na função de deformação longitudinal do AD para onda P (p=0,227) e para onda T (p=0,124). Houve redução abrupta da função do VD pelo TAPSE (p=0,032), pela deformação longitudinal (p=0,002) e pela ecocardiografia tridimensional (p=0,084). Não houve mudanças significativas da onda S\' (p=0,55) e da FAC (p=0789) do VD. Houve redução precoce do volume de ejeção do VD (p< 0,001) e aumento do volume de ejeção do VE (p=0,027). Houve redução da deformação longitudinal do VE (p=0,049) e não houve mudanças na função do VE pelo método de Simpson (p=0,462). Pacientes maiores que 60 anos (n=8) apresentaram valores iniciais maiores nas dimensões do AD e redução mais prolongada (p=0,0497). Pacientes com CIAs maiores que 20 mm (n=12) apresentavam valores iniciais maiores de TAPSE com redução mais retardada (p=0,013). Todas as mudanças observadas inicialmente nos primeiros 3 meses se sustentaram ao final do seguimento. Houve excelente concordância na análise intraobservador para todas as medidas repetidas (CCI> 0,9) com exceção da deformação do VE (CCI< 0,7). A concordância entre as medidas realizadas por diferentes observadores não foi tão boa, com apenas as variáveis TAPSE, deformação do VE e dimensões do AD possuindo CCI > 0,7. Conclusões: O fechamento percutâneo da CIA em adultos de meia idade leva a rápido remodelamento cardíaco tanto do ponto de vista anatômico como funcional com resultados sustentados dentro do primeiro ano de seguimento. Tais mudanças temporais são observadas independente do tamanho da CIA e da idade dos pacientes, denotando o efeito benéfico do procedimento sobre a geometria e o funcionamento cardíaco global em todos adultos portadores desta frequente cardiopatia congênita. / Introduction: The atrial septal defect of the secundum type (ASD) is a frequent congenital heart disease, being the most commonly encountered in the adult population. Treatment is indicated when there is hemodynamic burden characterized by increased dimensions of the right chambers on echocardiography, regardless of the presence of symptoms. In the last two decades, percutaneous closure of the ASD has emerged as the preferred therapeutic modality due to its high efficacy and lower morbidity when compared to surgical correction. Both surgical and percutaneous treatment of the ASD result in cardiac remodeling with progressive reduction in the size of the right chambers and increase in the left chambers. Recently, new echocardiographic techniques have been employed to assess the dimensions, geometry and function of the cardiac chambers including three-dimensional echocardiography (3D echo) and acoustic speckle tracking. Hypothesis and objectives: Hypothesizing that percutaneous closure, being a non-ivasive method, results in a fast cardiac remodeling even in adults with chronic volume overload of the right chambers, our aim was to assess the temporal pattern of cardiac remodeling and analyse possible differences between patients of different age groups and different ASD sizes. Material and methods: This was an observational, prospective, non randomized, single arm study of a cohort of adults submitted to percutaneous closure of the ASD with the Cera device (Lifetech, Shenzheng, China) followed along a year. Twenty nine adults with hemodynamicaly significant ASDs with suitable anatomy for percutaneous closure and no contra-indications for the procerdure were selected. Cardiac remodeling was assessed by various echocardiographic techniques including standard and new ones. Bidimensional echocardiography was used to measure the dimensions of the right atrium (RA), right ventricle (RV) and left ventricle (LV), to determine the fractional area of the RV (FAC) and the systolic anterior excursion of the tricuspid valve (TAPSE), and to analyse the volumes of the cardiac chambers. Three-D echo was employed for volumetric and functional analysis of the RV. Acoustic speckle tracking was utilized to assess the function of the RA, RV and LV. ANOVA tests were used to assess the observed changes in the repeated variables over time with multiple Boferoni comparison as applicable. An intra and interobserver analysis of the measurements was performed using concordance coefficients. Results: Mean age and weight was 45,2 ± 17,0 years and 68,8 ± 14,0 kgs, respectively. No patient had significant pulmonar arterial hypertension. The ASD dimension and the size of the device was a mean of 20,2 ± 5,0 mm and 22,9 ± 6,2 mm, respectively. In all patients the device was implanted successfully. There were no complications related to the procedure and in all patients the ASDs were successfully closed. After the procedure, there was an early (< 3 months) reduction of the RA (< 0.001) and RV (< 0.001) sizes and an increase of the LV dimensions (p< 0.014). There were no significant changes in the function of the RA as assessed by longitudinal strain (p=0.227 for the P wave and p=0.124 for the T wave). There was an abrupt reduction of the RV function assessed by TAPSE (p=0.032), longitudinal strain (p=0.002) and 3D echo (p=0.084). There were no changes in the S\' wave (p=0.55) and FAC (p=0.789) of the RV. There was an immediate decrease in the RV stroke volume (p< 0.001) and an increase in the LV srtroke volume (p=0.027). There was a reduction in LV longitudinal strain (p=0.049) and no change in LV function as assessed by the Simpson method (p=0.462). Patients older than 60 years of age (n=8) presented with larger RA dimensions, which decreased in a slower fashion (p=0.0497). Patients with ASDs larger than 20 mm (n=12) had initial higher TAPSE values, which decreased in a slower fashion (p=0.013). All changes observed earlier on endured after a year. There was excellent concordance in the intra observer analysis for all the repeated measures (CCI > 0.9) with the exception of LV strain (CCI < 0.7). The concordance between different observers was not as good with only TAPSE, LV strain, and RA dimensions variables having a CCI > 0.7. Conclusions: Percutaneous closure of the ASD in middle aged adults results in fast cardiac remodeling from both the anatomic and functional point of view with sustained results over the first year of follow up. These temporal changes are observed regardless of the ASD size and the age of the patients, which demonstrates the beneficial effect of the procedure over the cardiac geometry and global function in all adults who have this frequent congenital heart disease.

Thesis (M. Med.(Paediatrics and Child Health)) -- University of Limpopo, 2019 / Background: Congenital heart disease (CHD) is a significant contributor to Under 5 Mortality rate(U5MR) in Limpopo. Atrioventricular septal defect (AVSD) is the best ascertained lesion in Limpopo and is strongly associated with Down syndrome. Few children from Limpopo with CHD including AVSD access cardiac diagnostic and surgical services. Objectives: The study aimed to enumerate, describe syndromes associated with AVSD and outcomes of children with AVSD at Pietersburg hospital. Methods: This is a Retrospective study of all children (n=80) diagnosed with AVSD from 1 January 2010 to 31 December 2014 at Pietersburg hospital. Data were drawn from echocardiogram reports and patient records. District Health Information Software (DHIS) data was used to obtain the number of live births per district during the 5 years study period. Results: Eight hundred and sixty six (n=866) patients had a first diagnosis of CHD confirmed on echocardiography and 80 (9.2%) of these had AVSD (an estimated 31.5% of expected cases of AVSD). Eighty four per cent (84%) of AVSD patients were associated with Down syndrome. 42/67 (63%) AVSD patients were referred for surgical assessment and of those 15/42 (36%) had surgery. The median interval between diagnosis and surgery was 13 months. Seventy five percent (n=50/67) of patients defaulted follow up. Conclusion: The study confirmed that most children with AVSD had associated Down syndrome and that the majority of children with AVSD from Limpopo do no access surgery. There is under referral of children with Down syndrome for screening of CHD.

The transmission of sex pheromone-mediated signals is essential for goldfish reproduction. However, the neural pathways underlying this reproductive signalling pathway in the goldfish brain is not well described. Lesioning experiments have shown previously that two brain areas, the preoptic area (POA) and the ventral telencephali pars ventralis (Vv) in particular, are important for reproduction. We used patch clamp electrophysiology to study the electrical activities of POA and Vv neurons. Based on the intrinsic properties of these neurons, we suggest there are five different functional classes of POA neurons and a single class of Vv neurons. In addition, by electrically stimulating the olfactory bulb (OB), we were able to show that this primary sensory structure makes monosynaptic glutamatergic connections with both POA and Vv neurons. While electrophysiology measures signalling events occurring at short time scales on the order of milliseconds to minutes, we were also interested in studying sex pheromone signalling in the goldfish brain over a long time scale. Thus, we describe changes in gene expression in male goldfish exposed to waterborne sex pheromones (17alpha,20beta dihydroxy-4-pregene-3-one and Prostaglandin-F2alpha) over 6 hours. We perform cDNA microarrays on Prostaglandin-F2alpha-treated fish to study the rapid modulation of transcription and define the signalling pathways affected. Our microarrays showed that 71 genes were differentially regulated (67 up and 4 down). Through gene ontology enrichment analysis, we found that these genes were involved in various biological processes such as RNA processing, neurotransmission, neuronal development, apoptosis, cellular metabolism and sexual reproduction. RT-PCRs were performed to validate our microarrays and to facilitate direct comparisons of the effects of the two sex pheromones, 17alpha,20beta dihydroxy-4-pregene-3-one and Prostaglandin-F2alpha. By combining electrophysiology and gene expression analyses, we were able to study sex-pheromone signalling on two different time scales. One short, occurring on the order of milliseconds to minutes, that involves electrical activities in the brain through the glutamatergic amino-3-hydroxy-5-methylisoxazole-4-propionate and N-methyl-D-aspartate receptors; and the other long occurring several hours later that involves changes in the gene expression levels of calmodulin and ependymin among other genes underlying neuroplasticity. Reproductive neuroplasticity in the goldfish may therefore require the activation of glutamatergic receptors which then activate downstream signals like calmodulin and ependymin to transform the sex pheromones-mediate signal into gene expression.

Le cœur des vertébrés est un organe modulaire qui requiert le " patterning " complexe des champs morphogénétiques cardiogènes et la convergence coordonnée des diverses sous-populations de progéniteurs cardiogéniques. Au moins 7 facteurs de transcription de la famille T-box coopèrent au sein de ces nombreuses sous-populations de progéniteurs cardiogéniques afin de réguler la morphogenèse et l’agencement de multiples structures le long de l’ébauche cardiaque, ce qui explique que les mutations humaines de ces gènes engendrent diverses malformations congénitales cardiaques (MCCs). L’un de ces gènes T-box, Tbx5, dont l’haploinsuffisance génère le syndrome de Holt-Oram (SHO), intervient dans une grande variété de réseaux de régulation géniques (RRGs) qui orchestrent la morphogenèse des oreillettes, du ventricule gauche, de la valve mitrale, des septums inter-auriculaire et inter-ventriculaire, ainsi que du système de conduction cardiaque. La diversité des RRGs impliqués dans la formation de ces structures cardiaques suggère que Tbx5 détient une profusion de fonctions qui ne seront identifiables qu’en répertoriant ses activités moléculaires dans chaque lignée cardiaque examinée isolément. Afin d’aborder cette problématique, une ablation génétique de Tbx5 dans l’endocarde a été réalisée. Cette expérience a démontré le rôle crucial de Tbx5 dans la survie des cellules endocardiques bordant le septum primum et des cardiomyocytes au sein de cette structure embryonnaire qui contribuera à la morphogenèse du septum inter-auriculaire. En outre, cette étude a révélé l’existence d’une communication croisée entre la sous-population de cellules endocardiques Tbx5+ et le myocarde au niveau du septum primum, afin d’assurer la survie des cardiomyocytes, et ultimement de garantir la maturation du septum inter-auriculaire. Nos résultats confirment aussi l’importance de l’interdépendance génétique (Tbx5 et Gata4 ainsi que Tbx5 et Nos3) entre différents loci dans la morphogenèse de la cloison inter-auriculaire, et particulièrement de l’influence que peut avoir l’environnement sur la pénétrance et l’expressivité des communications inter-auriculaires (CIAs) dans le SHO. En outre, puisque les fonctions d’un gène dépendent ordinairement des différents isoformes qu’il peut générer, une deuxième étude a focalisé davantage sur l’aspect transcriptionnel de Tbx5. Cette approche a mené à la découverte de 6 transcrits alternatifs exhibant des fonctions à la fois communes et divergentes. La caractérisation de 2 de ces isoformes a révélé le rôle de l’isoforme long (Tbx5_v1) dans la régulation de la croissance des cardiomyocytes durant la cardiogénèse, tandis que l’isoforme court (Tbx5_v2), préférentiellement exprimé dans le cœur mature, réprime la croissance cellulaire. Il est donc entièrement concevable que les mutations de TBX5 entraînant une troncation de la région C-terminale accroissent la concentration d’une protéine mutée qui, à l’instar de Tbx5_v2, interfère avec la croissance de certaines structures cardiaques. En revanche, la divergence de fonctions de ces isoformes, caractérisée par les disparités de localisation subcellulaire et de d’interaction avec d’autres cofacteurs cardiaques, suggère que les mutations affectant davantage un isoforme favoriseraient l’émergence d’un type particulier de MCC. Finalement, un dernier objectif était d’identifier le ou les mécanisme(s) moléculaire(s) par le(s)quel(s) Tbx5 régule son principal gène cible, Nppa, et d’en extraire les indices qui éclairciraient sa fonction transcriptionnelle. Cet objectif nécessitait dans un premier lieu d’identifier les différents modules cis-régulateurs (MCRs) coordonnant la régulation transcriptionnelle de Nppa et Nppb, deux gènes natriurétiques dont l’organisation en tandem et le profil d’expression durant la cardiogénèse sont conservés dans la majorité des vertébrés. L’approche d’empreinte phylogénétique employée pour scanner le locus Nppb/Nppa a permis d’identifier trois MCRs conservés entre diverses espèces de mammifères, dont un (US3) est spécifique aux euthériens. Cette étude a corroboré que la régulation de l’expression du tandem génique Nppb/Nppa requérait l’activité transcriptionnelle d’enhancers en complément aux promoteurs de Nppa et Nppb. La concordance quasiment parfaite entre les profils d’expression de Tbx5 et de ces deux gènes natriurétiques chez les mammifères, suggère que le gradient d’expression ventriculaire de Tbx5 est interprété par le recrutement de ce facteur au niveau des différents enhancers identifiés. En somme, les études présentées dans cette thèse ont permis de clarifier la profusion de fonctions cardiaques que possède Tbx5. Certaines de ces fonctions émanent de l’épissage alternatif de Tbx5, qui favorise la synthèse d’isoformes dotés de propriétés spécifiques. Les diverses interactions combinatoires entre ces isoformes et d’autres facteurs cardiaques au sein des diverses sous-populations de progéniteurs cardiogènes contribuent à l’émergence de RRGs cardiaques divergents. / The vertebrate heart is a modular organ, which requires the complex patterning of the morphogenetic heart fields and the coordinated convergence of the diverse subpopulations of cardiogenic progenitors. At least 7 transcription factors of the T-box family cooperate within these numerous subpopulations of cardiogenic progenitors to regulate the morphogenesis and the layout of multiple structures along the primordial heart tube, which explains that the human mutations of these genes induce various congenital heart defects (CHDs). One of these T-box genes, Tbx5, whose haploinsufficiency generates the Holt-Oram syndrome (HOS), intervenes in a wide variety of gene regulatory networks (GRNs) that orchestrate the morphogenesis of the atria, the left ventricle, the mitral valve, the inter-atrial and inter-ventricular septa, as well as the cardiac conduction system. The diversity of GRNs involved in the formation of these cardiac structures suggests that Tbx5 holds a profusion of functions which will be identifiable only by indexing its molecular activities in each separately examined cardiac lineage. To address this problem, a conditional knockout of Tbx5 in the endocardium was generated. This experiment demonstrated a crucial role of Tbx5 in the survival of the endocardial cells lining the septum primum and the cardiomyocytes within this embryonic structure, which will contribute to the morphogenesis of the inter-atrial septum. Moreover, this study revealed a crosstalk between the Tbx5-positive endocardial cells subpopulation and the myocardium at the level of the septum primum to ensure the survival of cardiomyocytes, and ultimately to guarantee the maturation of the inter-atrial septum. Our results also confirmed the importance of genetic interdependence (Tbx5 and Gata4 as well as Tbx5 and Nos3) between different loci in the morphogenesis of the inter-atrial septum, and particularly the influence that the environment can have on the penetrance and the expressivity of atrial septal defects (ASDs) in the HOS. Besides, since the functions of a gene usually depend on the different isoforms it can generate, a second study focused more on the transcriptional aspect of Tbx5. This approach led to the discovery of 6 alternative transcripts exhibiting both common and specific functions. The characterization of 2 of these isoforms revealed the role of the long isoform (Tbx5_v1) in the regulation of cardiomyocytes growth during cardiogenesis, whereas the short isoform (Tbx5_v2), preferentially expressed in the mature heart, represses cell growth. It is thus entirely conceivable that TBX5 mutations leading to a C-terminal truncation increase the concentration of a mutated protein, which, like Tbx5_v2, interferes with the growth of certain cardiac structures. On the other hand, the divergence of functions of these isoforms, characterized by the disparities of subcellular localization and interaction with other cardiac cofactors, suggests that mutations affecting more one isoform would favor the emergence of a particular type of CHD. Finally, a last objective was to identify one or several molecular mechanism(s) by which Tbx5 regulates its main target gene, Nppa, and to extract clues that might clarify its transcriptional function. This objective required in a first place to identify the various cis-regulatory modules (CRMs) coordinating the transcriptional regulation of Nppa and Nppb, two natriuretic genes whose tandem organization and expression pattern during cardiogenesis are preserved in most vertebrates. The phylogenetic footprint approach employed to scan the Nppb/Nppa locus allowed the identification of three CRMs evolutionary conserved between different mammals species, one of which (US3) is specific to eutherians. This study confirmed that the regulation of the tandem genes Nppb/Nppa required the transcriptional activity of enhancers in complement to Nppa and Nppb promoters. The almost perfect concordance between the expression profiles of Tbx5 and these two natriuretic genes in mammals, suggests that the ventricular expression gradient of Tbx5 is interpreted by the recruitment of this factor to the identified enhancers. Altogether, the studies presented in this thesis allowed clarifying the profusion of Tbx5 cardiac functions. Some of these functions emanate from the alternative splicing of Tbx5, which favors the synthesis of isoforms endowed with specific properties. The diverse combinatorial interactions between these isoforms and other cardiac factors within the various cardiogenic progenitor subpopulations contribute to the emergence of distinct cardiac RRGs.

Les malformations cardiaques congénitales (CHM) représentent 28 % de toutes les malformations congénitales majeures et touchent 8 pour 1000 naissances à terme. Elles sont la cause de mortalité et de morbidité non infectieuse la plus fréquente chez les enfants de moins d’une année de vie. Les communications interventriculaires (VSD) forment le sous-type de CHM le plus fréquent et l’aggrégation familiale est extrêmement rare. Le but de cette étude était d’identifier les facteurs génétiques et les régions chromosomiques contribuant aux VSD. Une grande famille ségréguant diverses formes de pathologies septales, incluant des VSD, des anévrysmes du septum interventriculaire (VSA) et des communications interauriculaires (ASD), a été examinées et caractérisées cliniquement et génétiquement. Dix-huit membres de la famille, sur trois générations, ont pu être étudiés. (10 affectés : 4 VSD, 3 VSA, 2 ASD et une tétralogie de Fallot). L’analyse de liaison multipoint paramétrique démontre un logarithme des probabilités maximal (LOD) de 3.29 liant significativement le chromosome 10p15.3-10p15.2 aux traits observés dans cette famille. Le pointage LOD oriente vers une région pauvre en gènes qui a déjà été associée aux malformations du septum interventriculaire, mais qui est distincte de la région du syndrome de DiGeorge de type 2 sur le chromosome 10p. De plus, plusieurs scénarios d’analyse de liaison suggèrent que la tétralogie de Fallot est une phénocopie et qu’elle est donc génétiquement différente des autres pathologies du septum observées dans cette famille. En bref, cette étude associe une forme rare de VSD/VSA au chromosome 10p15 et permet d’étendre le spectre de l’hétérogénéité des pathologies septales. Mots-clés : Malformations cardiaques congénitales, malformations du septum, tétralogie de Fallot, analyse de liaison, chromosome 10p15, génétique moléculaire / Cardiac malformations represents 28 % of all major congenital malformation and affect 8 per 1000 live birth. They are the most frequent cause of non infectious mortality and morbidity in childen of less then 1 year of life. Although ventricular septal defects (VSD) are the most common congenital heart lesion, familial clustering has been described only in rare instances. The aim of this study was to identify genetic factors and chromosomal regions contributing to VSD. A unique, large kindred segregating various forms of septal pathologies—including VSD, ventricular septal aneurysms, and atrial septal defects (ASD)—was ascertained and characterized clinically and genetically. Eighteen family members in three generations could be studied, out of whom 10 are affected (2 ASD, 3 septal aneurysm, 4 VSD, and 1 tetralogy of Fallot). Parametric multipoint LOD scores reach significance on chromosome 10p15.3-10p15.2 (max. 3.29). The LOD score support interval is in a gene-poor region where deletions have been reported to associate with septal defects, but that is distinct from the DiGeorge syndrome 2 region on 10p. Multiple linkage analysis scenarios suggest that tetralogy of Fallot is a phenocopy and genetically distinct from the autosomal dominant form of septal pathologies observed in this family. This study maps a rare familial form of VSD/septal aneurysms to chromosome 10p15 and extends the spectrum of the genetic heterogeneity of septal pathologies. Fine mapping, haplotype construction, and resequencing will provide a unique opportunity to study the pathogenesis of septal defects and shed light on molecular mechanisms of septal development.

The transmission of sex pheromone-mediated signals is essential for goldfish reproduction. However, the neural pathways underlying this reproductive signalling pathway in the goldfish brain is not well described. Lesioning experiments have shown previously that two brain areas, the preoptic area (POA) and the ventral telencephali pars ventralis (Vv) in particular, are important for reproduction. We used patch clamp electrophysiology to study the electrical activities of POA and Vv neurons. Based on the intrinsic properties of these neurons, we suggest there are five different functional classes of POA neurons and a single class of Vv neurons. In addition, by electrically stimulating the olfactory bulb (OB), we were able to show that this primary sensory structure makes monosynaptic glutamatergic connections with both POA and Vv neurons. While electrophysiology measures signalling events occurring at short time scales on the order of milliseconds to minutes, we were also interested in studying sex pheromone signalling in the goldfish brain over a long time scale. Thus, we describe changes in gene expression in male goldfish exposed to waterborne sex pheromones (17alpha,20beta dihydroxy-4-pregene-3-one and Prostaglandin-F2alpha) over 6 hours. We perform cDNA microarrays on Prostaglandin-F2alpha-treated fish to study the rapid modulation of transcription and define the signalling pathways affected. Our microarrays showed that 71 genes were differentially regulated (67 up and 4 down). Through gene ontology enrichment analysis, we found that these genes were involved in various biological processes such as RNA processing, neurotransmission, neuronal development, apoptosis, cellular metabolism and sexual reproduction. RT-PCRs were performed to validate our microarrays and to facilitate direct comparisons of the effects of the two sex pheromones, 17alpha,20beta dihydroxy-4-pregene-3-one and Prostaglandin-F2alpha. By combining electrophysiology and gene expression analyses, we were able to study sex-pheromone signalling on two different time scales. One short, occurring on the order of milliseconds to minutes, that involves electrical activities in the brain through the glutamatergic amino-3-hydroxy-5-methylisoxazole-4-propionate and N-methyl-D-aspartate receptors; and the other long occurring several hours later that involves changes in the gene expression levels of calmodulin and ependymin among other genes underlying neuroplasticity. Reproductive neuroplasticity in the goldfish may therefore require the activation of glutamatergic receptors which then activate downstream signals like calmodulin and ependymin to transform the sex pheromones-mediate signal into gene expression.