Orientador: Diana Rodrigues de Pina / Resumo: A enterocolite necrosante (NEC – do inglês necrotizing enterocolitis) é caracterizada pela necrose isquêmica da mucosa intestinal de recém-nascidos prematuros. Uma vez que existe a suspeita de NEC, é instituída uma rotina de radiografias abdominais de acordo com a gravidade da doença. Os principais achados radiológicos de pacientes com NEC são: distensão abdominal generalizada, pneumatose intestinal, pneumoperitônio. Entretanto, a interpretação dessas radiografias é um processo difícil devido à falta de especificidade da maioria dos achados radiológicos. O objetivo desse estudo foi desenvolver uma ferramenta computacional que auxilie o corpo clínico na análise de radiografias abdominais para a diferenciação de alças normais e alças inflamadas em recém-nascidos prematuros. Para o desenvolvimento desta pesquisa foi utilizado um banco de dados composto por 45 radiografias abdominais e algoritmos computacionais desenvolvidos em ambiente MatLab. As espessuras das alças intestinais foram quantificadas através da ferramenta computacional Largura a Meia Altura (FWHM – do inglês Full Width at Half Maximum), e classificadas como alças edemaciadas ou alças normais. Para a análise de textura e extração de características, a fim de diferenciar regiões de pneumatose, aplicamos a técnica de Transformada Wavelet. Com a utilização do algoritmo, as alças intestinais normais apresentaram mediana igual a 10,30 pixels, enquanto as alças edemaciadas, foram estatisticamente maiores (Mann Whitney, p... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

INTRODUÇÃO: O conhecimento de valores de normalidade do fluxo sanguíneo da artéria mesentérica superior (AMS) em recém-nascidos prematuros (RNPT) saudáveis pode prevenir quadros de intolerância alimentar e a ocorrência da enterocolite necrosante. MÉTODOS: Com o objetivo de descrever a evolução dos índices de avaliação da dopplerfluxometria da AMS em RNPT saudáveis de idade gestacional entre 27 e 34 semanas completas, no primeiro, no terceiro, no sétimo e semanalmente (14, 21, 28, 35 e 42 dias de vida), foi realizado este estudo coorte prospectivo em RNPT de idade gestacional ao nascimento entre 27 e 34 semanas completas. O exame dopplerfluxométrico foi realizado, após o consentimento livre e esclarecido dos responsáveis pelos RNPT, através do aparelho Logic Book 8C-RS (General Eletric EUA); obtendo-se as seguintes medidas: pico de velocidade sistólica (PVS), pico de velocidade diastólica final (PVDF) e média de velocidade de fluxo; sendo, após, calculadas o Índice de Pourcelot, sendo: [pico de velocidade sistólico pico diastólico final] / pico de velocidade sistólico, que representa um índice de resistência (IR); e índice de pulsatilidade (IP). Foram excluídos: recém-nascidos com instabilidade hemodinâmica; em ventilação assistida com altos parâmetros; síndromes mal-formativas; intolerância alimentar ou enterocolite necrosante; fototerapia; presença de cateteres umbilicais, persistência de canal arterial e pequenos para a idade gestacional. O exame pré-prandial foi realizado antes da alimentação (até 30 minutos) e pós-prandial entre 15 e 60 minutos após a alimentação. Foram realizados no primeiro dia (entre 6 a 24 horas de vida), no terceiro, no sétimo, e após, semanalmente até 42 dias de vida. Os resultados foram expressos em médias e desvios-padrão e descritos de maneira evolutiva. RESULTADOS: Ao total, foram estudados 77 RNPT e realizados 125 exames. Os valores em média±desvio-padrão são descritos na seqüência do primeiro, terceiro, sétimo e, consecutivamente a cada semana, até 42 dias de vida; sendo: IR pré-prandial de 0,69±0,09; 0,67±0,15; 0,75±0,07; 0,74±0,07; 0,75±0,07; 0,76±0,07; 0,79±0,03; 0,78±0,05 e IR pós-prandial de 0,66±0,10; 0,70±0,21; 0,74±0,07; 0,73±0,08; 0,75±0,06; 0,76±0,06; 0,77±0,04; 0,77±0,03. Os resultados de IP pré-prandial foram: 1,45±0,30; 1,35±0,28; 1,68±0,29; 1,50±0,23; 1,47±0,22; 1,52±0,20; 1,62±0,09; 1,68±0,06 e IP pós-prandial: 1,38±0,39; 1,40±0,29; 1,58±0,26; 1,46±0,26; 1,45±0,24; 1,50±0,27; 1,58±0,10; 1,64±0,04. Obtivemos PVS pré-prandial: 60,51±22,24; 55,24±26,04; 90,61±12,74; 95,33±18,11; 92,89±15,40; 96,96±12,18; 63,18±14,08; 58,12±9,78 e pós-prandial: 59,60±24,14; 110,82±32,45; 118,10±20,15; 121,95±24,18; 124,15±25,16; 126,07±18,17; 96,68±11,12; 96,12±8,98. Quanto a PVDF pré-prandial, obtivemos: 18,85±6,09; 18,66±10,01; 20,99±8,12; 22,02±8,50; 23,04±7,89; 22,24±8,02; 11,99±6,15; 12,05±5,12 e PVDF pós-prandial: 20,63±6,89; 30,15±12,78; 27,98±9,72; 29,02±10,05; 34,56±9,00; 32,02±8,45; 19,02±4,95; 21,15±3,43. A partir dos resultados acima, demonstra-se que o fluxo sanguíneo da AMS em RNPT saudáveis apresenta uma evolução peculiar a partir do nascimento tanto dos valores basais quanto após a estimulação com a dieta, representados por uma evolução característica dos índices de resistência, melhora dos picos de velocidades sistólica e diastólica e melhora da resposta vasodilatadora após a alimentação enteral. CONCLUSÕES: RNPT saudáveis de idade gestacional ao nascimento de 27 a 34 semanas completas apresentam uma evolução do fluxo sanguíneo da artéria mesentérica superior de maneira peculiar, do nascimento até 42 dias de vida, tanto dos valores basais quanto em resposta à alimentação. O conhecimento destes valores pode indicar a dopplerfluxometria como um método preventivo de avaliação específico de cada RNPT para a introdução e progressão mais segura da alimentação, reduzindo a ocorrência de quadros gastrintestinais, melhorando os índices de morbi-mortalidade neonatal. / INTRODUCTION: The knowledge of the normal values of indices of Doppler velocimetry of the superior mesenteric artery in healthy premature neonates may help to prevent feeding intolerance situations and necrotizing enterocolitis. METHODS: In order to describe the indices for evaluation of Doppler velocimetry of the superior mesenteric artery in healthy premature neonates with gestational age between 27 and 34 weeks, on the first, third, seventh days, and then weekly, until six weeks of life; this is a prospective cohort study. The Doppler velocimetric examination was done by means of the Logic Book 8C-RS (General Electric USA), using a 8 MHz imaging transducer, with the pulsed color Doppler readings being obtained by sonographic waves at 4 MHz. The neonate was kept in a supine position, with the transducer positioned in the epigastric region, immediately below the xyphoid appendix, obtaining two-dimensional images of the celiac trunk and of the superior mesenteric artery, a few millimeters after its emergence from the aorta in the sagittal plane. The flux measurements were obtained in the longitudinal direction of the vessel and at an angle of insonation between 0 and 20 degrees. The blood flow curves were recorded after a sequence of five stable measurements, with respect to the quality of the waves, and with respect to their audible characteristics; thus obtaining the following measurements: peak systolic velocity (PSV), end diastolic velocity (EDV) and average flow velocity; with the Pourcelot Index being calculated subsequently, that is: [peak of systolic velocity end diastolic velocity / peak of systolic velocity, which represents a resitance index (RI); and pulsatility index (PI). The values obtained were expressed as averages and standard deviations. The results were stored in an Excel database, with blind analysis after the conclusion of data gathering. Uncomplicated and appropriate for gestational age premature neonates with gestational age between 27 and 34 weeks at birth were included in the study. We adopted as criteria for exclusion from the study: neonates in unstable hemodynamic conditions; needing assisted ventilation with high parameters; large deformations or clinical syndromes; feeding intolerance or diagnosis of necrotizing enterocolitis; conditions that alter the mesenteric flow, such as: phototherapy, presence of umbilical catheters, patent ductus arteriosus and sepsis. The exams were done prior to feeding (up to 30 minutes) and after feeding (between 15 and 60 minutes). If the neonate was fasting, only one of the above parameters was measured, in order to establish behavior of the basal mesenteric flow at that moment. The exams were done on the first day (between the 6th and 24th hours of life), third, seventh days, and then weekly, until six weeks of life. Data are shown as the mean ± standard deviation and described for each postnatal age group. RESULTS: A total of 77 neonates were studied and realized 125 exams. The values of the resistance and pulsatility indices (RI and PI); peaks of systolic (PSV) and final diastolic velocity (EDV) on the first, third, seventh days, and then, on sequentially for each week until six weeks of postnatal life; as mean and standard deviations, was described: RI prior to feeding were 0,69±0,09; 0,67±0,15; 0,75±0,07; 0,74±0,07; 0,75±0,07; 0,76±0,07; 0,79±0,03; 0,78±0,05 and RI after feeding were 0,66±0,10; 0,70±0,21; 0,74±0,07; 0,73±0,08; 0,75±0,06; 0,76±0,06; 0,77±0,04; 0,77±0,03. The results of PI prior to feeding: 1,45±0,30; 1,35±0,28; 1,68±0,29; 1,50±0,23; 1,47±0,22; 1,52±0,20; 1,62±0,09; 1,68±0,06 and PI after feeding: 1,38±0,39; 1,40±0,29; 1,58±0,26; 1,46±0,26; 1,45±0,24; 1,50±0,27; 1,58±0,10; 1,64±0,04. The values of PSV prior to feeding were: 60,51±22,24; 55,24±26,04; 90,61±12,74; 95,33±18,11; 92,89±15,40; 96,96±12,18; 63,18±14,08; 58,12±9,78 and after feeding: 59,60±24,14; 110,82±32,45; 118,10±20,15; 121,95±24,18; 124,15±25,16; 126,07±18,17; 96,68±11,12; 96,12±8,98. And the results of EDV prior to feeding: 18,85±6,09; 18,66±10,01; 20,99±8,12; 22,02±8,50; 23,04±7,89; 22,24±8,02; 11,99±6,15; 12,05±5,12 and EDV after feeding: 20,63±6,89; 30,15±12,78; 27,98±9,72; 29,02±10,05; 34,56±9,00; 32,02±8,45; 19,02±4,95; 21,15±3,43. These results shows that healthy premature neonates with gestational age between 27 and 34 weeks presents a peculiar evolution in blood flow in the superior mesenteric artery after birth, represented by the resistance patterns caracteristics, improvement in peaks of systolic and diastolic velocity, and improvement in vasodilation in response to feeding. CONCLUSION: These results suggest for the Doppler velocimetry as specific and preventive evaluation method for each premature neonate, as a way to a safer introduction and progression of feeding, reducing the prevalence of gastrointestinal inflammatory diseases in neonates, and improving the indices of neonatal morbidity and mortality. Knowledge of blood-flow velocity in the superior mesenteric artery in uncomplicated preterm infants might provide a clue in investigating the maturation of intestinal circulation and the pathogenesis or pathophysiology of gastrointestinal diseases in newborn infants.

INTRODUÇÃO: O conhecimento de valores de normalidade do fluxo sanguíneo da artéria mesentérica superior (AMS) em recém-nascidos prematuros (RNPT) saudáveis pode prevenir quadros de intolerância alimentar e a ocorrência da enterocolite necrosante. MÉTODOS: Com o objetivo de descrever a evolução dos índices de avaliação da dopplerfluxometria da AMS em RNPT saudáveis de idade gestacional entre 27 e 34 semanas completas, no primeiro, no terceiro, no sétimo e semanalmente (14, 21, 28, 35 e 42 dias de vida), foi realizado este estudo coorte prospectivo em RNPT de idade gestacional ao nascimento entre 27 e 34 semanas completas. O exame dopplerfluxométrico foi realizado, após o consentimento livre e esclarecido dos responsáveis pelos RNPT, através do aparelho Logic Book 8C-RS (General Eletric EUA); obtendo-se as seguintes medidas: pico de velocidade sistólica (PVS), pico de velocidade diastólica final (PVDF) e média de velocidade de fluxo; sendo, após, calculadas o Índice de Pourcelot, sendo: [pico de velocidade sistólico pico diastólico final] / pico de velocidade sistólico, que representa um índice de resistência (IR); e índice de pulsatilidade (IP). Foram excluídos: recém-nascidos com instabilidade hemodinâmica; em ventilação assistida com altos parâmetros; síndromes mal-formativas; intolerância alimentar ou enterocolite necrosante; fototerapia; presença de cateteres umbilicais, persistência de canal arterial e pequenos para a idade gestacional. O exame pré-prandial foi realizado antes da alimentação (até 30 minutos) e pós-prandial entre 15 e 60 minutos após a alimentação. Foram realizados no primeiro dia (entre 6 a 24 horas de vida), no terceiro, no sétimo, e após, semanalmente até 42 dias de vida. Os resultados foram expressos em médias e desvios-padrão e descritos de maneira evolutiva. RESULTADOS: Ao total, foram estudados 77 RNPT e realizados 125 exames. Os valores em média±desvio-padrão são descritos na seqüência do primeiro, terceiro, sétimo e, consecutivamente a cada semana, até 42 dias de vida; sendo: IR pré-prandial de 0,69±0,09; 0,67±0,15; 0,75±0,07; 0,74±0,07; 0,75±0,07; 0,76±0,07; 0,79±0,03; 0,78±0,05 e IR pós-prandial de 0,66±0,10; 0,70±0,21; 0,74±0,07; 0,73±0,08; 0,75±0,06; 0,76±0,06; 0,77±0,04; 0,77±0,03. Os resultados de IP pré-prandial foram: 1,45±0,30; 1,35±0,28; 1,68±0,29; 1,50±0,23; 1,47±0,22; 1,52±0,20; 1,62±0,09; 1,68±0,06 e IP pós-prandial: 1,38±0,39; 1,40±0,29; 1,58±0,26; 1,46±0,26; 1,45±0,24; 1,50±0,27; 1,58±0,10; 1,64±0,04. Obtivemos PVS pré-prandial: 60,51±22,24; 55,24±26,04; 90,61±12,74; 95,33±18,11; 92,89±15,40; 96,96±12,18; 63,18±14,08; 58,12±9,78 e pós-prandial: 59,60±24,14; 110,82±32,45; 118,10±20,15; 121,95±24,18; 124,15±25,16; 126,07±18,17; 96,68±11,12; 96,12±8,98. Quanto a PVDF pré-prandial, obtivemos: 18,85±6,09; 18,66±10,01; 20,99±8,12; 22,02±8,50; 23,04±7,89; 22,24±8,02; 11,99±6,15; 12,05±5,12 e PVDF pós-prandial: 20,63±6,89; 30,15±12,78; 27,98±9,72; 29,02±10,05; 34,56±9,00; 32,02±8,45; 19,02±4,95; 21,15±3,43. A partir dos resultados acima, demonstra-se que o fluxo sanguíneo da AMS em RNPT saudáveis apresenta uma evolução peculiar a partir do nascimento tanto dos valores basais quanto após a estimulação com a dieta, representados por uma evolução característica dos índices de resistência, melhora dos picos de velocidades sistólica e diastólica e melhora da resposta vasodilatadora após a alimentação enteral. CONCLUSÕES: RNPT saudáveis de idade gestacional ao nascimento de 27 a 34 semanas completas apresentam uma evolução do fluxo sanguíneo da artéria mesentérica superior de maneira peculiar, do nascimento até 42 dias de vida, tanto dos valores basais quanto em resposta à alimentação. O conhecimento destes valores pode indicar a dopplerfluxometria como um método preventivo de avaliação específico de cada RNPT para a introdução e progressão mais segura da alimentação, reduzindo a ocorrência de quadros gastrintestinais, melhorando os índices de morbi-mortalidade neonatal. / INTRODUCTION: The knowledge of the normal values of indices of Doppler velocimetry of the superior mesenteric artery in healthy premature neonates may help to prevent feeding intolerance situations and necrotizing enterocolitis. METHODS: In order to describe the indices for evaluation of Doppler velocimetry of the superior mesenteric artery in healthy premature neonates with gestational age between 27 and 34 weeks, on the first, third, seventh days, and then weekly, until six weeks of life; this is a prospective cohort study. The Doppler velocimetric examination was done by means of the Logic Book 8C-RS (General Electric USA), using a 8 MHz imaging transducer, with the pulsed color Doppler readings being obtained by sonographic waves at 4 MHz. The neonate was kept in a supine position, with the transducer positioned in the epigastric region, immediately below the xyphoid appendix, obtaining two-dimensional images of the celiac trunk and of the superior mesenteric artery, a few millimeters after its emergence from the aorta in the sagittal plane. The flux measurements were obtained in the longitudinal direction of the vessel and at an angle of insonation between 0 and 20 degrees. The blood flow curves were recorded after a sequence of five stable measurements, with respect to the quality of the waves, and with respect to their audible characteristics; thus obtaining the following measurements: peak systolic velocity (PSV), end diastolic velocity (EDV) and average flow velocity; with the Pourcelot Index being calculated subsequently, that is: [peak of systolic velocity end diastolic velocity / peak of systolic velocity, which represents a resitance index (RI); and pulsatility index (PI). The values obtained were expressed as averages and standard deviations. The results were stored in an Excel database, with blind analysis after the conclusion of data gathering. Uncomplicated and appropriate for gestational age premature neonates with gestational age between 27 and 34 weeks at birth were included in the study. We adopted as criteria for exclusion from the study: neonates in unstable hemodynamic conditions; needing assisted ventilation with high parameters; large deformations or clinical syndromes; feeding intolerance or diagnosis of necrotizing enterocolitis; conditions that alter the mesenteric flow, such as: phototherapy, presence of umbilical catheters, patent ductus arteriosus and sepsis. The exams were done prior to feeding (up to 30 minutes) and after feeding (between 15 and 60 minutes). If the neonate was fasting, only one of the above parameters was measured, in order to establish behavior of the basal mesenteric flow at that moment. The exams were done on the first day (between the 6th and 24th hours of life), third, seventh days, and then weekly, until six weeks of life. Data are shown as the mean ± standard deviation and described for each postnatal age group. RESULTS: A total of 77 neonates were studied and realized 125 exams. The values of the resistance and pulsatility indices (RI and PI); peaks of systolic (PSV) and final diastolic velocity (EDV) on the first, third, seventh days, and then, on sequentially for each week until six weeks of postnatal life; as mean and standard deviations, was described: RI prior to feeding were 0,69±0,09; 0,67±0,15; 0,75±0,07; 0,74±0,07; 0,75±0,07; 0,76±0,07; 0,79±0,03; 0,78±0,05 and RI after feeding were 0,66±0,10; 0,70±0,21; 0,74±0,07; 0,73±0,08; 0,75±0,06; 0,76±0,06; 0,77±0,04; 0,77±0,03. The results of PI prior to feeding: 1,45±0,30; 1,35±0,28; 1,68±0,29; 1,50±0,23; 1,47±0,22; 1,52±0,20; 1,62±0,09; 1,68±0,06 and PI after feeding: 1,38±0,39; 1,40±0,29; 1,58±0,26; 1,46±0,26; 1,45±0,24; 1,50±0,27; 1,58±0,10; 1,64±0,04. The values of PSV prior to feeding were: 60,51±22,24; 55,24±26,04; 90,61±12,74; 95,33±18,11; 92,89±15,40; 96,96±12,18; 63,18±14,08; 58,12±9,78 and after feeding: 59,60±24,14; 110,82±32,45; 118,10±20,15; 121,95±24,18; 124,15±25,16; 126,07±18,17; 96,68±11,12; 96,12±8,98. And the results of EDV prior to feeding: 18,85±6,09; 18,66±10,01; 20,99±8,12; 22,02±8,50; 23,04±7,89; 22,24±8,02; 11,99±6,15; 12,05±5,12 and EDV after feeding: 20,63±6,89; 30,15±12,78; 27,98±9,72; 29,02±10,05; 34,56±9,00; 32,02±8,45; 19,02±4,95; 21,15±3,43. These results shows that healthy premature neonates with gestational age between 27 and 34 weeks presents a peculiar evolution in blood flow in the superior mesenteric artery after birth, represented by the resistance patterns caracteristics, improvement in peaks of systolic and diastolic velocity, and improvement in vasodilation in response to feeding. CONCLUSION: These results suggest for the Doppler velocimetry as specific and preventive evaluation method for each premature neonate, as a way to a safer introduction and progression of feeding, reducing the prevalence of gastrointestinal inflammatory diseases in neonates, and improving the indices of neonatal morbidity and mortality. Knowledge of blood-flow velocity in the superior mesenteric artery in uncomplicated preterm infants might provide a clue in investigating the maturation of intestinal circulation and the pathogenesis or pathophysiology of gastrointestinal diseases in newborn infants.

La recherche en bactériologie et virologie est à la fois de nature cognitive et appliquée. Elle consiste à fédérer et mettre en place une capacité de recherche multidisciplinaire et pouvoir l'intégrer sur un champ très vaste de microorganismes et de maladies. Les nouvelles avancées conceptuelles et technologiques dans le domaine de la génomique, notamment les avancées dans les techniques à haut débit (séquençage, PCR...) permettent actuellement d’avoir rapidement des génomes bactériens et viraux entiers, ou seulement sur quelques gènes d’une grande population. Les progrès dans ce domaine permettent l’accès à ces informations en évitant une combinaison de plusieurs méthodologies, et à moindre coûts. Dans notre travail de thèse, nous avons été porté à analyser et traiter les données de deux études genomiques et métagenomiques, mettant en évidence avantages, limites et attentes liés à ces techniques. La première étude porte sur l'analyse génomique de nouveaux virus géants et chlamydia infectant Vermamoeba vermiformis. La deuxième étude concerne le pyroséquençage 16S de microbiote intestinal de nouveau-nés atteint de l'entérocolite nécrosante. Pour le premier projet du travail de thèse, nous avons analysé les génomes de trois nouvelles espèces de Chlamydiae et onze virus giants (premiers membres de deux probables nouvelles familles) qui se multiplient naturellement dans Vermamoeba vermiformis. L'objectif étant de mettre en évidence les caractéristiques génétiques spécifiques à ces micro-organismes. La deuxième partie a été consacrée à l'analyse des données de pyroséquençage 16S des selles de nouveau-nés atteints de l'entérocolite nécrosante. / Research in bacteriology and virology is both cognitive and applied. It involves federating and developing a multidisciplinary research capacity and being able to integrate it into a very broad field of microorganisms and diseases. New genomic and conceptual advances in genomics, including advances in high-throughput techniques, now permit rapid bacterial and viral genomes, or only a few genes of a large population. Progress in this area allows access to this information by avoiding a combination of several methodologies and at lower costs. In our thesis work, we were led to analyze and process the data of two genomic and metagenomic studies, highlighting advantages, limitations and expectations related to these techniques. The first study focuses on the genomic analysis of new giant viruses and chlamydia infecting Vermamoeba vermiformis. The second study concerns the 16S pyrosequencing of intestinal microbiota of neonates with necrotizing enterocolitis. The first project of the thesis work analyzed the genomes of three new species of Chlamydiae and eleven giant viruses (first members of two probable new families) which naturally multiply in Vermamoeba vermiformis. The objective is to highlight the genetic characteristics specific to these microorganisms. The second part was devoted to the analysis of 16S pyrosequencing data from neonatal enterocolitis neonatal stools. The goal was to identify an agent responsible for this disease.

La dysbiose est une cause importante dans la survenue de maladies, favorisant la prolifération de pathogènes ou induisant l’inflammation. L’étude de ce phénomène est devenue possible grâce aux approches d’analyse génomique (AG) associé avec d’autres techniques. L’entérocolite nécrosante (ECN) et l’infection du pied diabétique (IPD) demeurent deux maladies associées à la dysbiose dans lesquels différents bactéries ont été décrites, notamment C. butyricum dans l’ECN, E. coli et S. aureus dans l’IPD. Dans le cadre de l’ECN, C. butyricum demeure l’espèce la plus fréquente chez les ECN. L’identification clusters liés géographiquement et en fonction du temps. Le portage asymptomatique est suggéré par une similarité génomique des souches patients et contrôles. La prédiction d’un gène de β-hémolysine ainsi leur effet cytotoxique sur les cellules Jurkat avait été observé. De même, sur les cellules Caco-2 malgré le KO du gène de β-hémolysine. En se basant sur l’analyse physico-chimique du surnageant bactérien, nous avons suggéré que la fraction cytotoxique est protéique. La purification de la fraction cytotoxique a permis de trouver une protéine codant pour PspC family possédant un domain conservé commun avec celui de la toxine A/B. L’inactivation du gène codant pour cette protéine n’a pas supprimé l’effet cytotoxique, suggérant la présence d’une combinaison gènes. En parallèle, nous avons ciblé l’impact de C. neonatale par qRPC spécifique rpoB. Cette espèce était plus fréquente chez les patients, ainsi de clones géographiques ont été identifiées. Enfin, des SNPs ont été observés dans des gènes de virulence dans le cas des E. coli et S. aureus isolés de l’IPD. / Dysbiosis remains a main cause during the establishment of several diseases, by promoting bacterial translocation, leading to inflammation process. Specific microorganisms were involved in the pathogenesis of dysbiosis-associated diseases, notably necrotizing enterocolitis (NEC) and diabetic foot (DF). This was possible by the implication of whole-genome analysis (WGA) in association with other techniques. In case of NEC, C. butyricum was significantly associated with in NEC; tested on a South-East French cohort. Geographical and/or temporal clusters were identified, thus genomic relationship between NEC-associated isolates and controls, suggesting the presence of asymptomatic carriage. Genes encoding for β-hemolysin was detected and C. butyricum supernatant exhibited cytotoxic effect on Jurkat cells. Cytotoxic effect was also presented on Caco-2 cells. Supernatant of β-hemolysin-mutant C. butyricum showed enterotoxic effect. Basing on physico-chemical data, we assumed that the evaluated fraction was a protein. Proteomics analysis revealed that PspC family was the cytotoxic protein. This protein owned a glucan-binding domain, shared by C. difficile toxin A/B. The KO of PspC gene was enterotoxic, suggesting by this the existence of a combination of genes. In parallel, a specific rpoB-based qPCR was developed to identify C. neonatale. We found that, C. neonatale was more prevalent in NEC than in controls. Although co-identified in association with C. butyricum. C. neonatale clones were distinguished especially in strains isolated from the same hospital. Regarding to DF infection, SNPs were identified within S. aureus and E. coli genomes, especially in virulent genes.

Contexte : Près de 2,5 millions de nouveau-nés meurent chaque année et plus de 80 % d'entre eux ont un petit poids à la naissance (PPN). Le PPN est une entité clinique complexe impliquant le retard de croissance in utero (RCIU) et la naissance prématurée (NPM). Les nouveau-nés survivants sont exposés à un risque élevé de morbidités périnatales graves (telles que la dysplasie broncho-pulmonaire, l'entérocolite nécrosante, l'encéphalopathie néonatale) en raison des effets dévastateurs de l'inflammation utéro-fœtale sur les organes fœtaux vulnérables. Il n'existe actuellement aucun traitement efficace pour la protection fœtale ante partum. Parmi les nombreux médiateurs pro-inflammatoires, l'IL-1β se distingue par ses effets délétères. Notre laboratoire a conçu un nouvel antagoniste allostérique du récepteur de l'IL-1, Rytvela, qui s'est avéré efficace contre la NPM lorsqu'il est administré en prophylaxie. Objectif : Cette étude vise à mieux caractériser Rytvela en évaluant son efficacité dans la prévention de la NPM et du RCIU lorsqu'il est administré après l'insulte inflammatoire initiale selon un cadre clinique plus réaliste. Méthodes : Des souris gravides CD-1 ont reçu une injection d'agents pro-inflammatoires/pro-travail, soit l’IL-1β (1 μg i.u.) ou le LPS (10 μg i.p.) aux jours 16-17 de la gestation. Rytvela (2 mg/kg/jour s.c.) a été administré à différents intervalles de temps (0,5h, 2h, 4h, 6h) après l’induction inflammatoire. Le taux de NPM, la survie et le poids des souriceaux ont été évalués. Des analyses histologiques des poumons, intestins et cerveau des nouveau-nés ont été réalisées. Résultats : Toutes les grossesses traitées avec Rytvela ont été menées à terme dans le modèle de l’IL-1β, alors que le taux de NPM était de 57 % dans le groupe non traité. La survie, la croissance et le poids des souriceaux ont été considérablement améliorés avec Rytvela administré 0,5 h post-inflammation (avec une survie presque doublée des portées). L'analyse histologique a révélé dans tous les modèles une morphogenèse fœtale protégée, y compris une alvéolarisation pulmonaire préservée, des villosités intestinales intactes et un arbre cérébrovasculaire protégé associé à une masse cérébrale préservée. Conclusion : Rytvela est efficace dans la prévention de la NPM et du RCIU lorsqu'il est administré en post-inflammatoire. Il présente un effet maximal lorsqu'il était administré rapidement (0,5 h après IL-1β/LPS) et maintenait des effets protecteurs fœtaux significatifs avec une administration retardée (jusqu'à 6 h après IL-1β/LPS). Rytvela améliore la survie et la santé néonatale en préservant l'intégrité et la croissance des tissus fœtaux. Par conséquent, Rytvela est un nouveau prototype thérapeutique prometteur et sécuritaire pour le traitement de la NPM et du RCIU. / Background: Over 2.5 million newborns die yearly and more than 80% of them are of low birthweight (LBW). LBW is a complex clinical entity involving fetal growth restriction (FGR) and preterm birth (PTB). Surviving neonates face a higher risk of serious perinatal morbidities (such as bronchopulmonary dysplasia, necrotizing enterocolitis, neonatal encephalopathy) due to the devastating effects of utero-fetal inflammation on vulnerable fetal organs. There is currently no efficient treatment for fetal antepartum protection. Among the many proinflammatory mediators, IL-1β stands out for its detrimental effects. The host lab has designed a novel allosteric IL-1 receptor antagonist, Rytvela, which has been shown to be effective against PTB when administered prophylactically. Objective: This study aims to further characterize Rytvela by evaluating its efficacy in preventing PTB and FGR when administered after the initial inflammatory insult according to a more realistic clinical setting. Methods: Pregnant CD-1 mice were injected with proinflammatory/prolabour agents, either IL-1β (1 μg i.u.) or LPS (10 μg i.p.) on days 16-17 of gestation. Rytvela (2 mg/kg/day s.c.) was administered at different time intervals (0.5, 2, 4, 6 h) after initial inflammatory insults. PTB rate, neonatal survival, and weight were assessed. Histological analyses of the lungs, intestines, and brain of the neonates were performed. Results: All pregnancies treated with Rytvela were carried to term in the IL-1β model, while the PTB rate was 57% in the untreated group. Pup survival, growth and weight were considerably improved with Rytvela administered 0.5h post-inflammatory insults (with a nearly 2-fold increase in litters survival). Histological analysis revealed in all models a protected morphogenesis of vulnerable fetal organs including preserved lung alveolarization, intact intestinal villi integrity, and protected cerebrovascular tree associated with preserved brain mass. Conclusion: Rytvela is efficient in preventing PTB and FGR when administered post-inflammatory insults. It exhibited maximum effect when administered promptly (0.5h post-IL-1β/LPS) and maintained significant fetal protective effects with delayed administration (up to 6h post- IL-1β/LPS). Rytvela improved birth outcome by preserving fetal tissue integrity and growth. Hence, Rytvela is a promising new and safe therapeutic prototype for treatment of PTB and FGR.

In der vorliegenden Arbeit wurde anhand der im Rahmen des Vermont-Oxford-Neonatal-Networks erhobenen Daten an der Berliner Klinik für Neonatologie der Charité Campus Mitte und der Abteilung für neonatologische Intensivmedizin der Universitätskinderklinik in Innsbruck ein Benchmarking-Projekt für die Jahre 1997 bis 2001 durchgeführt. Nach der Analyse des Outcomes wurde eine Analyse der externen Evidenz anhand von Literatursuche in PubMed und der Cochrane Datenbank für systematische Reviews durchgeführt. Danach wurde ein Fragebogen entworfen, der gezielt Handlungsstrategien und -richtlinien bezüglich der relevanten Outcome-Parameter erfragt. Für das Benchmarking-Projekt wurden das Atemnotsyndrom, die nekrotisierende Enterokolitis und die bakteriellen Infektionen ausgewählt. Die Analyse der Handlungsstrategien durch den Fragebogen zeigte, dass in den drei Bereichen respiratorische Interventionen, Nahrung und Ernährung sowie im Infektionsmanagement Unterschiede vorlagen. In der Diskussion zeigte sich, dass in vielen Bereichen noch Bedarf nach guter externer Evidenz und weiterer Forschung besteht. / This dissertation presents the results of a 1997 - 2001 benchmark project in co-operation with the "Berliner Klinik für Neonatologie der Charité Campus Mitte" and the "Abteilung für neonatologische Intensivmedizin der Universitätskinderklinik" in Innsbruck. The study is based on the Vermont-Oxford-Neonatal-Network''s data. After analysing the results, further evidence was analysed by way of literary research in PubMed and the Cochrane Database of Systematic Reviews. Afterwards, a questionnaire was created, lining out the clinical guidelines of the relevant outcome parameters. The respiratory distress syndrom, the necrotising enterocolitis and the bacterial infections were selected for the benchmark. The internal guidelines'' analysis showed that there were differences between the two clinics'' results in respiratory interventions, feeding and the management of infections. The discussion made clear that research based on further evidence is necessary in many fields.