CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPEMIG - Fundação de Amparo a Pesquisa do Estado de Minas Gerais / Muitos microorganismos desenvolveram a habilidade de adquirir ferro a partir do seu hospedeiro para seu próprio metabolismo. Toxoplasma gondii, um parasito intracelular obrigatório, produz proteinas de roptrias que são capazes de ligar ao transportador de ferro. Além disso, quelante de ferro foi capaz de controlar o parasitismo em células intestinais de rato. Logo, este trabalho teve como objetivo investigar a relação do efeito da adição ou privação de ferro na multiplicação de T. gondii e resultado da infecção quando o parasito é administrado por via oral e em cultura celular. Camundongos C57BL/6 foram infectados oralmente com T. gondii e tratados com um quelante de ferro, deferoxamina ou suplementados com ferro e o parasitismo, parâmetros imunológicos e histológicos foram analisados. Observou-se que a infecção aumentou a deposição de ferro no intestino delgado, pulmão e fígado e também sistematicamente. O tratamento com deferoxamina foi capaz de diminuir os níveis de ferro nas amostras de soro. O tratamento com deferoxamina diminuiu a carga parasitária no intestino delgado e alterações inflamatórias no órgão, preservando seu comprimento e também diminuiu o parasitismo pulmonar. Além disso, a suplementação com ferro aumentou a carga parasitária no intestino delgado, pulmão e fígado, associado com alterações inflamatórias nesses órgãos, bem como houve aumento da proliferação parasitária em cultivo celular com células Caco-2. A infecção oral modulou o perfil de expressão de marcadores de absorção de ferro, aumentando os níveis de IL-6 sistêmicos. Juntos, nossos resultados sugerem que a quelação de ferro comumente usada para tratar a sobrecarga de ferro pode ser um medicamento promissor para controlar a proliferação de T. gondii e consequentemente a inflamação causada por infecção e que o ferro é um nutriente essencial para o metabolismo de T. gondii. / Many microorganisms have developed the ability to acquire iron from their host to their own metabolism. Toxoplasma gondii, an obligate intracellular parasite, produces rhoptry proteins that are capable of binding to the iron transporter. In addition, iron chelator was able to control parasitism in rat intestinal cells. Therefore, the objective of this work was to investigate the relationship of the effect of iron addition or deprivation on the multiplication of T. gondii and the result of infection when the parasite is administered orally and in cell culture. C57BL/6 mice were orally infected with T. gondii and treated with an iron chelator, deferoxamine or iron supplemented and parasitism, immunological and histological parameters were analyzed. It was observed that the infection increased the deposition of iron in the small intestine, lung and liver and also systematically. Treatment with deferoxamine was able to decrease iron levels in the serum samples. Treatment with deferoxamine reduced parasite burden in the small intestine and inflammatory changes in the organ, preserving its length and also decreased pulmonary parasitism. In addition, iron supplementation increased parasite burden in the small intestine, lung and liver, associated with inflammatory changes in these organs, as well as increased parasite proliferation in cell culture with Caco-2 cells. Oral infection modulated the expression profile of iron absorption markers, increasing systemic IL-6 levels. Together, our results suggest that iron chelation commonly used to treat iron overload may be a promising drug to control T. gondii proliferation and consequently inflammation caused by infection and that iron is an essential nutrient for the metabolism of T. gondii. / Tese (Doutorado)

Foi avaliado o fornecimento do colostro bovino liofilizado como alternativa de fonte inicial de imunoglobulinas para caprinos recém-nascidos bem como as características histológicas, considerando avaliações histomorfométricas, estereológicas, imunohistoquímicas e o número de células caliciformes no epitélio intestinal destes animais. Foram utilizados dois grupos de 15 cabritos, que receberam 5% do peso vivo de colostro bovino liofilizado (CBL) ou colostro caprino (CC), com 55 mg/mL de imunoglobulina G, às 0, 7 e 14 horas de vida. Amostras do duodeno, jejuno médio e íleo foram coletadas às 18, 36 e 96 horas de vida para análises histológica e imunohistoquímica. Três animais foram sacrificados logo após o nascimento, constituindo o grupo zero hora. O delineamento experimental foi inteiramente casualizado, sendo as variáveis morfométricas consideradas em arranjo fatorial 2x3+1, tendo como efeitos principais os dois tipos de colostro, os três horários de abate e o grupo zero hora. A concentração de IGF-I no colostro bovino liofilizado e colostro caprino foi de 158,71 e 356,32 ng/mL, respectivamente. A altura das vilosidades no jejuno mostrou-se superior (P<0,05), apresentando vilosidades 30,7% e 24,2% mais altas do que o duodeno e o íleo às 36h, respectivamente. O duodeno, às 18h, apresentou profundidade das criptas 48,4% maior que o jejuno e, às 96h as criptas duodenais mostraram-se 23,2% maiores que as criptas do íleo (P<0,05). No jejuno, as criptas apresentaram-se mais profundas às 96h (P<0,05) do que nos outros horários. A espessura da túnica muscular no jejuno apresentou-se mais espessa às 36 e 96h do que à zero h e mais espessa às 36h do que às 18h (P<0,05). No geral, jejuno e íleo, apresentaram túnica muscular menos espessa (P<0,05) que a encontrada no duodeno. Independente dos segmentos, às 96h a túnica muscular foi mais espessa que às 18h. O Vv da mucosa absortiva foi maior no jejuno, enquanto no íleo às 36h o Vv foi maior do que nos demais horários experimentais. Independente do segmento, às 36h o Vv foi 17,3% maior que às 96h. O número de células caliciformes no jejuno foi 78,6% menor às 18h (P<0,05) nos cabritos que ingeriram colostro caprino e 54,6% maior às 96h (P<0,05) nos animais que ingeriram colostro bovino liofilizado. Colostro bovino liofilizado pode ser utilizado como fonte de proteção passiva alternativa para os caprinos recémnascidos. O IGF-I presente no colostro não influenciou a morfometria entérica. O jejuno mostrou-se o segmento mais importante no processo absortivo de imunoglobulinas. A distribuição dos anticorpos internalizados pelo epitélio entérico mostrou-se relacionada aos segmentos do intestino e tempo de vida dos recém-nascidos, independente da fonte de anticorpos, bovina liofilizada ou caprina. / It was evaluated the supply of the lyophilized bovine colostrum as an alternative source of immunoglobulin for newborn goat kids and the histological characteristics, through histomorphometric, stereological, immunohistochemical evaluations and goblet cells number in the intestinal epithelium in this animals. Two groups of fifteen newborn goat kids received 5% of body weight of lyophilized bovine colostrum (LBC) or caprine colostrum (CC) with 55 mg/mL of immunoglobulin G at 0, 7 and 14 hr of life. Samples of duodenum, medium jejunum and ileum were collected at 18, 36 and 96 hr of life for histological and immunohistochemical analysis. Three animals were sampled just after birth representing the zero hr group. A completely randomized designing was used, and the variable morphometric was considered as 2x3+1 factorial arrangement, having as the main factors colostrum supply, the three slaughter hours and the zero hr group. The IGF-I concentration in lyophilized bovine colostrum or caprine colostrum was 158,71 and 356,32 ng/mL, respectively. Villous height was superior in the jejunum (P<0.05) with villous 30.7% and 24.2% higher than duodenum and ileum at 36hr, respectively. The duodenum showed at 18hr crypts 48.4% deeper than in the jejunum and at 96hr, the duodenal crypts was 23.2% deeper than ileum crypts (P<0.05). In the jejunum, crypts were deeper at 96hr compared to the other experimental hours. The muscularis submucosal thickness in the jejunum was thicker at 36 and 96hr than zero hr and was thicker at 36hr than 18hr. Jejunum and ileum, in general thinner (P<0.05) tunica muscular layer than found in the duodenum. Regardless of the segments, at 96hr, the muscularis submucosal was thicker than 18hr. The absorptive mucosa partial volume (Vv) was higher in the jejunum, while in the ileum the Vv was higher at 36hr than all other experimental dates. Regardless of the segments, at 36hr, the Vv was 17.3% higher than at 96hr. The goblet cells number was different in jejunum with 78.6% less cells at 18hr (P<0.05) in the goat kids fed caprine colostrum and 54.6% higher at 96hr (P<0.05) in the goat kids fed lyophilized colostrum. Lyophilized bovine colostrum can be used as an alternative source of passive protection for the newborn kids. The IGF-I present in colostrum didnt influence the enteric morphology. The jejunal epithelium was the most important segment related to absorption process. The internalized antibody distribution in the enteric epithelium showed related to the small intestine segments and post-partum period, regardless of the antibodies source, lyophilized bovine or caprine.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2015-05-25T13:09:16Z No. of bitstreams: 2 Tese - Felix André Sanches Penhavel - 2014.pdf: 2537517 bytes, checksum: 2edff02f8a1f3fa50c180c2222a53b17 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-05-25T13:34:49Z (GMT) No. of bitstreams: 2 Tese - Felix André Sanches Penhavel - 2014.pdf: 2537517 bytes, checksum: 2edff02f8a1f3fa50c180c2222a53b17 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-05-25T13:34:49Z (GMT). No. of bitstreams: 2 Tese - Felix André Sanches Penhavel - 2014.pdf: 2537517 bytes, checksum: 2edff02f8a1f3fa50c180c2222a53b17 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-12-15 / American Tripanosomiasis is one of the most prevalente tropical disease in Latin America. One third of brazilian territory is considered endemic for Chagas disease. Vectorial transmission is not complitely interrupted. In Central Brazil, specially Goiás State, digestive forms with megasyndormes have a high frequence. The involvement of small bowel has been discribed with functional disturbances. Patients with acquired megacolon may have normal bowel movements or long lasting constipation. Until now the influence of small bowel on the frequence of bowel movements is not clear. This prospective study addressed the small bowel motility measuring the orocecal transit time (OCTT) and the occurence of small bowel intestinal bacterial overgrowth (SIBO) by hydrogen breath test. 45 patients with positive serology for Chagas disease were divided into four groups: A – without megacolon and no constipation (17); B – with megacolon and no constipation (8); C – with megacolon and constipation (10) and D – without megacolon and constipation (10). Constipation was defined by at least 7 days without bowel movements. 15 healthy volunteers were taken as a control group (CG). Non hydrogen producers: 10/45 patients and 1/15 controls. The OCTT (medium time in minutes) was longer in patients than in controls: A=108.18, B=108.0, C=112.5, D=130.0 and CG=68.46. Patients together showed difference compared to controls (P=0.001). No difference was found among groups (P>0.05). The prevalence of SIBO was: 66.7% in constipated patients (C and D), 25% in non constipated patients (A and B) and 8.3% in controls. Significant statistical difference was found only comparing constipated patients and controls (P=0.017). The presence of megacolon did not show influence the frequence of SIBO (P=0.181). Lactose and D-xylose malabsorption was higher in controls. The number of patients with symptoms during the test was the same for chagasic and controls independently of the test result. Patients with Chagas disease have a prolonged OCTT and those with constipation showed a higher prevalence of SIBO and both factors are not related to megacolon. Chagasic patients showed a less frequency of lactose and D- xylose malabsorption. / A Tripanossomíase Americana é uma das doenças tropicais mais prevalentes na América Latina. Mais de um terço do território brasileiro é área endêmica, e a transmissão vetorial ainda não se interrompeu em todos os estados. Em Goiás, as megassíndromes do tubo digestivo são muito frequentes. O comprometimento do tubo digestivo não se restringe à víscera dilatada e alterações funcionais têm sido descritas no intestino delgado. O objetivo geral deste estudo foi investigar alterações de motilidade e de absorção de carboidratos no intestino delgado de pacientes chagásicos com e sem constipação e megacólon. Avaliou-se 45 pacientes com sorologia positiva para doença de Chagas, divididos em quatro grupos: A - sem constipação, sem megacólon (17); B - sem constipação, com megacólon (8); C - com constipação, com megacólon (10) e D - com constipação, sem megacólon (10). Quinze voluntários sadios foram usados como controles (GC). Todos os pacientes e controles realizaram três testes respiratórios com os substratos: lactulose, lactose e D-xilose. Identificou-se não produção de hidrogênio em 10 pacientes e um controle. O tempo de trânsito orocecal (TTOC) (média em minutos) foi maior nos pacientes do que nos controles: A=108,18; B=108,0; C=112,5; D=130,0 e GC=68,46 (P=0,001). Não houve diferença no TTOC entre os grupos de pacientes (P<0,05). A prevalência de supercrescimento bacteriano no intestino delgado (SBID) foi de 66,7% nos pacientes constipados (C e D), 25% nos não constipados (A e B) e de 8,3% nos controles (P=0,017). A presença de megacólon não influenciou a frequência de SBID (P=0,181). A má absorção de lactose foi maior entre os controles. O número de pacientes que apresentava sintomas ou não durante o teste da lactose foi igual para pacientes e controles, independentemente do resultado do teste. A má absorção de D-xilose foi mais frequente no grupo controle. Concluiu-se que os pacientes chagásicos têm o tempo de trânsito orocecal prolongado e que aqueles com constipação apresentam uma maior prevalência de SBID, ambos os fatores não sofrem influência da presença do megacólon. A má absorção de lactose e D-xilose foi maior entre controles.

Le macaque rhésus infecté par le virus de l’immunodéficience simienne (SIV) fait l’objet de nombreuses études en tant que modèle de la pathogenèse induite par le virus de l’immunodéficience humaine de type 1 (VIH-1). Il existe deux sous-espèces de macaque rhésus définies notamment d’après leur origine géographique. Le macaque rhésus indien montre une progression pathologique particulièrement rapide, caractérisée par une déplétion massive de la population lymphocytaire T CD4+ intestinale les jours suivant l’infection. Cette déplétion a été associée à la translocation des bactéries commensales à travers l’épithélium intestinal en phase chronique. En revanche, chez le macaque rhésus chinois la vitesse de développement de la maladie est comparable à celle des patients infectés par le VIH-1. En périphérie, les données virales et immunologiques sont également plus proches de ce qui est documenté chez l’Homme infecté. Toutefois, la cinétique de dégradation de la muqueuse intestinale les jours suivant l’infection reste peu explorée dans ce modèle. Dans un premier temps, mes travaux de doctorat ont permis de confirmer la dissémination rapide du SIV dans le tractus gastro-intestinal du macaque rhésus d’origine chinoise. L’intestin grêle, notamment l’iléon, est la cible d’une réplication virale soutenue et très précoce. Malgré une réplication virale intense, le nombre de lymphocytes T CD4+ dans la muqueuse de l’iléon reste constant durant les deux premières semaines suivant l’infection par le virus dans ce modèle. Nous observons en revanche une augmentation conséquente du nombre de cellules T cytotoxiques et de macrophages, suggérant la mise en place d’une forte réponse immune in situ. Nous démontrons que l’augmentation du nombre de ces cellules et le maintien du nombre de lymphocytes T CD4+ dans la muqueuse iléale sont certainement liés, du moins en partie, au recrutement de cellules circulantes. En effet, nous décrivons pour la première fois une augmentation significative de l’expression de nombreuses chimiokines par cette muqueuse dès les premiers jours suivant l’infection. En parallèle nous décrivons, dans le sang périphérique, une diminution transitoire du nombre de lymphocytes T CD4+ et CD8+. Enfin, nous avons décelé une augmentation de l’expression d’interleukine 7 (IL-7) après infection. Cette augmentation, spécifiquement observée dans la muqueuse de l’intestin grêle, est corrélée à l’expression des chimiokines. Ces résultats apportent de nouveaux éléments sur la contribution de l’IL-7 dans la régulation de l’expression des chimiokines par la muqueuse intestinale suite à l’infection par le SIV. L’ensemble de nos résultats démontre que la population de lymphocytes T CD4+ de l’intestin grêle est préservée au cours de l’infection aiguë par le SIV chez le macaque rhésus chinois. En parallèle, l’exacerbation de l’expression locale de chimiokines laisse supposer une relocalisation des cellules du système immunitaire vers la muqueuse intestinale. Ces migrations pourraient avoir des effets délétères pour l’hôte en apportant de nouvelles cibles nourrissant la réplication virale. A l’opposé, le recrutement localisé de cellules immunitaires clés pour le déclenchement des réponses antivirales innées et adaptatives pourrait limiter la réplication du virus. Il est donc crucial de mieux définir l’impact de ce recrutement sur l’immunité muqueuse et la progression de la maladie. Nos découvertes apportent également de nouveaux arguments en faveur de l’utilisation du macaque rhésus d’origine chinoise en tant que modèle de choix pour l’étude de la physiopathologie de l’infection humaine par le VIH-1. / As a model to study type 1 human immunodeficiency virus (HIV-1) pathogenesis, rhesus macaques infected with the simian immunodeficiency virus (SIV) are under extensive investigation. Two subspecies of rhesus macaques have been defined, based on a different geographic origin. Indian rhesus macaques exhibit a rapid disease progression and acute infection is characterized by a massive CD4 T-cell loss in the intestinal mucosa. This was associated to the translocation of bacterial products through the gut epithelium during the chronic stage. Contrary to the animals of Indian origin, the pathogenesis of Chinese rhesus macaques infected with SIV is similar to HIV-1 infected patients. Viral and immunological settings in periphery are also closer to what is described in infected humans. However, the kinetics of mucosal disruption is poorly documented in this model. As a first step, I confirmed the rapid SIV dissemination in the gastro-intestinal tract of Chinese rhesus macaques. The small intestine, in particular the ileum, undergoes an early and high viral replication. Despite this high viral load, the numbers of CD4+ T cells in the ileum mucosa remains unchanged during the first two weeks following infection in this model. On the other hand, we noticed a substantial augmentation of cytotoxic T-cell numbers and macrophages, suggesting the establishment in situ of a strong immune response. We demonstrated that this augmentation of CD8+ T cells and macrophages together with the maintenance of helper T-cell numbers in the ileum mucosa are most probably related, at least in part, to the recruitment of circulating cells. Indeed, we describe for the first time a significant augmentation of numerous chemokine expressions by this mucosa the first days post-infection. At the same time, we described a transient diminution of CD4+ and CD8+ T-cell numbers in the blood. Finally, we detected a significant upregulation of interleukine 7 (IL-7) expression after SIV infection. This increase, specifically observed in the small intestine mucosa, is correlated to chemokine expressions. These results highlight new evidences on IL-7 contribution in the regulation of chemokines expression following SIV infection. All together, our results revealed the preservation of CD4+ T cell population in the small intestine mucosa during the acute phase of SIV infection in Chinese rhesus macaques. Furthermore, the exacerbation of local chemokine expressions let us think that immune cells are relocated to the intestinal mucosa the first days after infection. These migrations could have deleterious effects to the host, bringing new targets for viral replication. On the other side, this localized recruitment of immune cells that are key players in intestinal immunity could restrict the replication of the virus. Consequently, it is of major importance to better define the impact of immune cells trafficking on intestinal mucosa integrity and disease progression. Our findings bring new arguments in favor of Chinese rhesus macaque as a suitable model to study HIV-1 pathogenesis.

Results of recent studies indicate that dendritic cells are capable of transporting commensal intestinal bacteria into the mammary glands, which ultimately leads to their occurrence in breast milk. We have therefore decided to evaluate the phenotype of immunologically relevant antigen presenting cells (APCs) present in the mammary glands and the small intestine, respectively and perform a comparison study. We also studied plasticity of these populations during lactation. In situ immunodetection and flow cytometry methods were used to determine phenotype. We succeeded in optimising the methods for preparation of samples for flow cytometry and microscopy. We thoroughly tested protocols for 3D visualisation of APC populations and quantitative image analysis for correlation with flow cytometry, further optimization is nevertheless needed. We found out that during lactation large numbers of MHC II+ cells cluster around the alveoli and milk ducts. These cells are of a distinctly dendritic shape and their phenotype does not correspond to the APCs in the surrounding tissue. A pronounced increase of APC cells in the mammary glands between the fourth and sixth days of lactation was observed, with the majority of these cells expressing the CD103 antigen typical for cell populations of immune cells of the...

Résumé confidentiel / Résumé confidentiel

Plusieurs méthodes de capture antigénique ont été décrites dans l’intestin grêle, surtout en cas d’infection: échantillonnage direct par les cellules dendritiques (DC), capture par les macrophages qui délivrent ensuite l’antigène aux DC du stroma, passage des antigènes à travers les cellules caliciformes. Des travaux antérieurs in vitro dans le laboratoire ont montré l’importance de la myosine IIA dans la coordination de la migration des DC avec la capture et de l’apprêtement antigénique. L’objectif de ma thèse était de combiner plusieurs méthodes d’imagerie telle que la microscopie intravitale, la microscopie confocale ex vivo et l’immunofluorescence sur tissus à la cytométrie en flux pour déterminer l’impact de la myosine IIA sur la capture antigénique in vivo. Cette étude montre que les DC patrouillent en permanence dans l’épithélium de l’intestin grêle, y compris hors conditions infectieuses. Elles sont recrutées dans la lamina propria (LP) et pénètrent dans l’épithélium par transmigration à travers la membrane basale qui sépare ces deux compartiments. La myosine IIA est indispensable à la transmigration de CD103+CD11b+DC. Ces événements de transmigration surviennent plus fréquemment dans les parties proximales de l’intestin grêle, duodénum and jéjunum, que dans l’iléon. Chez les souris adultes, ces DC ne sont pas recrutées sous l’influence du microbiote mais sont sensibles au rétinal, un métabolite de la vitamine A qu’elles transforment en une molécule active l’acide trans-rétinoïque (AtRA). D’après notre analyse transcriptomique, les DC intra-épithéliales constituent une population homogène dont le profil est distinct de celui de leurs homologues de la LP. Elles sont enrichies en ARN des voies liées à l’apprêtement antigénique, l’autophagie et les lysosomes. Ces résultats suggèrent qu’elles ont une fonction différente des CD103+CD11b+DC de la LP: elles n’agissent pas sur la prolifération ni la différenciation des lymphocytes T mais contrôlent spécifiquement l’effectif des lymphocytes intra-épithéliaux CD8+αβ. Ces découvertes reflètent l’importance de l’épithélium comme première ligne de défense contre les pathogènes. Elles soulèvent également de nouvelles questions concernant la régulation de la réponse immune dans l’épithélium et les interactions mutuelles entre la lumière intestinale, l’épithélium et le stroma des villosités. / Several routes for antigen capture have been described in the small intestine, mainly upon pathogenic infection: direct sampling by Dendritic Cells (DCs), sampling by macrophages that deliver antigens to DCs in the stroma, antigenic passage through goblet cells. Previous in vitro work in the lab showed that myosin IIA is essential to coordinate antigen uptake and processing with DC migration. The objective of my thesis was to combine several imaging methods including intravital microscopy, ex vivo confocal microscopy and immunofluorescence on gut tissue to flow cytometry in order to unravel the impact of myosin IIA on DC physiology in vivo. My work shows that CD103+CD11b+ DCs, which are unique to the gut, constantly patrol the epithelium of the small intestine at steady state: they are recruited from the lamina propria (LP) and penetrate into the epithelium by transmigrating through the basal membrane that separates these two compartments. DC transmigration requires myosin IIA in vivo. Remarkably, we found that DC transmigration into the epithelium occurs mainly in the upper parts of the small intestine, the duodenum and the jejunum, but is not observed in the ileum. DC transmigration does not require the gut microbiota but relies on retinal, a vitamin A metabolite of that they convert into its active form all-trans retinoic acid (AtRA). Strikingly, single cell RNA-seq showed that intra-epithelial CD103+CD11b+ DCs constitute a homogenous cell population with a distinct transcriptomic signature from their LP counterpart. They are enriched with RNA related to antigen presentation, autophagy and lysosome pathways. Our results further suggest that these cells have a different function from LP CD103+CD11b+ DCs, as they do not significantly impact proliferation or differentiation of T helper lymphocytes but control the CD8+αβ intraepithelial lymphocytes (IELs) pool. These findings highlight the importance of the epithelial tissue as a first line of defense against pathogens in the upper parts of the small intestine. They also raise new questions about the regulation of the immune response in the epithelium and the mutual influences between lumen, epithelium and intestinal lamina propria.

Es wird vermutet, dass das Zusammenspiel von NF-κB-Signalen und Autophagie die Entzündung in verschiedenen zellulären Kontexten und als Reaktion auf unterschiedliche Stimuli reguliert. Der molekulare Mechanismus, durch den diese beiden Signalwege bei der Regulierung der Entzündungsreaktion zusammenwirken, ist jedoch noch nicht bekannt. Mithilfe biochemischer Analysen und bildgebender Verfahren haben wir zum ersten Mal die Interaktion zwischen dem autophagischen Marker LC3 und der NF-κB/p65-Untereinheit als Reaktion auf verschiedene Stressbedingungen charakterisiert. Wir konnten zeigen, dass die Anhäufung von LC3 im Zellkern nach der NF-κB-Aktivierung mit p65 interagiert, was durch die Ubiquitinierung des p65-Proteins gefördert und durch den Cargo-Rezeptor p62 erkannt wird. Zusammengenommen weisen diese Daten auf eine neue Rolle von p62 beim Transport von im Kern ubiquitiniertem p65 zu Autophagosomen hin, wo es abgebaut wird, um die entzündungsbedingte NF-κB-Hyperaktivierung zu kontrollieren. Diese Erkenntnisse sind wichtig für die Entwicklung neuer therapeutischer Strategien gegen Krankheiten, die mit einer gestörten Autophagie und einer konstitutiven NF-κB-Aktivität einhergehen. Die NF-κB-Signalübertragung spielt nicht nur eine entscheidende Rolle bei Entzündungen und der Tumorbildung, sondern ist auch für Entwicklungsprozesse wichtig. Durch die Etablierung von 3D-Organoid-Kulturen aus dem Dünndarm und unter Verwendung verschiedener Mauslinien weisen wir im zweiten Teil der Arbeit nach, dass NF-κB eine wichtige Funktion bei der Zelldifferenzierung und der Erhaltung von Stammzellen in vivo und ex-vivo spielt. Wir konnten zeigen, dass die Proliferation und das Absterben von Darmepithelzellen (IEC) bei Mäusen mit ubiquitärer Unterdrückung der NF-κB-Aktivität unverändert sind, während die Zahl der Becherzellen auf Kosten der Paneth-Zellen zunimmt. Zusammenfassend lässt sich sagen, dass unsere Ergebnisse eine neue IEC-immanente Rolle von NF-κB bei Entscheidungen über das Zellschicksal und die Differenzierung aufzeigen, die über die Regulierung der Wnt-Signale und der Sox9-Expression stromabwärts von NF-κB erfolgt. Die hier beschriebenen Erkenntnisse verbessern unser Verständnis der NF-κB-Funktionen in der Stammzellbiologie, die, wenn sie dereguliert sind, auch Auswirkungen auf die Entzündung des Darms und die Tumorentstehung haben. / The interplay between NF-κB signaling and autophagy has been suggested to regulate inflammation in different cellular contexts and in response to different stimuli. However, the molecular mechanism by which these two pathways interact to regulate the inflammatory response remains elusive. By using biochemical analysis and imaging techniques, we characterized for the first time the interaction of autophagic marker LC3 and NF-κB/p65 subunit in response to different stress conditions. We demonstrated that the accumulation of LC3 within the nucleus interacts with p65 following NF-κB activation, which is promoted by ubiquitination of p65 protein and recognized by the cargo receptor p62. Together, these data identify a novel role for p62 in trafficking nuclear-ubiquitinated p65 to autophagosomes for degradation to control inflammation-driven NF-κB hyperactivation. These findings are important for developing novel therapeutic strategies against diseases involving defective autophagy and constitutive NF-κB activity. In addition to its critical role in inflammation and tumor formation, NF-κB signaling is essential in developmental processes. Establishing 3D organoid culture from the small intestine and using different mouse lines, we prove in the second part of the thesis that NF-kB plays an important function in cell differentiation and stem cell maintenance in vivo and in ex-vivo. We demonstrated that while intestinal epithelial cell (IEC) proliferation and death are unaltered in mice with ubiquitous suppression of NF-κB activity, goblet cell numbers increase at the expense of Paneth cells. In summary, our results revealed a novel IEC-intrinsic role of NF-κB in cell fate decisions and differentiation which occur via regulation of Wnt signaling and Sox9 expression downstream of NF-κB. The findings described here improve our understanding of NF-κB functions in stem cell biology which, when deregulated, also have an impact on intestinal inflammation and tumorigenesis.

Organoids retain the morphological and molecular patterns of their tissue of origin, are self-organizing, relatively simple to handle and accessible to genetic engineering. Thus, they represent an optimal tool for studying the mechanisms of tissue maintenance and aging. Long-term expansion under standard growth conditions, however, is accompanied by changes in the growth pattern and kinetics. As a potential explanation of these alterations, epigenetic drifts in organoid culture have been suggested. Here, we studied histone tri-methylation at lysine 4 (H3K4me3) and 27 (H3K27me3) and transcriptome profiles of intestinal organoids derived from mismatch repair (MMR)-deficient and control mice and cultured for 3 and 20 weeks and compared them with data on their tissue of origin. We found that, besides the expected changes in short-term culture, the organoids showed profound changes in their epigenomes also during the long-term culture. The most prominent were epigenetic gene activation by H3K4me3 recruitment to previously unmodified genes and by H3K27me3 loss from originally bivalent genes. We showed that a long-term culture is linked to broad transcriptional changes that indicate an ongoing maturation and metabolic adaptation process. This process was disturbed in MMR-deficient mice, resulting in endoplasmic reticulum (ER) stress and Wnt activation. Our results can be explained in terms of a mathematical model assuming that epigenetic changes during a long-term culture involve DNA demethylation that ceases if the metabolic adaptation is disturbed.

Background: The use of stapling equipment for intestinal surgery in cats is rarely reported, and appropriate staple sizes for cat intestine are unknown. Objective: To determine staple cartridge sizes for thoracoabdominal (TA) and endoscopic gastrointestinal anastomosis (EndoGIA) that will simultaneously prevent leakage of small intestinal contents while also allowing for sufficient vascular permeability past the staple lines for intestinal healing. Methods: Two sizes of EndoGIA cartridges (2.0/2.5/3.0mm and 3.0/3.5/4.0mm) and two sizes of TA cartridges (2.5mm and 3.5mm), applied in a transverse manner across fresh cadaveric cat jejunum, were evaluated via intestinal burst pressure testing for maximum intraluminal pressure prior to leaking, and via infusion of an intravascular dye at normal arterial pressures to determine percentage of vascular patency past the staple lines. Vascular patency was compared not only from pre-and post-staple segments of the same intestinal sample, but also EndoGIA vascular patency was evaluated against TA vascular patency. Results: Two cats met study criteria. All samples had intraluminal burst pressures over twice the chosen minimum (of 30mmHg). Vascular patency post- staple line ranged from 0-90.8%, with the most consistently high numbers noted with the TA 3.5mm cartridges. No EndoGIA cartridge had a post- staple line vascular patency higher than 31.1%, and no intravascular dye was noted in any post- staple line sample in the EndoGIA 2.0/2.5/3.0mm group. Conclusions: While statistical analysis of the dataset was unable to be performed due to low numbers of samples for comparison, both intestinal intraluminal burst pressure trends and intravascular dye patterns suggested both the TA 3.5mm cartridge and (to a lesser extent) the 3.0/3.5/4.0mm EndoGIA cartridge could provide the ideal combination of intraluminal seal without restriction of vascular access for healing. The intravascular dye infusion technique, developed during this research, shows promise as a future instrument to determine vascular patterns around intestinal implants in cadaveric cat specimens. / Master of Science / Despite the regularity of feline small intestinal surgery, few reports exist of stapled anastomoses in cats, in part due to stapler size limitations. However, the recently developed endoscopic gastrointestinal anastomosis (EndoGIA) stapler shows promise as a future surgical tool for cats because it fits into cat intestine. In dogs, 3.5mm staples are often chosen for intestinal surgery; however, dog intestine is considerably thicker than cat intestine. The study goal was to evaluate not only intestinal burst pressures (the pressure at which repaired intestine leaks), but also the ability of fluids to flow through blood vessels that cross the staple lines of four stapler cartridge types from two staple lineages (EndoGIA 2.0/2.5/3.0mm, EndoGIA 3.0/3.5/4.0mm, TA 2.5mm, and TA 3.5mm). The central hypothesis was twofold. First, smaller stapler cartridge sizes (the EndoGIA 2.0/2.5/3.0mm and TA 2.5mm) would have higher intraluminal burst pressures when compared to the larger sizes (the EndoGIA 3.0/3.5/4.0mm). Second, larger stapler cartridges (the EndoGIA 3.0/3.5/4.0mm and the TA 3.5mm) would allow for increased flow of fluids in blood vessels past the stapler lines compared to the smaller cartridges (the EndoGIA 2.0/2.5/3.0mm and the TA 2.5mm). Two cats were included in the study. Trends in the data suggested that all components of the hypothesis might be proven with further data. However, due to the low number of cats acquired during the study period, the hypotheses could not be verified with statistics. The dye infusion technique to evaluate flow of fluids in blood vessels, developed during this research, shows promise as a future instrument to determine vascular patterns around intestinal implants. Future research should focus on acquiring more cats to have the ability able to perform statistical analyses (and prove the hypothesis), before proceeding with additional related studies.