京都大学 / 新制・課程博士 / 博士(医学) / 甲第24796号 / 医博第4988号 / 新制||医||1066(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 上野 英樹, 教授 椛島 健治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Human pluripotent stem cells (hPSCs) have the ability to self-renew and differentiate into all specialized body cells, providing material suitable for studying basic biology, modeling disease, and for regenerative medicine. The differentiation of hPSCs into functional cell types has been further enhanced by the production of organoids, miniature 3D organ-like structures that mimic the architecture and function of their in vivo counterparts, representing more physiologically relevant models of native tissues than monolayer cultures. Our initial aim was to differentiate hPSCs into lung epithelial organoids in vitro, and we hypothesized that applying knowledge of signaling cues during embryonic development to the dish would produce lineage-specific tissue. Using a multi-stage differentiation strategy, we derived organoids sharing properties with the developing lung as well as intestine. From this work, we learned the importance of purification, selection, and using singularized precursor cells to produce populations of bona fide lineage-restricted organoids. Upon developing a type of intestinal organoid technology from hPSCs not reported before, we shifted our focus to the intestine. We generated cystic intestinal epithelial organoids called enterospheres (hEnS) in vitro from hPSCs, which mimic structural and cell type properties of the native small intestinal epithelium. hEnS growth, differentiation, and long-term culture can be controlled by modulating media conditions. Importantly, hEnS are functional in that they elicit an innate immune response upon treatment with enteric pathogens. We established hEnS as an attractive in vitro model system for studying human gastrointestinal biology. We then developed an automated hEnS imaging assay to measure responses to growth factors, bacterial products, and enteric bacteria themselves. In doing so, we demonstrated the utility of hEnS as a germ-free system for studying host-microbe interactions and intestinal maturation. Finally, we investigated the expression of protein markers of intestinal maturation in tissue sections of primary human intestine spanning gestation, and made observations that are different from those reported in mice. Overall, our work provides new and important insights into hPSC differentiation, organoid technologies, and intestinal development in humans. / Thesis / Doctor of Philosophy (PhD)

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-01-21T13:29:23Z No. of bitstreams: 1 fernandomendoncavidigal.pdf: 1080288 bytes, checksum: 1512d97ed552f3d14c85e421c3f6c0b9 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-01-25T18:45:58Z (GMT) No. of bitstreams: 1 fernandomendoncavidigal.pdf: 1080288 bytes, checksum: 1512d97ed552f3d14c85e421c3f6c0b9 (MD5) / Made available in DSpace on 2016-01-25T18:45:58Z (GMT). No. of bitstreams: 1 fernandomendoncavidigal.pdf: 1080288 bytes, checksum: 1512d97ed552f3d14c85e421c3f6c0b9 (MD5) Previous issue date: 2014-12-12 / Introdução: Pacientes com Doença de Crohn (DC) suboclusiva que receberam tratamento com azatioprina (AZA) tiveram menores taxas de re-hospitalização devido a todas as causas e para tratamento operatório da DC quando comparados àqueles tratados com mesalazina durante um período de três anos. Nós investigamos se a AZA também foi efetiva para a prevenção da obstrução intestinal recorrente. Material e Métodos: Taxas de obstrução intestinal recorrente foram comparadas entre pacientes tratados com AZA e aqueles tratados com mesalazina. Nós avaliamos o intervalo de tempo livre de obstrução intestinal assim como a sobrevida livre de oclusão para ambos os grupos. Resultados: Houve uma taxa cumulativa significativamente mais baixa de pacientes com suboclusão recorrente no grupo da AZA (43,8%) comparado ao grupo da mesalazina (79,4%; OR 3,34, 95% IC 1,67-8,6; p = 0,003) com o número necessário para prevenir um episódio de suboclusão de 3,7 a favor da AZA. O intervalo de tempo livre de oclusão foi maior no grupo da AZA comparado ao grupo da mesalazina (28,8 vs. 18,3 meses, p = 0,000). A sobrevida livre de oclusão aos 12, 24 e 36 meses foi significativamente maior no grupo da AZA (91%, 81%, e 72%, respectivamente) do que no grupo da mesalazina (64,7%, 35,3%, e 23,5%, respectivamente; p < 0.05 para todas as comparações). Conclusão: Em uma análise exploratória de pacientes com DC ileocecal suboclusiva, a terapia de manutenção com AZA é mais efetiva que a mesalazina para evitar ou postergar a obstrução intestinal recorrente durante um período de três anos de tratamento. / Background: Patients with subocclusive Crohn’s disease (CD) who received azathioprine (AZA) therapy had lower re-hospitalization rates due to all causes and for surgical management of CD compared to those treated with mesalazine during a 3-year period. We investigated whether AZA also was effective for prevention of recurrent bowel obstruction. Material and Methods: Rates of recurrent bowel occlusion were compared between patients treated with AZA and those treated with mesalazine. We assessed the time interval-off intestinal obstruction as well as the occlusion-free survival for both groups. Results: There was a significantly lower cumulative rate of patients with recurrent subocclusion in the AZA group (43.8%) compared with the mesalazine group (79.4%; OR 3.34, 95% CI 1.67-8.6; P= 0.003) with a number needed to treat in order to prevent one subocclusion episode of 3.7 favoring AZA. The occlusion-free time interval was longer in AZA compared with the mesalazine group (28.8 vs. 18.3 months; P=0.000).The occlusion-free survival at 12, 24, and 36 months was significantly higher in the AZA group (91%, 81%, and 72%, respectively) than in the mesalazine arm (64.7%, 35.3%, and 23.5%, respectively; P<0.05 for all comparisons). Conclusions: In an exploratory analysis of patients with subocclusive ileocecal CD maintenance therapy with AZA is more effective than mesalazine for eliminating or postponing recurrent intestinal obstruction through 3 years of therapy.

<p>Intestinal microbiota are key determinants of gut homeostasis and affect various gut physiological and immune processes. Co-evolution has enabled the host and intestinal microbes to exist in a mutualistic relationship. However, interactions between the host and its intestinal microbiota exist in a delicate balance between mutualism and pathogenicity. Maintenance or disruption of this balance depends on a complex interplay between the microbiota and the host, as well as other gut luminal factors, including diet, that are poorly understood. The main goal of this thesis has been to study the host-gut luminal interactions that regulate gut physiology and immunity. In particular, <strong>Chapter 2</strong> centers on investigating the effect of perturbing the intestinal barrier using a non-steroidal inflammatory drug on host-microbial and dietary interactions in a mouse model of gluten sensitivity. I demonstrated that indomethacin-induced increase in intestinal permeability is associated with altered intestinal microbiota composition, systemic antibody development against intestinal bacteria and a shift in immune responses to the dietary antigen, gluten. <strong>Chapter 3</strong> focuses on investigating whether modulation of the intestinal microbiota can affect the host’s susceptibility to intestinal injury. I used mice with defective intracellular bacterial receptor signaling because discrimination between commensals and pathogens is, in part, achieved by a family of receptors that recognize conserved bacterial components. I demonstrated that the microbiota with which these mice are colonized influences the expression of RegIII-γ, a type of antimicrobial peptide, and susceptibility to intestinal injury. To gain further insight on the effect of microbiota on antimicrobial peptides, in <strong>Chapter 4</strong> we conducted a combination of gnotobiotic and <em>in-vitro</em> experiments where we identified that specific components of the microbiota differentially regulate RegIII expression. Further examination showed that <em>MyD88 a</em>nd <em>Ticam1 </em>genes, which are signaling adaptor proteins of pattern recognition receptors, are essential regulators of microbial–induced RegIII expression by intestinal epithelial cells. Collectively, the work presented in this thesis provides novel insight on the bi-directional interaction between the host and the gut luminal content as well as of potential beneficial effects of microbiota-modulating strategies in maintaining homeostasis and preventing disease.</p> / Doctor of Philosophy (Medical Science)

Les BMPs (Boue Morphogenetic Proteins) sont des morphogènes membres de la superfamille du TGF-[bêta] qui interagissent avec des récepteurs de surface cellulaire à activité sérine/thréonine kinase. L'interaction des BMPs avec ces récepteurs entraîne l'activation d'une cascade de signalisation cellulaire impliquant les facteurs de types R-Smads (Smad1, 5 et 8). La voie de signalisation des BMPs joue des rôles cruciaux dans des processus biologiques tels que l'embryogenèse et l'organogenèse des tissus ainsi que des processus cellulaires tels que la prolifération, la migration et la différenciation cellulaire. De plus, ces derniers semblent aussi impliqués dans les processus de mort cellulaire et dans la tumorigenèse. Malgré un intérêt grandissant pour la signalisation des BMPs, il existe très peu d'études sur les rôles spécifiques joués individuellement par les différents Smads dans la morphogenèse de l'intestin in vivo . Ceci est principalement dû au fait que la délétion classique des différents effecteurs de la voie des BMPs entraîne la mort in utero à cause de multiples défauts dans l'embryogenèse. Le système Cre/loxP, sous le contrôle d'un promoteur tissu spécifique, a été utilisé dans notre laboratoire, dans le but de générer une lignée murine possédant une délétion conditionnelle de l'effecteur Smad5 strictement au niveau de l'épithélium intestinal. Une analyse comparative à l'aide d'un modèle de délétion conditionnelle du récepteur BmpR1a au niveau de l'épithélium intestinal a été effectuée afin de décortiquer spécifiquement le rôle de l'effecteur Smad5 dans le développement de cet organe. Afin de valider les résultats obtenus in vivo nous avons généré un modèle cellulaire nous permettant de mimer l'effet de la délétion de l'effecteur Smad5 à l'aide de la technologie du shRNA.Les résultats obtenus dans les deux modèles suggèrent que Smad5 serait un facteur clé impliqué dans la régulation de la migration cellulaire des entérocytes. En effet, l'invalidation de la voie des BMPs et plus particulièrement de l'effecteur Smad5 dans les souris entraîne une augmentation de la vitesse de migration des cellules le long de l'axe crypte villosité. Ces résultats sont corroborés dans un modèle cellulaire dans lequel l'expression de Smad5 a été inhibée par interférence d'ARN. Dans ce modèle, la migration se fait de façon beaucoup plus compacte en comparaison aux cellules contrôles. L'augmentation de la vitesse de migration cellulaire pourrait être due à un phénomène de relocalisation des protéines de jonctions adhérentes ainsi qu'à une modulation de l'actine filamenteuse. Ce phénomène pourrait faire intervenir les petites protéines G Rho/Rac ainsi que les kinases Src. En plus de l'actine filamenteuse, différentes protéines impliquées dans la formation des complexes de jonctions adhérentes semblent relocalisées dans nos deux modèles (E-cadhérine/[bêta]-caténine). En conclusion, les différents résultats recueillis au cours de mes travaux de maîtrise dans le laboratoire du Dr. Nathalie Perreault nous suggèrent que l'effecteur Smad5 de la voie des BMPs serait un facteur impliqué dans la stabilité des complexes de jonctions adhérentes, régulant ainsi la migration des cellules le long de l'axe crypte villosité.

PTEN est une protéine dotée d'une activité phosphatase qui déphosphoryle les phosphatidylinositols issus de l'activation de la phosphatidylinositol 3-kinase (PI3K). Des mutations germinales du gène PTEN ont été mises en évidence dans le syndrome de Cowden, une maladie caractérisée par le développement de polypes le long du tube digestif et associée à un risque accru de cancer. De plus, la perte d'un allèle de Pten chez la souris conduit à la formation d'hyperplasie et de dysplasie du tractus gastro-intestinal ainsi qu'à des tumeurs notamment au niveau du côlon. Ces observations suggèrent que PTEN joue un rôle important dans le tube digestif. Cependant, ses mécanismes d'action dans les cellules épithéliales intestinales sont peu connus. Nos travaux nous ont permis de mieux caractériser les rôles de PTEN dans ces cellules. Pour ce faire, nous avons d'abord analysé l'effet d'un shRNA inhibant spécifiquement l'expression de PTEN dans les cellules Caco-2/15, une lignée cancéreuse colorectale qui a la particularité de se différencier suite à l'atteinte de la confluence en adoptant un phénotype semblable aux cellules absorbantes de l'intestin grêle. La perte d'expression de PTEN stimule la prolifération de ces cellules. Cette augmentation de la prolifération semble résulter d'une diminution de l'expression de p21 et de p27 ainsi que d'une hausse des cyclines D2 et E. De plus, le shRNA contre PTEN inhibe la différenciation fonctionnelle et morphologique des Caco-2/15. Cela découle partiellement de l'inhibition de l'expression des facteurs de transcription CDX2, HNF-1? et HNF-4?. Les jonctions serrées sont également altérées dans ces cellules. En effet, une réduction importante de l'expression des claudines et une augmentation de la perméabilité transépithéliale a été observée. Une augmentation de la synthèse protéique a aussi été remarquée. De plus, nos résultats laissent également croire que PTEN pourraient jouer un rôle dans la carcinogenèse colorectale. Nous avons effectivement constaté une augmentation du potentiel tumorigénique des Caco-2/l5 exprimant le shRNA contre PTEN suite à l'injection des cellules dans des souris nues. De plus, ces cellules ont des capacités de migration et d'invasion accrues. Nous avons également constaté que les niveaux de PTEN sont plus faibles dans plusieurs lignées cancéreuses colorectales comparativement aux cellules épithéliales intestinales normales. Nous avons aussi analysé le phénotype d'une lignée de souris possédant une délétion du gène Pten exclusivement au niveau de l'épithélium intestinal, générée à l'aide du système Cre/loxP. Macroscopiquement, une organomégalie de l'intestin grêle et du côlon a été observé chez les souris déficientes pour Pten. Histologiquement, nous avons constaté une désorganisation de l'architecture épithéliale intestinale caractérisée par un allongement des villosités et par la présence d'embranchements villositaires. De plus, un épaississement important des couches musculaires a été remarqué. Il y a également une augmentation du nombre de cellules prolifératives au niveau des cryptes intestinales corrélant avec une augmentation des niveaux de ?-caténine et des cyclines D. Finalement, une augmentation du contenu protéique par cellule a également été observée ainsi qu'une activation de la voie mTOR. En conclusion, nos résultats montrent que la phosphatase PTEN est impliquée dans l'établissement de l'architecture générale de l'intestin et qu'elle contrôle la synthèse protéique, la migration, le cycle cellulaire ainsi que la différenciation des cellules épithéliales intestinales. De plus, nos résultats indiquent que la perte d'expression de PTEN pourrait influencer la progression des cancers colorectaux. [Symboles non conformes]

Thesis (MScMedSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Introduction: Chronic HIV-1 infection is characterized by immune activation and dysregulation of immune homeostasis, which impacts on multiple immune cell types. The B-cell compartment, which plays an important role in the producing neutralizing antibodies, is also dysregulated in HIV- 1 infection. In this study we investigated peripheral blood B-cell subset distribution, and changes in expression of cellular activation, inhibition, and apoptosis signaling markers in both untreated chronic HIV-1 infected individuals and healthy uninfected controls. The neuropeptide immune modulator, vasoactive intestinal peptide (VIP) is known to selectively down-regulate activation of CD4+ T-cells in various disease settings including HIV-1, however to our knowledge, no studies have investigated the effect of VIP inhibition on B-cell activation. Materials & Methods: A total of 21 HIV+ve (CD4 count >250 cells/µl), and 19 HIV-ve individuals were recruited from the Emavundleni voluntary testing and counseling clinic in Crossroads, Western Province, South Africa. Whole blood was stained to distinguish B-cell subsets (activated memory (AM: CD21-CD27+), resting memory (RM: CD21+CD27+), mature naïve (MN: CD21+CD27-), or tissue-like memory (TLM: CD21loCD27lo). In addition expression of markers of B-cell activation (CD126, CD86, CD38, CD284, CD287), inhibition (CD72, CD85j, CD300a, CD305, CD307d), and apoptosis signaling (CD95), was assessed ex vivo by flow cytometry (BD FACSCanto II). For determination of functional responsiveness isolated B-cells (RosetteSep, Stemcell Technologies) were cultured for 18h (37°C, 5%CO2) without stimulation or stimulated with TLR ligands (LPS or R848). Stimulation experiments were also performed in the presence or absence of VIP. Results: Chronic HIV-1 infection affected B-cell subset distribution. The percentage (%) of TLM was increased by 59.24%, and %RM was decreased by 22.73% (both p<0.01). Total expression of the VIP receptor VPAC2 was decreased by 47.35% (p=0.0296). Subsets had a mixed phenotype ex vivo; HIV infection upregulated CD38 (by 59.56%, p=0.0004), CD72 (by 60.70%, p=0.0396), CD307d (by 68.63%, p=0.0015) on AM, while RM B-cells had increased expression of TLR4 (by 107.04%, p=0.0057) and TLR7 (by 208.14%, p=0.0199). TLM B cells (i.e. exhausted phenotype) displayed upregulated TLR7 (by 550%, p=0.0128) and CD307d (by 72.40% p=0.045) expression. MN B-cells had increased CD72 expression (by 70.98%, p=0.0026). R848 upregulated CD86 expression by 42.20% on AM (p<0.01), and by 56.06% on RM B-cells (p<0.01), which was significantly downregulated with VIP inhibition (both p<0.05). Similarly, CD95 expression on RM, TLM, and MN B-cells increased by 31.10% (p<0.001), 21.46% (p<0.01), and 39.92% (p<0.01) with R848 stimulation respectively, which was also significantly downregulated with VIP inhibition. Conclusion: These data indicate that B-cells in untreated HIV infection display increased levels of activation, and also the potential for increased susceptibility to apoptosis as evidenced by increased FAS (CD95) expression. VIP significantly down-regulated markers of activation, inhibition, and apoptosis signaling. Dysregulation of B-cells is thus apparent in asymptomatic stable chronic HIV-1 infection, which may impact on both inefficient neutralizing antibody production and hypergammaglobulinemia. The ability of VIP to prevent stimulationassociated marker upregulation may indicate that VIP is a potential therapeutic agent. Its immuno-modulatory properties were demonstrated to limit B-cell hyperactivation, and selectively down-regulate apoptosis and mark it out for further investigation. / AFRIKAANSE OPSOMMING: Inleiding: Immunaktivering en ongekoppelde immuun-homeostase is kenmerke van chroniese MIVinfeksie. Ons het perifere bloed B-sel subgroep-verspreiding, en veranderinge in die uitdrukking van merkers van aktivering, inhibisie, en apoptose in 'n onbehandelde MIV-1 besmettende groep ondersoek (in vergelyk met 'n gesonde onbesmettende kontrole). Die immuun-moduleerder, vasoaktiewe intestinale peptied (VIP) is bekend om aktivasie van geaktiveerde CD4+ T-selle te verminder, maar tot ons kennis, is daar geen studies wat die effek van VIP-inhibisie op B-sel aktivering ondersoek het, in die konteks van MIV-1 infeksie. Materiaal & Metodes: MIV+we individue (CD4-telling >250 selle/µl) , en MIVwe kontroles is gewerf uit die vrywillige toetsing en berading Emavundleni kliniek, Crossroads, Westelike Provinsie, Suid-Afrika. Bsel subgroepe is gedefinieer as geaktiveerde geheue (AM: CD21- CD27+ ), rusende geheue (RM: CD21+ CD27+ ), volwasse naïef (MN: CD21+ CD27- ), of weefsel-agtige geheue (TLM: CD21loCD27lo). Merkers van aktivering (CD126, CD86, CD38, CD284, CD287), inhibisie (CD72, CD85j, CD300a, CD305, CD307d), en apoptose signalering (CD95) is via vloeisitometrie (BD FACSCanto II) op B-selle ex vivo en ook op geïsoleerde B-selle (RosetteSep, Cell Technologies) ondersoek. Vir die bepaling van funksionele responsiwiteit, geïsoleerde B-selle (RosetteSep, StemCell Technologies) was vir 18h (37°C, 5%CO2) gekweek, sonder stimulasie of gestimuleer met TLR ligande (LPS of R848). Stimulasie eksperimente het ook in die teenwoordigheid of afwesigheid van VIP plaasgevind. Resultate: Chroniese MIV-1 infeksie het B-sel subset verspreiding geraak. Die persentasie (%) van TLM is verhoog deur 59,24%, en% RM het met 22.73% afgeneem (beide p <0,01). Totale uitdrukking van die VIP reseptor VPAC2 het met 47,35% afgeneem (p = 0,0296). Subgroepe het 'n gemengde ex vivo fenotipe; MIV-infeksie het CD38 (deur 59,56%, p=0,0004), CD72 (deur 60,70%, p=0,0396), CD307d (deur 68,63%, p=0,0015) op AM verhoog, terwyl RM Bselle het verhoogde uitdrukking van TLR4 (deur 107,04%, p=0,0057) en TLR7 (deur 208,14%, p=0,0199). TLM B-selle (die uitgeputtende fenotiep) het verhoogde TLR7 (deur 550%, p=0,0128) en CD307d (deur 72,40% p=0.045) uitdrukking gewys. MN B-selle het verhoogde uitdrukking van CD72 (deur 70,98%, p = 0,0026). R848 het CD86 uitdrukking op AM deur 42,20%, en op RM deur 56,06% toegeneem (beide p <0,01). Dit het met VIP inhibisie beduidend afgeneem (beide p <0.05). CD95 uitdrukking was soortgelyk verhoog op RM, TLM, en MN B-selle met 31.10% (p <0.001), 21,46% (p <0,01), en 39,92% (p <0,01) met R848 stimulasie. Al drie het beduidend afgeneem met VIP inhibisie. Gevolgtrekking: Hierdie data dui daarop dat B-selle in onbehandelde MIV-infeksie vertoon verhoogde aktiveringsvlakke, en ook die potensiaal vir verhoogde vatbaarheid vir apoptose soos bewys deur verhoogde uitdrukking van FAS (CD95). VIP het beduidend merkers van aktivering, inhibisie, en apoptose af-gereguleer. Wanfunksie van B-selle is dus in asimptomatiese stabiele chroniese MIV-1 infeksie duidelik, wat impak kan hê op beide oneffektiewe neutraliserende teenliggaampie produksie, en hiepergammaglobulinimie. Die vermoë van VIP stimulasie-verwante merker opregulasie te voorkom kan aandui dat VIP 'n potensiële terapeutiese agent is. VIP se immuno-moduleerende eiendomme is gedemonstreer om Bsel hieperaktiveering te beperk, en selektief apoptose afreguleer, en merk dit vir verdere ondersoek.

Long term consumption of a high fat diet (HFD) increases the risk of developing Metabolic Syndrome and type 2 diabetes. This led us to hypothesize that long term HFD consumption impairs immune tolerance to the intestinal bacteria. Our studies had two goals. First, we characterized the effect of long term HFD consumption on the systemic immune response by comparing C57BL6 mice fed a HFD and low fat diet (LFD). Plasma immunoglobulin G (IgG) against Escherichia coli (LF-82), E. coli (Nissle 1917), Bacteroides thetaiotaomicron and Lactobacillus acidophilus were measured by a lab-developed ELISA. Fasting blood glucose and inflammation were measured in LFD mice and HFD mice. To test whether our findings were clinically relevant, anti-bacterial IgG and TNF-α were measured in plasma samples from lean healthy individuals, obese non-diabetics and obese diabetics. Our second aim was to investigate the relationship between HFD consumption and intestinal immunity. The effect of HFD consumption on immune responses in the GI tract was assessed by measuring fecal IgA levels in HFD mice and LFD mice. HFD mice had higher plasma IgG against the LF82 strain of Escherichia coli as well as higher plasma TNF-α, neutrophil percentage and fasting blood glucose levels. Obese diabetics had higher plasma IgG against the LF82 strain of E. coli than lean healthy controls. Studies on the effect of HFD on intestinal immunity revealed that HFD mice had lower fecal IgA than LFD mice. Our findings are novel in that they show an association between long term HFD consumption, systemic inflammatory immune responses to pathogenic intestinal bacteria and insulin resistance. These studies also showed that HFD consumption may impair intestinal immunity.

Memoria para optar al Titulo Profesional de Médico Veterinario / La microbiota intestinal corresponde a las poblaciones bacterianas que habitan en el sistema gastrointestinal de los animales y humanos, esta participa directamente en la estimulación del sistema inmune, en la síntesis de vitaminas y en la inhibición de poblaciones bacterianas nocivas para el huésped. En la producción avícola destaca el uso de variados antimicrobianos para el tratamiento de diversas enfermedades infecciosas, el efecto de estos antimicrobianos sobre la microbiota intestinal en términos de resistencia y variaciones poblacionales ha sido escasamente estudiado en aves de postura. Por esto es que el objetivo del presente trabajo fue estudiar la variación de poblaciones intestinales de Escherichia coli, Enterococcus spp. y Lactobacillus spp. en aves de postura expuestas a un tratamiento con enrofloxacino y además medir la variación en los niveles de resistencia bacteriana en las poblaciones de E. coli y Enterococcus spp. en su rol de bacterias indicadoras de resistencia. Se utilizaron 30 gallinas ponedoras raza leghorn las cuales fueron separadas en un grupo tratado (n=15) y control (n=15), el grupo tratado recibió enrofloxacino 5% por vía oral y el grupo control un placebo (agua destilada) durante 10 días. A los 3, 7 y 15 días posteriores al tratamiento se obtuvieron muestras de contenido intestinal a partir de íleon y ciego de los animales, las cuales se sembraron y cultivaron en los diferentes medios de cultivo para el posterior conteo de colonias intestinales. A partir de estas muestras se aislaron 99 cepas de E. coli y 90 cepas de Enterococcus spp. para el análisis de la variación en los niveles de resistencia frente a amoxicilina, cefotaxima, enrofloxacino, ácido nalidíxico, estreptomicina, oxitetraciclina y sulfametoxazol + trimetoprim para E. coli; y frente a amoxicilina, enrofloxacino, eritromicina y oxitetraciclina para Enterococcus spp. En el caso del conteo de colonias bacterianas solo se encontraron diferencias significativas posteriores al tratamiento en las colonias de Lactobacillus spp. En el caso de los niveles de resistencia posteriores al tratamiento con enrofloxacino para las cepas de E. coli, estos aumentaron respecto del grupo control frente a amoxicilina, enrofloxacino, ác nalidíxico, estreptomicina y sulfametoxazol + trimetoprim. En el caso de las cepas de Enterococcus spp. la resistencia se incrementó solo en el caso de enrofloxacino, mientras que para eritromicina y oxitetraciclina disminuyeron respecto al control. De acuerdo a estos resultados se puede concluir que la utilización de antimicrobianos afecta directamente a la microbiota intestinal tanto en términos de variación en las poblaciones bacterianas como también en relación a los niveles de resistencia antimicrobiana de estas mismas

<p>Transferrin receptor 1 (Tfr1) serves as a receptor for transferrin, an iron-binding protein in the blood, in its canonical role of iron assimilation. Tfr1 is expressed ubiquitously in many tissues and is believed to be required for iron uptake by most cells. </p><p>The Tfr1 global knockout mouse highlights the requirement for Tfr1 in erythrocyte precursors. The erythron is the tissue with the highest iron requirement, to enable hemoglobin production. Tfr1-null embryos die by embryonic day 12.5 with anemia, which has been assumed to cause lethality of the knockout mice. Due to the embryonic lethality of the mice, the role of Tfr1 has not been well characterized in other tissues in vivo. This thesis examines the role of Tfr1 in other tissues through the generation and characterization of conditional knockout mouse models of Tfr1 deletion in the intestinal epithelium, pancreas, and skin.</p><p>Tfr1 is expressed on the basolateral surface of proliferating cells in the intestinal epithelium. Deletion of Tfr1 specifically in the intestinal epithelium resulted in the loss of intestinal epithelial homeostasis, loss of proliferation, lipid accumulation, gene expression indicating epithelial to mesenchymal transition of intestinal epithelial cells, and early neonatal lethality. These phenotypes were mostly alleviated by forced expression of a mutant Tfr1 allele which is unable to bind to iron-loaded transferrin, suggesting that Tfr1 has a novel role independent of its canonical iron-assimilatory ability.</p><p>Deletion of Tfr1 in the pancreas resulted in juvenile death due to perturbed homeostasis of both endocrine and exocrine tissues, resulting in symptoms associated with pancreatitis and diabetes. No diabetic phenotype was detected in the conditional knockout mouse model of Tfr1 deletion specifically in &#946;-cells, suggesting that the primary effect of the loss of Tfr1 was limited to the exocrine tissue.</p><p>Deletion of Tfr1 in the epidermis of the skin caused neonatal lethality with abnormal hair follicle morphology and a significant reduction in dermal adipocytes.</p><p>These results indicate that the loss of Tfr1 has pleiotropic effects, depending on the cell type affected. Furthermore, Tfr1 appears to have non-canonical functions in the intestinal epithelium, a novel discovery.</p> / Dissertation