A doença do enxerto versus hospedeiro (DEVH) é uma grave complicação do transplante de órgãos sólidos, com alta mortalidade. Seu estudo tem sido limitado pela carência de modelos experimentais apropriados. Descreve-se um modelo de DEVH baseado no aumento do quimerismo, sua evolução clínica, histopatológica, do número das células quiméricas, do perfil das citocinas e da tolerância imunológica. Ratos Lewis (LEW) foram submetidos a transplante simultâneo de intestino delgado e medula óssea provenientes de ratos ACI (grupo de estudo - E) ou LEW (grupo controle - C), tratados com FK-506 (1 mg/Kg/dia) entre o 0 e 13o PO, e uma dose semanal daí por diante. Os ratos foram divididos nos seguintes grupos: E1- 6 ratos sacrificados no 120o PO. E2- 8 ratos após apresentarem sinais clínicos graves de DEVH entre o 189o e o 271o PO. Como controle, ratos LEW foram receptores dos mesmos tipos de enxertos provenientes de ratos LEW, submetidos à mesma imunossupressão e foram assim divididos: C1- 6 ratos sacrificados no 120o PO, C2- 5 ratos sacrificados entre o 223o e o 270o PO. A citometria de fluxo foi realizada para quantificar a porcentagem das células linfóides de ACI doadores no sangue periférico nos E1, E2 em 6 períodos: 30o PO, 65o PO, 95o PO, 120o PO, 160o PO, 200o PO. Os animais foram examinados 2 vezes por semana à procura de sinais de DEVH (rash cutâneo, perda de peso, de pelo e hiperqueratose). No sacrifício dos animais do grupo E1 e C1, foram colhidas amostras de língua (LI), de linfonodos cervicais (LC), intestino delgado do receptor e do enxerto para análise das citocinas IL-2, IL-4, IL-6, IL-10, IFN-gama e TNF-alfa por meio da reação em cadeia da polimerase. Em todos os grupos foram também colhidas amostras destes órgãos para histopatologia e nos animais do grupo E2 linfonodos cervicais foram processados para análise da reatividade celular por meio da reação mista dos linfócitos (MLR). A evolução clínica e histopatológica foi graduada de 0 a 3 de acordo com a severidade dos sintomas e do infiltrado mononuclear das amostras. Os ratos dos grupos E1 e E2 iniciaram sinais da DEVH entre o 84o e 115o PO. Os ratos dos grupos C1 e C2 não apresentaram evidência de DEVH. Amostras de LI e LC dos ratos do grupo E1 apresentaram alterações histopatológicas grau 2 e do grupo E2 apresentaram alterações histopatológicas grau 3, respectivamente. Nenhuma alteração histopatológica foi encontrada nos ratos do grupo controle e em amostras do ID. Nenhuma alteração histopatológica foi encontrada no intestino delgado do receptor e do enxerto. O aumento da porcentagem de células do doador no sangue periférico do receptor foi progressivo chegando a 5,4±2.3% no 10o período, 21±4,6% no 3o período e 39,3±4% no 6o período. IL-2, IL-6, IL-10, IFN-gma e TNF-alfa estiveram aumentados em língua e IL-4, IL-6, IL-10, IFN-gama e TNF-alfa em linfonodos cervicais. Os linfócitos de ratos do grupo E2 mostraram hiporreatividade aos de ratos ACI e hiperreatividade aos de ratos PVG (terceira parte) denotando tolerância imunológica. Neste modelo experimental há uma inexorável evolução imunológica para DEVH; existe correlação direta entre o aumento do quimerismo em sangue periférico e da expressão de citocinas em língua e linfonodos cervicais e a severidade da DEVH, além da indução de tolerância imunológica do rato do grupo E2 quimérico ao rato ACI normal. / Graft-versus-host disease (GVHD) has been a major concern after small bowel transplantation (SBTX) and the lack of suitable experimental models has limited the study of GVHD after solid organ transplantation. Here we describe a re1evant experimental model of GVHD after fully allogeneic SBTX based on chimerism augmentation, its clinical and histophatological evolution, cytokine involvement, responsible donor cell and immunologic tolerance analysis. LEW rat recipients received orthotopic SBTX and simultaneous donor bone marrow cell infusion (250x106), from ACI rats (experimental group - E) or LEW (control group C). FK-506 was administered dayly at a dose of 1 mg/kg on day 0 to 13, then continued as a weekly injection of same dose until the experimental end point. The recipients were divided in the following groups: E1 - 6 rats sacrificed at 120° POD. E2 - 8 rats sacrificed with critical GVHD between DPO 189 to 271. LEW recipient of LEW grafts, under the same immunossupression were used as control and divided as: C1 - 6 rats sacrificed at POD 120; C2- 5 rats sacrificed between 223 and 270 POD the number of donor cell in the recipient circulation was determined by flowcytometry in 6 pos-operative time: 30, 65, 95, 120, 160, 200. The rats were analyzed twice a week for body weigh and searching for signs of GVHD (cutaneous rush, hiperkeratosis and loss of hair and body weigh). At the sacrificed, samples from tongue (TG), cervical lymph node (CLN), donor (SBD) and recipient (SBR) small bowel were taken from all animals for histophatology and from E1 and C1l animals for IL-2, IL-4, IL-6, IL-10, IFN-gama e TNF-alfa cytokines analysis using reverse transcription polymerase chain reaction. Samples from cervical lynph nodes of 5 animals from group E2 were used for mixed lymphocyte reaction for tolerance analysis. The clinical and histophatological evolution of the disease were evaluated from degree 0 to 3 according to the severity. GVHD in E1 and E2 animals started between 84 and 115 POD. Histophatological analysis of TG and CLN showed that E1 animals present GVHD grade 2 and E2 animals grade 3. The increase of donors cells in the recipient circulation was progressive and account for 5.4± 2.3% at POD 30, 21.4±4.6% at POD 95 and 39.3±4% at POD 200. IL-4, IL-6, IL-10, IFN-gama e TNF-alfa were upregulated in CLN and IL-2, IL-6, IL-10, IFN-gama e TNF-alfa were upregulated in TG when compared with the respective controls. The lymphocytes from E2 group showed hyporeactivety to lymphocytes of normal ACI and hypereactivety to those of PVG, meaning tolerance. No cytokines alteration was noted in SBD neither SBR. Animals from group C1 and C2 did not present any sign of disease. This result show that GVHD is a inexoravel evolution under the experimental conditions of this study and the evolution of the disease is near correlated with the augmentation of the donor cells in the recipient circulation and upregulation of cytokines gene expression in target organs. Tolerance to the same donor strain lynphocytes was also noted.

The series of experiments described in this thesis were designed to investigate the role of suckling or late weaning in the response of young lambs to nematode infection. All experiments were conducted outdoors with grazing animals and no supplementation but for suckled groups of lambs whose counterparts were weaned to ryegrass – white clover swards. The parasite of interest was mainly Teladorsagia circumcincta solely but with mixed infection of Trichostrongylus colubriformis in one instance. In Chapter 3 (first experiment), the hypothesis that milk per se may have a direct effect on nematode development, rather than an indirect effect through enhancement of host immunity by superior nutrient supply was tested. Sixty, twinborn lambs were used, allocated to one of eight groups formed by either dosing lambs from 42 days of age or not with the equivalent of 1000 or 250 L₃ T. circumcincta larvae d⁻¹ until five days before necropsy, while a twin was either weaned at 39 days of age, suckled as single or twin until necropsy on day 84. The possibility that weaning one of a twin set onto pasture in close proximity to the ewe would cause abnormal ewe and lamb behaviour was tested by replicating the work in twins maintained as twins but in which one twin received equivalent of 250 and the other 1000 L₃ T. circumcincta larvae d⁻¹. This showed no abnormal ewe nursing or lamb suckling behaviour as a result of weaning a twin in a set. Relatively low faecal egg counts (FEC) and a two to three fold lower worm burdens suggest suckling could reduce larval establishment. Inability to detect peripheral titres of immunoglobulins supports this conclusion. An intra worm-population regulation of T. circumcincta, indicated by a pattern of greater egg-laying by a numerically smaller but physiologically better developed nematode population in suckled lambs measured in eggs 'in utero' and worm length made interpretation of FEC difficult. Suckling significantly improved weight gain and carcass weights, but early weaning did not reduce resilience to infection. In Chapter 4 (second experiment), 40 pairs of twin lambs, average age of 39 days, were either infected with the equivalent of 1000 L₃ T. circumcincta larvae d⁻¹ or not, while one twin was weaned and the other allowed to continue suckling. Necropsy was carried out on groups of five and six lambs from each of the uninfected and infected treatments, respectively, at mean age of 84, 112, and on six lambs from each group at 140 days of age. This serial slaughter allowed further confirmation of the hypothesis in Chapter 3 but also investigated the long-term effect of suckling on resistance or resilience of lambs at the trial when immune responses were anticipated to be developing. An in vitro direct larval challenge (IVDC) study, to monitor larval establishment, was carried out on tissue explants from necropsied lambs. Suckled lambs consistently showed lower FEC (P < 0.05) and worm burdens (P < 0.05) at every phase of the trial. Within the infected groups, % in vitro larval rejection suggested earlier immune responses in the weaned lambs by day 84, which was not consistent with lower worm burdens in suckled lambs but appeared similar in the subsequent necropsies. Lambs continued to show better growth due to suckling while weaning did not reduce the resilience of lambs confirming observations in Chapter 3. The immunoglobulin profile suggested the commencement of immune responses in lambs from the period after the 84th day necropsy, with significantly greater (P < 0.01) IgA titre in the infected groups, and the suckled lambs towards the end of the trial on day 140. A vaccinating effect of early exposure to parasites was coincidentally revealed as a result of unintentional pasture larval contamination, seen in suckled non-infected lambs shedding fewer eggs and harbouring fewer worms during the later necropsies compared with their weaned non-infected counterparts. In Chapter 5 (third trial), 93 pairs of twin lambs, 47 pairs of which received a vaccinating mixed infection of T. circumcincta and T. colubriformis larvae (60 L₃ / kg W / d) at ratio 40:60, respectively during the period 36 – 103 days of age, were either weaned early on day 51 or later on day 108. All lambs were drenched on day 108 and groups received challenge infections from day 116, at same rate with the vaccinating infection, or not, which ceased five days before respective necropsies. Necropsies were carried out on selected lambs on days 108, 184 and 218. The direct effect of milk on larval establishment appeared to feature only in the T. circumcincta populations on slaughter day 108. The long-term benefit of late weaning for development of resistance was conditional on lambs receiving the vaccinating infection, and appeared to be more pronounced in the small intestine, reflected by a greater reduction of T. colubriformis populations in that organ than of T. circumcincta populations in the abomasum. A negative consequence of enhanced immune response was the suggestion of an increased metabolic cost in reduced performance of lambs. In conclusion, the work provides support to the hypotheses that: (a.) suckling may reduce the establishment of nematode larvae through the direct effect of milk, (b.) may enhance rapid development of host immunity to infection, and (c.) it further suggests that lack of larval experience during suckling may have long term negative implications for host resistance. Finally, it suggests that milk may play little role in the enhancement of host resilience to infection and, on the contrary, that additional metabolic cost may be associated with a more rapid development of immunity resulting from larval challenge while suckling.

En lien avec l’augmentation constante de l’obésité, de plus en plus de personnes sont atteintes de résistance à l’insuline ou de diabète de type 2. Ce projet doctoral s’est surtout intéressé à l’une des conséquences majeures des pathologies cardiométaboliques, soit la dyslipidémie diabétique. À cet égard, les gens présentant une résistance à l’insuline ou un diabète de type 2 sont plus à risque de développer des perturbations lipidiques caractérisées essentiellement par des taux élevés de triglycérides et de LDL-cholestérol ainsi que de concentrations restreintes en HDL-cholestérol dans la circulation. Les risques de maladies cardiovasculaires sont ainsi plus élevés chez ces patients. Classiquement, trois organes sont connus pour développer l’insulino-résistance : le muscle, le tissu adipeux et le foie. Néanmoins, certaines évidences scientifiques commencent également à pointer du doigt l’intestin, un organe critique dans la régulation du métabolisme des lipides postprandiaux, et qui pourrait, conséquemment, avoir un impact important dans l’apparition de la dyslipidémie diabétique. De façon très intéressante, des peptides produits par l’intestin, notamment le GLP-1 (glucagon-like peptide-1), ont déjà démontré leur potentiel thérapeutique quant à l’amélioration du statut diabétique et leur rôle dans le métabolisme intestinal lipoprotéinique. Une autre évidence est apportée par la chirurgie bariatrique qui a un effet positif, durable et radical sur la perte pondérale, le contrôle métabolique et la réduction des comorbidités du diabète de type 2, suite à la dérivation bilio-intestinale. Les objectifs centraux du présent programme scientifique consistent donc à déterminer le rôle de l’intestin dans (i) l’homéostasie lipidique/lipoprotéinique en réponse à des concentrations élevées de glucose (à l’instar du diabète) et à des peptides gastro-intestinaux tels que le PYY (peptide YY); (ii) la coordination du métabolisme en disposant de l’AMPK (AMP-activated protein kinase) comme senseur incontournable permettant l’ajustement précis des besoins et disponibilités énergétiques cellulaires; et (iii) l’ajustement de sa capacité d’absorption des graisses alimentaires en fonction du gain ou de la perte de sa sensibilité à l’insuline démontrée dans les spécimens intestinaux humains prélevés durant la chirurgie bariatrique. Dans le but de confirmer le rôle de l’intestin dans la dyslipidémie diabétique, nous avons tout d’abord utilisé le modèle cellulaire intestinal Caco-2/15. Ces cellules ont permis de démontrer qu’en présence de hautes concentrations de glucose en basolatéral, telle qu’en condition diabétique, l’intestin absorbe davantage de cholestérol provenant de la lumière intestinale par l’intermédiaire du transporteur NPC1L1 (Niemann Pick C1-like 1). L’utilisation de l’ezetimibe, un inhibiteur du NPC1L1, a permis de contrecarrer cette augmentation de l’expression de NPC1L1 tout comme l’élévation de l’absorption du cholestérol, prouvant ainsi que le NPC1L1 est bel et bien responsable de cet effet. D’autre part, des travaux antérieurs avaient identifié certains indices quant à un rôle potentiel du peptide intestinal PYY au niveau du métabolisme des lipides intestinaux. Toutefois, aucune étude n’avait encore traité cet aspect systématiquement. Pour établir définitivement l’aptitude du PYY à moduler le transport et le métabolisme lipidique dans l’intestin, nous avons utilisé les cellules Caco-2/15. Notre étude a permis de constater que le PYY incubé du côté apical est capable de réduire significativement l’absorption du cholestérol et le transporteur NPC1L1. Puisque le rôle de l'AMPK dans l'intestin demeure inexploré, il est important non seulement de définir sa structure moléculaire, sa régulation et sa fonction dans le métabolisme des lipides, mais aussi d'examiner l'impact de l’insulino-résistance et du diabète de type 2 (DT2) sur son statut et son mode d’action gastro-intestinal. En employant les cellules Caco-2/15, nous avons été capables de montrer (i) la présence de toutes les sous-unités AMPK (α1/α2/β1/β2/γ1/γ2/γ3) avec une différence marquée dans leur abondance et une prédominance de l’AMPKα1 et la prévalence de l’hétérotrimère α1/β2/γ1; (ii) l’activation de l’AMPK par la metformine et l’AICAR, résultant ainsi en une phosphorylation accrue de l’enzyme acétylCoA carboxylase (ACC) et sans influence sur l'HMG-CoA réductase; (iii) la modulation négative de l’AMPK par le composé C et des concentrations de glucose élevées avec des répercussions sur la phosphorylation de l’ACC. D’autre part, l’administration de metformine au Psammomys obesus, un modèle animal de diabète et de syndrome métabolique, a conduit à (i) une régulation positive de l’AMPK intestinale (phosphorylation de l’AMPKα-Thr172); (ii) la réduction de l'activité ACC; (iii) l’augmentation de l’expression génique et protéique de CPT1, supportant une stimulation de la β-oxydation; (iv) une tendance à la hausse de la sensibilité à l'insuline représentée par l’induction de la phosphorylation d'Akt et l’inactivation de la phosphorylation de p38; et (v) l’abaissement de la formation des chylomicrons ce qui conduit à la diminution de la dyslipidémie diabétique. Ces données suggèrent que l'AMPK remplit des fonctions clés dans les processus métaboliques de l'intestin grêle. La preuve flagrante de l’implication de l’intestin dans les événements cardiométaboliques a été obtenue par l’examen des spécimens intestinaux obtenus de sujets obèses, suite à une chirurgie bariatrique. L’exploration intestinale nous a permis de constater chez ceux avec un indice HOMA élevé (marqueur d’insulinorésistance) (i) des défauts de signalisation de l'insuline comme en témoigne la phosphorylation réduite d'Akt et la phosphorylation élevée de p38 MAPK; (ii) la présence du stress oxydatif et de marqueurs de l'inflammation; (iii) la stimulation de la lipogenèse et de la production des lipoprotéines riches en triglycérides avec l’implication des protéines clés FABP, MTP et apo B-48. En conclusion, l'intestin grêle peut être classé comme un tissu insulino-sensible et répondant à plusieurs stimuli nutritionnels et hormonaux. Son dérèglement peut être déclenché par le stress oxydatif et l'inflammation, ce qui conduit à l'amplification de la lipogenèse et la synthèse des lipoprotéines, contribuant ainsi à la dyslipidémie athérogène chez les patients atteints du syndrome métabolique et de diabète de type 2. / In relation with the constant increase in obesity, more and more people suffer from insulin resistance and type 2 diabetes (DT2). This doctoral research program especially emphasizes lipid disorders, one of the major consequences of cardiometabolic diseases. In this respect, people with insulin resistance or DT2 are at higher risk of developing lipid disturbances characterized mainly by high levels of triglycerides and LDL-cholesterol concentrations and HDL cholesterol in the blood circulation. The risks of cardiovascular disease are higher in these patients. Classically, three organs are known to develop insulin resistance: muscle, adipose tissue and liver. Nevertheless, important studies begin to point out the small intestine as a major organ in the regulation of postprandial lipids, which may have a significant impact on the development of diabetic dyslipidemia. In addition, the intestine produces peptides, including GLP-1 (glucagon-like peptide-1), that have already demonstrated their therapeutic potential with regard to diabetic status and intestinal lipoprotein metabolism. Further evidence is also is provided by the advent of bariatric surgery that has a positive effect on radical and sustainable weight loss, metabolic control and reduction of comorbidities of DT2, following biliopancreatic diversion. The central objectives of this scientific program are therefore to determine the role of the intestine in (i) lipid/ lipoprotein homeostasis in response to high concentrations of glucose (mimicking diabetes) and to gastrointestinal peptides such as PYY; (ii) the coordination of metabolism by involving AMPK (AMP-activated protein kinase) as an essential sensor for fine tuning of cellular energy needs; and (iii) adjusting absorption capacity of dietary fat in the gain or loss of insulin sensitivity demonstrated in intestinal specimens collected during bariatric surgery. In order to confirm the role of the intestine in diabetic dyslipidemia, we first used the intestinal Caco-2/15 cell model. The use of this epithelial cell line has shown a marked stimulation of cholesterol uptake via the transporter NPC1L1 (Niemann-Pick C1-like 1) in the presence of high glucose concentrations (as is the case in diabetic conditions) in basolateral compartment (compared to apical). The use of ezetimibe, an inhibitor of NPC1L1, helped to counteract this elevation of cholesterol absorption, thus proving that NPC1L1 is indeed behind this effect. If previous reports have identified some clues as to the potential role of intestinal PYY (peptide YY) in lipid metabolism disorders, no study has yet addressed this issue systematically. To definitively establish the ability of PYY to modulate lipid transport and metabolism in the intestine, we have used Caco-2/15 cells. Our recent investigation has shown that PYY (administered in the apical compartment) is able to significantly reduce cholesterol absorption via NPC1L1 transporter. Since the role of AMPK in the intestine remains unexplored, it is important to define not only its molecular structure, regulation and function in lipid metabolism, but also its impact on insulin resistance and T2D on its status and mode of action in the gastrointestinal tract. Using Caco-2/15 cells, we have been able to show (i) the presence of all AMPK subunits (α1/α2/β1/β2/γ1/γ2/γ3) with a marked difference in their abundance, but with a predominance of AMPKα1 and the prevalence of α1/β2/γ1 heterotrimer; (ii) the activation of AMPK by metformin and AICAR, resulting in increased phosphorylation of the downstream target acetylCoA carboxylases (ACC) without no influence on HMG-CoA reductase; (iii) the negative modulation of AMPK by compound C and glucose concentrations with high impact on ACC phosphorylation. On the other hand, administration of metformin to Psammomys obesus with insulin resistance and T2D led to (a) an upregulation of intestinal AMPK signaling pathway essentially typified by ascending AMPKα-Thr172 phosphorylation; (b) a reduction in ACC activity; (c) an elevation in the gene and protein expression of CPT1, supporting a stimulation of β-oxidation; (d) a trend of increase in insulin sensitivity portrayed by augmentation of Akt and GSK3β phosphorylation; (e) an inactivation of the stress-responsive p38-MAPK and /ERK1/2 exemplified by their phosphorylation lessening; and (f) a decrease in diabetic dyslipidemia following lowering of intracellular events that govern lipoprotein assembly. Therefore these data suggest that AMPK fulfills key functions in metabolic processes in the small intestine. The clear evidence for the involvement of the gut in cardiometabolic events has been obtained through the scrutiny of intestinal specimens obtained from obese subjects after bariatric surgery. Intestine of insulin-resistant subjects shows defects in insulin signaling as demonstrated by reduced Akt phosphorylation but increased p38 MAPK phosphorylation. These defects were accompanied with increased oxidative stress and inflammation markers in intestine of insulin-resistant subjects. Enhanced de novo lipogenesis rate and apo B-48 biogenesis along with increased triglyceride-rich lipoprotein production was also observed in the intestine of insulin-resistant subjects. Concomitantly, fatty acid binding proteins (FABP) and microsomal transfer protein (MTP) expression was increased in the intestine of insulin-resistant subjects. In conclusion, the small intestine may be classified as an insulin-sensitive tissue. Its deregulation, possibly triggered by oxidative stress and inflammation, may lead to amplification of lipogenesis and lipoprotein synthesis and may therefore represent a key mechanism for atherogenic dyslipidemia in patients with metabolic syndrome and T2D.

La stéatose hépatique et la détérioration du profil lipidique plasmatique sont des pathologies métaboliques favorisées par la carence œstrogénique post-ménopausique. Cependant les mécanismes à la base de ces pathologies n’ont été que très peu étudiés. Le but de cette thèse a été d’investiguer les mécanismes moléculaires possibles à l’origine de l’hypercholestérolémie et de l’accumulation des lipides (triglycérides : TG et cholestérol) dans le foie en utilisant un modèle animal de la ménopause, la rate Sprague Dawley ovariectomisée (Ovx). Nous avons également examiné si le changement des habitudes de vie comme la pratique de l’exercice physique pouvait prévenir ou corriger les modifications induites par l’Ovx. Enfin, rosuvastatine (statine) a été utilisée comme thérapie pharmacologique de l’hypercholestérolémie dans le but de comprendre son effet au niveau moléculaire chez la rate Ovx. L’objectif de la première étude était de déterminer comment l’Ovx peut affecter les niveaux de TG et de cholestérol dans le foie des rates nourries avec une diète riche en lipides (HF : 42% gras). Les rates ont été soumises à la diète HF ou normale pendant 6 semaines avant d’être Ovx ou Sham (ovariectomie simulée), puis maintenues aux mêmes conditions diététiques pour 6 autres semaines. L’Ovx a provoqué une accumulation de TG dans le foie, mais pas la diète HF seule. Cependant, lorsque l’Ovx était combinée à la diète HF, l’accumulation des TG était beaucoup plus importante comparé à ce qui était observé chez les rates Ovx soumises à la diète normale. L’expression génique (ARNm) de CPT1 (Carnitine palmitoyltransferase 1), PGC1α (Peroxisome proliferator-activated receptor gamma, coactivator 1) et PPARα (Peroxysome proliferetor activated receptor alpha) intervenant dans l’oxydation des acides gras dans le foie était augmentée par la diète HF (p ˂ 0.001; p ˂ 0.01; p ˂ 0.05 respectivement) ; mais atténuée (p ˂ 0.05; p ˂ 0.05; p ˂ 0.07 respectivement) lorsque les rates ont été Ovx, favorisant ainsi l’accumulation des TG dans le foie. La combinaison de la diète HF à l’Ovx a également provoqué une hypercholestérolémie et une accumulation de cholestérol dans le foie malgré la diminution de l’expression de la HMGCoA-r (3-hydroxy-3-methylglutaryl-CoA reductase), enzyme clé de la synthèse du cholestérol. Ceci était associé à l’inhibition de l’expression génique de CYP7a1 (Cytochrome P450, family 7, subfamily a, polypeptide 1), suggérant une diminution de la synthèse des acides biliaires. Ayant constaté dans la première étude que l’Ovx élevait les niveaux de cholestérol hépatique et plasmatique, nous nous sommes fixés comme objectif dans la deuxième étude d’évaluer les effets de l’Ovx sur l’expression génique des transporteurs et enzymes responsables du métabolisme du cholestérol et des acides biliaires dans le foie et l’intestin, et de vérifier si l’exercice sur tapis roulant pouvait prévenir ou corriger les changements causés par l’Ovx. L’hypercholestérolémie constatée chez les rates Ovx comparativement aux Sham était accompagnée de la diminution de l’expression génique des récepteurs des LDL (R-LDL), des résidus de lipoprotéines (LRP1), de SREBP-2 (Sterol regulatory element binding protein 2) et de PCSK9 (Proprotein convertase subtilisin/kexin type 9) dans le foie, suggérant une défaillance dans la clairance des lipoprotéines plasmatiques. L’Ovx a aussi inhibé l’expression génique de la MTP (Microsomal triglyceride transfer protein) et stimulé celle de SR-B1 (Scavenger receptor class B, member 1); mais aucun changement n’a été observé avec CYP7a1. Ces changements moléculaires pourraient par conséquent favoriser l’accumulation de cholestérol dans le foie. L’exercice physique n’a pas corrigé les modifications causées par l’Ovx sur l’expression génique de ces molécules au niveau hépatique à l’exception de SREBP-2. Par contre, au niveau intestinal (iléum), l’exercice sur tapis roulant a inhibé l’expression génique des marqueurs moléculaires intervenant dans l’absorption des acides biliaires (OSTα/β, FXR, RXRα, Fgf15) et du cholestérol (LXRα, NCP1L1) au niveau de l’iléum chez les rates Sham entraînées. Ces adaptations pourraient prévenir le développement de l’hypercholestérolémie protégeant en partie contre la survenue de l’athérosclérose. Au vue des effets délétères (hypercholestérolémie et diminution de l’expression du R-LDL, PCSK9, LRP1, SREBP-2 et HMGCOA-r dans le foie) causés par l’Ovx sur le métabolisme du cholestérol constatés dans l’étude 2, la 3ième étude a été conçue pour évaluer l’efficacité de rosuvastatine (Ros) sur l’expression génique de ces marqueurs moléculaires chez les rates Ovx sédentaires ou soumises à l’entraînement volontaire. Ros a été administrée aux rates Ovx pendant 21 jours par voie sous-cutanée à la dose de 5mg/kg/j à partir de la 9ième semaine après l’Ovx. Ros n’a pas diminué la concentration plasmatique de LDL-C et de TC chez les rates Ovx. Par contre, Ros a stimulé (P ˂ 0.05) l’expression génique de PCSK9, SREBP-2, LRP1, HMGCoA-r et ACAT2 (Acyl-CoA cholesterol acyltransferase) mais pas significativement (P = 0.3) celle du R-LDL dans le foie des rates Ovx sédentaires et entraînées. Ros n’a pas réduit la concentration plasmatique de LDL-C probablement à cause de l’induction plus importante de PCSK9 par rapport au R-LDL. Cependant, la stimulation de LRP1 par Ros protège partiellement contre la survenue des maladies cardiovasculaires. En conclusion, les études de cette thèse indiquent que la baisse du niveau des œstrogènes entraîne des changements radicaux du métabolisme hépatique des TG et du cholestérol provoqués par des altérations de l’expression des gènes clés des voies métaboliques associées. / Hepatic steatosis and plasma lipid profile deterioration are metabolic diseases favored by post-menopausal estrogen deficiency. However, mechanisms underlying these diseases have not been systematically adressed. The aim of this thesis was to investigate molecular mechanisms causing hypercholesterolemia and lipids (triglycerides: TG and cholesterol) accumulation in the liver using animal model of menopause, the ovariectomized (Ovx) Sprague Dawley rat. We also examined whether lifestyle modifications such as physical activity can prevent or correct changes induced by Ovx. Finally, rosuvastatin (statine) was used as a pharmacological therapy of hypercholesterolemia in order to understand its effect at the molecular level in Ovx rats. The first study was designed to determine how the Ovx may affect levels of TG and cholesterol in the liver of rats fed a high-fat diet (HF: 42% fat). Rats were submitted to a HF or a normal diet for 6 weeks prior to Ovx or being sham operated, and then kept on the same diets for another 6 weeks. The Ovx increased liver TG content, but not the HF diet alone. However, the combination of Ovx and HF diet resulted in a greater liver TG accumulation than that observed in Ovx submitted to normal diet. The mRNA levels of CPT-1, PGC1 and PPARα involved in liver lipid oxidation significantly increased in rats fed the HF diet (p ˂ 0.001; p ˂ 0.01; p ˂ 0.05 respectively); but this increase was substantially less if HF fed rats were Ovx (p ˂ 0.05; p ˂ 0.05; p ˂ 0.07 respectively), thus favouring TG accumulation in the liver. The combination of HF diet and Ovx also induced hypercholesterolemia and an increase in liver total cholesterol content, in spite of the reduction of liver HMGCoA-r gene expression, the key enzyme for cholesterol synthesis. This was also associated with a decrease of liver CYP7a1 gene expression, suggesting a reduction in bile acids synthesis. Having found in the first study that the Ovx increases liver and plasma cholesterol levels, we aimed in the second study at determining the effects of Ovx on gene expression of hepatic and intestinal transporters and enzymes involved in cholesterol and bile acids metabolism; and to verify whether treadmill exercise could prevent or correct changes induced by Ovx. The Ovx resulted in hypercholesterolemia associated with a reduction in gene expression of hepatic low-density lipoprotein receptor (LDL-R), lipoprotein remnants receptor (LRP1), SREBP-2 and PCSK9, suggesting a failure in the clearance of plasma lipoproteins particles. The Ovx also inhibited the expression of MTP and stimulated that of SR-B1 in the liver, but no change was observed with CYP7a1. These molecular changes might, therefore, favor cholesterol accumulation in the liver. Exercise training did not correct the deleterious effects caused by Ovx on gene expression of these molecular markers in the liver with the exception of SREBP-2. However, in the intestine (ileum) treadmill exercise reduced gene expression of molecular markers involved in the absorption of bile acids (OSTα/β, FXR, RXRα, Fgf15) and cholesterol (LXRα, NCP1L1) in Sham trained rats compared to sedentary rats. This could prevent the development of cholestasis and hypercholesterolemia protecting partially against the onset of atherosclerosis. In view of the deleterious effects (hypercholesterolemia and decreased in gene expression of LDL-R, PCSK9, LRP1, SREBP-2 and HMGCoA-r in the liver) caused by Ovx on cholesterol metabolism observed in the second study, the 3rd study was designed to test the effect of rosuvastatin (Ros) on gene expression of these molecular markers in Ovx sedentary rats or in Ovx rats submitted to voluntary training. Ros was injected to Ovx rats subcutaneously at dose of 5mg/kg/day during 21 days from the ninth week after ovariectomy. Ros failed to decrease plasma LDL-C and TC in Ovx rats. In contrast, Ros increased (P ˂ 0.05) PCSK9, SREBP-2, LRP1, HMGCoA-r and ACAT2 but not significantly (P ˂ 0.3) LDL-R mRNA in the Ovx sedentary and trained rat liver. Ros failed to decrease plasma LDL-C in Ovx rats probably because of a stronger induction of PCSK9 than LDL-R gene expression. However by increasing LRP1 expression, Ros could decrease circulating lipoprotein remnants and, therefore, protects partially against the onset of cardiovascular diseases. In conclusion, the studies of this thesis indicate that the decrease of ovarian estrogen levels causes radical changes in hepatic TG and cholesterol metabolism caused by alterations in the expression of key genes associated with metabolic pathways.

No description available.

INTRODUÇÃO: O estudo das vias moleculares e das alterações específicas responsáveis pela progressão desfavorável de pacientes com CCR parece essencial para o desenvolvimento de terapias mais efetivas. OBJETIVO: Comparar a expressão quantitativa dos genes TACSTD2, Ku70, KRAS mutante e SERIN1 em amostras de tecidos normal e tumoral de pacientes com CCR e relacionar sua expressão com variáveis clínico-patológicas. MÉTODOS: Foram estudados 37 pacientes com CCR submetidos à ressecção cirúrgica entre julho de 2005 e julho de 2009 e cujas amostras congeladas de tecidos tumoral e normal foram armazenadas em um banco de tecidos. Através da RT-PCR foi sintetizado o cDNA a partir do RNA extraído das amostras teciduais. A expressão dos genes TACSTD2, KRAS mutante, Ku70 e SERIN1 foi quantificada pela técnica de PCR em tempo real. RESULTADOS: A expressão do KRAS mutante foi maior no tecido tumoral do que no normal (p = 0,024). A expressão tumoral dos genes Ku70, TACSTD2 e SERIN1 foi respectivamente menor, igual e maior que o tecido normal, porém sem significância estatística. Associação estatisticamente significativa também foi observada entre idade e expressão de KRAS mutante no tecido normal e tumores pouco diferenciados e expressão de Ku70 no tecido normal. Não foram observadas outras associações estatisticamente significativas. CONCLUSÕES: A expressão do KRAS mutante no tecido tumoral é maior do que no tecido normal (p = 0,024) na casuística de 37 pacientes com CCR estudados através da técnica de PCR em tempo real. / INTRODUCTION: Knowledge of the molecular pathways and of the specific alterations responsible for the unfavorable progression of patients with CCR appears essential for the development of more effective therapies. PURPOSE: To compare the quantitative expression of the genes TACSTD2, mutant KRAS, Ku70 and SERIN1 in samples of normal and tumoral tissues of patients with CCR and to relate their expression to clinicopathologic characteristics. METHODS: 37 patients with CCR were studied. The patients had been operated on between July 2005 and July 2009, and their frozen samples of tumoral and normal tissues had been stored in a tissue bank. The expression of the genes TACSTD2, mutant KRAS, Ku70 and SERIN1 was quantified through the technique of real time polymerase chain reaction. RESULTS: The mutant KRAS expression was higher in the tumoral tissue than in the normal tissue (p = 0,024). Although not significant, the tumoral expression of the genes Ku70, TACSTD2 and SERIN1 was respectively lower, equal to, and higher than in the normal tissue. Statistically significant association was also observed between age and mutant KRAS expression in normal tissue and between poorly-differentiated tumors and Ku70 expression in normal tissue. No other statistically significant associations were observed. CONCLUSIONS: Tumoral tissues express mutant KRAS at higher levels than normal tissues in the casuistic of 37 patients with CCR studied through the technique of PCR real time.

INTRODUÇÃO: O estudo das vias moleculares e das alterações específicas responsáveis pela progressão desfavorável de pacientes com CCR parece essencial para o desenvolvimento de terapias mais efetivas. OBJETIVO: Comparar a expressão quantitativa dos genes TACSTD2, Ku70, KRAS mutante e SERIN1 em amostras de tecidos normal e tumoral de pacientes com CCR e relacionar sua expressão com variáveis clínico-patológicas. MÉTODOS: Foram estudados 37 pacientes com CCR submetidos à ressecção cirúrgica entre julho de 2005 e julho de 2009 e cujas amostras congeladas de tecidos tumoral e normal foram armazenadas em um banco de tecidos. Através da RT-PCR foi sintetizado o cDNA a partir do RNA extraído das amostras teciduais. A expressão dos genes TACSTD2, KRAS mutante, Ku70 e SERIN1 foi quantificada pela técnica de PCR em tempo real. RESULTADOS: A expressão do KRAS mutante foi maior no tecido tumoral do que no normal (p = 0,024). A expressão tumoral dos genes Ku70, TACSTD2 e SERIN1 foi respectivamente menor, igual e maior que o tecido normal, porém sem significância estatística. Associação estatisticamente significativa também foi observada entre idade e expressão de KRAS mutante no tecido normal e tumores pouco diferenciados e expressão de Ku70 no tecido normal. Não foram observadas outras associações estatisticamente significativas. CONCLUSÕES: A expressão do KRAS mutante no tecido tumoral é maior do que no tecido normal (p = 0,024) na casuística de 37 pacientes com CCR estudados através da técnica de PCR em tempo real. / INTRODUCTION: Knowledge of the molecular pathways and of the specific alterations responsible for the unfavorable progression of patients with CCR appears essential for the development of more effective therapies. PURPOSE: To compare the quantitative expression of the genes TACSTD2, mutant KRAS, Ku70 and SERIN1 in samples of normal and tumoral tissues of patients with CCR and to relate their expression to clinicopathologic characteristics. METHODS: 37 patients with CCR were studied. The patients had been operated on between July 2005 and July 2009, and their frozen samples of tumoral and normal tissues had been stored in a tissue bank. The expression of the genes TACSTD2, mutant KRAS, Ku70 and SERIN1 was quantified through the technique of real time polymerase chain reaction. RESULTS: The mutant KRAS expression was higher in the tumoral tissue than in the normal tissue (p = 0,024). Although not significant, the tumoral expression of the genes Ku70, TACSTD2 and SERIN1 was respectively lower, equal to, and higher than in the normal tissue. Statistically significant association was also observed between age and mutant KRAS expression in normal tissue and between poorly-differentiated tumors and Ku70 expression in normal tissue. No other statistically significant associations were observed. CONCLUSIONS: Tumoral tissues express mutant KRAS at higher levels than normal tissues in the casuistic of 37 patients with CCR studied through the technique of PCR real time.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Martin Huličiak Supervisor: PharmDr. Lukáš Červený, Ph.D Title of diploma thesis: Study of interactions of antiviral drugs with intestinal drug efflux ABC transporters P-gp, MRP2 and BCRP are efflux transporters, members of the family of ATP binding cassette (ABC) transporters. These transporters are located on the apical membrane of the intestinal epithelium, where they may limit absorption of orally administered drugs. Study of drug interactions with/on intestinal efflux transporters is necessary to provide safe and effective treatment. The Caco-2 cell line is FDA recommended in vitro model of intestinal barrier and it is used for bidirectional testing of substrates and inhibitors of ABC transporters in preclinical research. However, this methodology has several shortcomings, so the need of introduction of new experimental models is increasing and the ex vivo method based on human or rat intestine is a promising option. Precision-cut intestinal slices (PCIS) represent a mini-model of the organ and contain all types of cells of the tissue. We used both in vitro model using Caco-2 cell monolayers for drug transport study and in our lab established ex vivo method of PCIS for accumulation study...

A isquemia do intestino delgado ocorre nas oclusões arteriais dos vasos mesentéricos ou associada à baixa perfusão tecidual causada por choque circulatório. Seus efeitos deletérios locais e sistêmicos frequentemente agravam a evolução clínica de muitas doenças. Este estudo experimental investiga como a reposição do volume intravascular utilizando duas soluções salinas com diferentes concentrações de sódio (solução salina fisiológica e solução 7,5% de cloreto de sódio) modifica a resposta inflamatória e o estresse oxidativo causados pela isquemia do intestino delgado. Ratos Wistar, machos, peso corporal entre 250 e 300 g, número total =102, foram submetidos à oclusão transitória da artéria mesentérica superior durante 45 minutos. No protocolo utilizado, os animais foram sorteados para inclusão em um de quatro grupos experimentais: isquemia falsa (IF), isquemia intestinal seguida da infusão de solução salina hipertônica 7.5% em volume de 4 ml/kg de peso (SH), isquemia intestinal seguida da infusão de solução salina 0.9% em volume de 33 ml/kg de peso (SF) e isquemia intestinal sem reposição do volume intravascular (ST). Quando apropriado, as soluções foram administradas lentamente (5 minutos) pela veia jugular externa imediatamente antes da reperfusão intestinal. Em cada grupo experimental, logo após a reperfusão intestinal, os animais foram sorteados para tempo de sobrevida: 2 horas, 4 horas ou 6 horas após a reperfusão. Amostras de sangue foram colhidas pela veia jugular externa em vários períodos: imediatamente após a liberação da oclusão da artéria mesentérica, 2 horas, 4 horas e 6 horas após a reperfusão intestinal. O plasma foi separado e foram realizadas as dosagens de interleucinas (IL-6 e IL-10). No tempo determinado, os animais foram submetidos à eutanásia em condições humanamente aceitáveis e, então, nesse momento, foram colhidas amostras de tecidos (intestino, fígado e pulmão) para posterior quantificação das concentrações de malondialdeído (MDA) e interleucinas (IL-6 e IL-10). A atividade da mieloperoxidase (MPO) também foi avaliada nessas amostras. Os animais que não receberam tratamento apresentaram uma taxa de mortalidade maior do que os demais grupos. Os grupos de animais tratados com reposição de volume intravascular apresentaram uma taxa de mortalidade semelhante ao grupo de isquemia falsa. Os animais que receberam reposição de volume intravascular com soluções cristalóides (SH ou SF) apresentaram concentrações de MDA, MPO, IL-6 e IL-10 nos tecidos (intestino, fígado e pulmão) comparáveis ao grupo de animais com isquemia falsa. Em todos os momentos, esses valores foram mais elevados no grupo que não recebeu tratamento. As concentrações plasmáticas da IL-6 e da IL-10 foram mais elevadas nos animais tratados com SH. As análises mostram que a simples abertura da cavidade abdominal causa um trauma cirúrgico relevante aos animais e é responsável pelas alterações observadas no grupo de isquemia falsa. Os resultados sugerem que a isquemia intestinal transitória (45 minutos) realizada por oclusão da artéria mesentérica superior em ratos representa um modelo experimental de moderada gravidade. Dessa maneira, o modelo é adequado aos estudos das alterações bioquímicas e celulares que ocorrem a curto, médio e longo tempo de sobrevida. Este estudo foi elaborado para análise dos fatores relativos ao estresse oxidativo e reação inflamatória que ocorrem nas primeiras horas que seguem a reperfusão intestinal. De uma maneira geral, os animais foram beneficiados pela reposição do volume intravascular com soluções cristalóides. A solução salina fisiológica foi utilizada em volume aproximadamente oito vezes superior à solução hipertônica 7,5% de cloreto de sódio. Comparativamente, a atenuação similar das respostas deletérias após a reperfusão intestinal atingida com o uso de menor volume da solução hipertônica 7,5% de cloreto de sódio representa um fator positivo para a mesma. Considera-se que a maior concentração plasmática das interleucinas (IL-6 e IL-10) encontrada nos animais tratados com solução hipertônica7,5% de cloreto de sódio esteja relacionada ao aumento de permeabilidade da microcirculação associado às soluções hipertônicas / Gut ischemia is responsible for both local and systemic deleterious events. Since reperfusion occurs in a previous ischemic superior mesenteric artery territory (SMA), a succession of harmful mechanisms begins in the luminal epithelium that quickly lengthens the limits of the intestinal tract. Depending on the extension of the intestinal system involved in the ischemic/reperfusion injury there will be severe repercussion to distant organs in response to SMA occlusion. Several diseases could be associated with variables degrees of intestinal ischemia. Even minor intensity of intestinal ischemia had deleterious systemic effects and often aggravates the clinical outcome of many diseases. Our study investigates how different forms of volume restoration could modify two important mechanisms of injury after intestinal ischemia: oxidative stress and inflammatory responses. Wistar rats (n=102) were submitted to transient superior mesenteric artery occlusion (SMAo). After randomization, animals were divided in four groups: Sham intestinal ischemia; infusion of small volume of 7.5% hypertonic saline (HS), or infusion of high volume of 0.9% saline (NS) just prior reperfusion, and animals that did not receive intra vascular volume treatment (NT). At sequential times, the animals were euthanatized and tissue samples (lung, liver, and intestine) were collected to Malondialdehyde (MDA) dosage and myeloperoxidase (MPO) activity. Also, sequential plasmatic concentration of IL-6 and IL-10 were done. Animals treated with both forms of volume infusion showed lower levels of tissue MDA, MPO, IL-6, and IL-10 than found in NT group. Plasmatic concentration of IL-6 and IL-10 were higher in animals treated with HS. Positive correlation was found between tissue concentration of IL-10 and IL-6. The mortality rate was similar between the treated rats and the group of sham ischemia. The mortality rate was higher in the non treated animals. In this rat model of transient intestinal ischemia, adequate maintenance of intravascular volemia decreases oxidative stress and synthesis of inflammatory markers. Small volume of 7.5% HS (4ml/Kg body weight) and high amounts of NS ( 33 ml/Kg body weight) had similar effects in attenuation of these responses. In this study, 7.5% HS attenuates deleterious effects found after intestinal ischemia with the main advantage of the smallest volume utilized when compared with NS solution. Plasmatic concentrations of IL-6 and IL-10 were higher in HS treated animals. This observation is supported by action of hypertonic/hyperosmotic solutions at the microcirculatory level. These solutions increase the local vascular permeability. This characteristic of 7.5% HS solution could facilitate the passage of the locally produced interleukin to the systemic circulation

As lesões térmicas corporais (LTC) são um sério problema de saúde, atingindo principalmente crianças. A extensão e a profundidade da lesão são fatores que alteram várias estruturas. Alterações gastrintestinais também são relatadas, sendo a principal delas, a atrofia das mucosas, provocando ulcerações e a perda da barreira seletiva. Na presente pesquisa avaliou-se através de técnicas histoquímicas, imunohitoquimicas e de microscopia de luz, as alterações ocorridas nos componentes do plexo mioentérico e na espessura da mucosa do jejuno em três porções: oral (O), média (M) e aboral (A), de ratos adultos com 30% da superfície corpórea exposta ao escaldamento, 4 dias (q4) e 10 dias (q10) após a LTC. Verificou-se em q10 o não restabelecimento da massa corpórea, a diminuição da área do jejuno, bem como espessura de sua mucosa. No plexo mioentérico, a área média do perfil celular dos neurônios NADPH não variou, tendo estes uma menor densidade em q10, sendo estes corpos altamente reativos em q4 e q10. Varicosidades grandes destacaram-se em q4 e q10, quando pela SP e VIP. / Burn is a determinant factor to alter body structures as the striated muscle. It also determines gastrintestinal mucosal atrophy what produce loss of selective barrier. With histochemical, immunohistochemical and light microscopy methods the myenteric plexus (MP) of the jejunum was evaluated in rats submitted to burn injury. The scalding was performed in 30% of the body surface. The MP and the mucosa of the oral (O), middle (M) and aboral (A) parts of the jejunum were analyzed four (q4) and ten (q10) days post-lesion. The loss of weight due the burn is not recovered in q10 where the jejunal surface area and the thickness of the mucosa decreased. The neuronal profile of nitregic neurons was similar in q4, q10. The density of nitregic neurons was lower in q10 showing that the time post injury is an important factor able to alter this parameter. The q4 and q10 groups exhibited neuronal bodies highly reactive to NADPH. The immunoreactivity to SP and VIP in q4 and q10 was expressed mainly in large varicosities.