Abstract Necrotizing enterocolitis (NEC) is severe and life-threatening disease of infant gastrointestinal tract (GIT). It is a defect of GIT adaptation to extrauterine life, which affects mostly premature infants and infants afflicted by congenital developmental defects of GIT or heart. Gut barrier disruption and presence of abnormal gut microbiota has eminent role in NEC pathogenesis. Nowadays, the diagnosis of NEC is based on presence of clinical symptoms - abdominal distension, blood in stool and radiologic finding Pneumatosis intestini. However, the symptoms are in early stage of NEC non- specific and do not allow unambiguous distinguishing of NEC from other GIT disorders or sepsis. Aim of this thesis was to extend the understanding of inflammatory response in neonates with NEC and to find applicable biomarker for early diagnosis of NEC and thereby provide quick intervention. Cytokines have important role in immune response regulation. Although the connection between some cytokines and NEC was described, the inflammatory response during NEC was not yet properly stated. The levels of 40 cytokines related to inflammation was shown in this thesis. Intestinal - Fatty Acid Binding Protein (i- FABP) is a small...

Heart-type Fatty Acid-Binding Protein (H-FABP) is required for high rates of skeletal muscle long chain fatty acid (LCFA) oxidation and esterification. Here we assessed whether H-FABP affects soleus muscle glucose uptake when measured in vitro in the absence of LCFA. Wild type and H-FABP null mice were fed a standard chow or high fat diet before muscle isolation. With the chow, the mutation increased insulin-dependent deoxyglucose uptake by 141% (P<0.01) at 0.02 mU/ml of insulin, but did not cause a significant effect at 2 mU/ml insulin; skeletal muscle triglyceride and long chain acyl-CoA (LCACoA) levels remained normal. With the fat diet, the mutation increased insulin-dependent deoxyglucose uptake by 190% (P<0.01) at 2 mU/ml insulin, thus partially preventing insulin resistance, and completely prevented the threefold (P<0.001) diet-induced increase of muscle triglyceride levels; however, muscle LCACoA levels showed little or no reduction. With both diets, the mutation reduced the basal (insulinindependent) soleus muscle deoxyglucose uptake by 28% (P<0.05). These results establish a close relationship of FABP-dependent lipid pools with insulin sensitivity, and indicate the existence of a non-acute, antagonistic, and H-FABP-dependent fatty acid regulation of basal and insulin-dependent muscle glucose uptake. Liver fatty acid binding protein (L-FABP) has been proposed to limit the availability of chain LCFA for oxidation and for peroxisome proliferator-activated receptor (PPAR-alpha), a fatty acid binding transcription factor that determines the capacity of hepatic fatty acid oxidation. Here, we used L-FABP null mice to test this hypothesis. Under fasting conditions, this mutation reduced &#946;-hydroxybutyrate (BHB) plasma levels as well as BHB release and palmitic acid oxidation by isolated hepatocytes. However, the capacity for ketogenesis was not reduced: BHB plasma levels were restored by octanoate injection; BHB production and palmitic acid oxidation were normal in liver homogenates; and hepatic expression of key PPAR-alpha target (MCAD, mitochondrial HMG CoA synthase, ACO, CYP4A3) and other (CPT1, LCAD) genes of mitochondrial and extramitochondrial LCFA oxidation and ketogenesis remained at wild-type levels. These results suggest that under fasting conditions, hepatic L-FABP contributes to hepatic LCFA oxidation and ketogenesis by a nontranscriptional mechanism.

Necrotizing enterocolitis (NEC), a severe inflammatory disorder of the gastrointestinal tract with high morbidity and mortality, affects primarily preterm infants. The diagnosis represents a challenging task, and no biomarker has been found to aid early diagnosis with high accuracy. Microdialysis has been widely used to detect metabolites of anaerobic metabolism, enabling a local and early detection of ischemia. This thesis aims to evaluate the possibility of detecting intestinal ischemic stress in experimental and clinical  NEC, by use of rectal intraluminal microdialysis. Intraluminal rectal microdialysis was performed on rats subjected to total intestinal ischemia. Metabolites of ischemia were detectable in both ileum and rectum, with raised glycerol concentrations and lactate/pyruvate ratios. Elevated concentrations of glycerol correlated to increasing intestinal histopathological injury. Experimental early NEC was induced in newborn rat pups, by hypoxia/re-oxygenation treatment. Development of NEC was confirmed by histopathology. Elevated glycerol concentrations were detected by rectal microdialysis. The genetic alterations following experimental NEC in rat pups were studied with microarray. Immunohistochemistry staining was performed for tight junction proteins claudin-1 and claudin-8. Several genes were altered in experimental NEC, mainly genes regulating tight junctions and cell adhesion. Immunohistochemistry revealed reduced expression of claudin-1. A prospective study was conducted on preterm infants with a gestational age of less than 28 weeks. The infants were admitted to a neonatal intensive care unit, and observed during a 4-week period. Rectal microdialysis was performed twice a week, and blood was drawn for analysis of I-FABP. A total of 15 infants were included in the study, whereof four infants developed NEC, and 11 served as controls. Rectal glycerol and I-FABP displayed high concentrations, which varied considerably during the observation periods, both in NEC and controls. No differences in either glycerol or I-FABP concentrations were seen in the NEC-group vs. the controls. In conclusion, rectal microdialysis can detect metabolites of intestinal ischemia, both in experimental and clinical NEC. Rectal microdialysis is safe and could provide a valuable non-invasive aid to detect hypoxia-induced intestinal damage or ischemic stress in extremely preterm infants. In this study however, it was not possible to predict the development of clinical NEC using microdialysis or I-FABP.

Heart-type fatty acid binding protein (H-FABP) is a major fatty acid binding factor in skeletal muscles. Genetic lack of H-FABP severely impairs the esterification and oxidation of exogenous fatty acids in soleus muscles isolated from chow-fed mice (CHOW-solei) and high fat diet-fed mice (HFD-solei), and prevents the HFD-induced accumulation of muscle triglycerides. Here, we examined the impact of H-FABP deficiency on the relationship between fatty acid utilization and glucose oxidation. Glucose oxidation was measured in isolated soleus muscles in the presence or absence of 1 mM palmitate (simple protocol) or in the absence of fatty acid after preincubation with 1 mM palmitate (complex protocol). With the simple protocol, the mutation slightly reduced glucose oxidation in CHOW-muscles, but markedly increased it in HFDmuscles; unexpectedly, this pattern was not altered by the addition of palmitate, which reduced glucose oxidation in both CHOW- and HFD-solei irrespective of the mutation. In the complex protocol, the mutation first inhibited the synthesis and accumulation of triglycerides and then their mobilization; with this protocol, the mutation increased glucose oxidation in both CHOW- and HFD-solei. We conclude: (i) H-FABP mediates a non-acute inhibition of muscle glucose oxidation by fatty acids, likely by enabling both the accumulation and mobilidoes not mediate the acute inhibitory effect of extracellular fatty acids on muscle glucose oxidation; (iii) H-FABP affects muscle glucose oxidation in opposing ways, with inhibition prevailing at high muscle triglyceride contents.zation of a critical mass of muscle triglycerides; (ii) H-FABP

Las proteínas que unen ácidos grasos o FABPs (Fatty Acid Binding Proteins), componen una familia de proteínas citosólicas ubicuas, de aproximadamente 15 KD de peso molecular, con una estructura terciaria altamente conservada en toda la familia. En todos los casos, la estructura terciaria se compone de láminas beta antiparalelas empaquetadas formando un barril beta elipsoide y dos segmentos alfa-helicoidales cortos que formarían parte de un pequeño portal en uno de los extremos del barril. Es de destacar que, a pesar de la gran similitud de su estructura terciaria, la estructura primaria de FABPs de distintos tejidos no presenta una semejanza comparable. Las FABPs son capaces de unir ácidos grasos transportándolos desde y hacia las membranas celulares dentro de una cavidad hidrofóbica formada por el barril beta antes mencionado. Sin embargo, experiencias empíricas han evidenciado que existen diferencias en los mecanismos por los cuales desarrollan su labor. Por un lado, un grupo de estas proteínas interactuarían con las membranas celulares colisionando con las mismas para transferir sus ligandos, mientras que otras lo harían por una difusión acuosa del ligando. No obstante, publicaciones recientes proponen que la transferencia por difusión implicaría un contacto directo con la membrana, aunque con una conformación distinta y sin penetración en la bicapa lipídica. Los distintos mecanismos propuestos para la transferencia de ligandos desde la proteína hacia las membranas han resultado en la división teórica de la familia FABP en los grupos colisionales y difusionales. Distintos trabajos experimentales han demostrado que el dominio α-helicoidal de IFABP (perteneciente al grupo colisional) y LFABP (difusional) desempeña un rol crítico en la determinación del mecanismo de transferencia de los ácidos grasos, aún cuando la composición de las hélices es muy distinta. Tomando como base estas diferencias estructurales, de unión y de cinética de transferencia del ligando, se ha propuesto que las FABPs colisionales y difusionales tendrían funciones diferentes, tal vez contribuyendo al transporte diferencial y compartimentación de los lípidos. Si bien los mecanismos propuestos fueron y son intensamente estudiados, no se ha propuesto aún una diferencia estructural de estas proteínas que explique acabadamente la existencia de los mismos. En la presente tesis, utilizando diferentes técnicas de la biología computacional, se estudiaron distintos aspectos de la interacción de FABP con membrana con el objetivo de describir los distintos mecanismos. Las técnicas utilizadas permitieron el estudio estructural de FABP, la comprobación de la influencia de la energía electrostática en la interacción, la importancia de residuos conservados y la descripción de la dinámica del sistema. Del estudio estructural de las FABPs de mamíferos, se desprende la existencia de residuos de carga neta distinta de cero, conservados en polos opuestos de las proteínas. Los resultados señalan la conservación del signo de la carga en regiones determinadas para FABPs con igual mecanismo de interacción descripto. El análisis electrostático determinó que es esta energía la que dirige la interacción. Se confirmó además, que los aminoácidos cargados altamente conservados poseen un rol fundamental en la selección del mecanismo, ya que su mutación por residuos de carga opuesta resulta en una inversión del perfil electrostático. La dinámica de los mecanismos señala nuevamente a los residuos cargados conservados como los primeros en tomar contacto con la membrana durante la interacción, confirmando nuevamente los resultados de los estudios estructurales. En suma, los resultados obtenidos permitieron generar un modelo computacional que explica en detalle la existencia de los mecanismos colisional y difusional para FABPs de mamíferos. / Fatty Acid Binding Proteins (FABP) are a huge family of ubiquitous citosolic proteins of 15 KD of molecular weigth, with a highly conserved tertiary structure. In all cases, tertiary structure is composed by antiparallel beta strands that form an elliptic barrel and two alpha helices on one end of the barrel, forming a putative gate. It is a significant fact that, in spite of highly conserved tertiary structure, FABPs from different tissues have no primary structure similarity. FABPs are able to bind fatty acids and to carry them from and to cellular membranes into a hydrophobic cavity in their beta barrel. Nevertheless, empirical evidence suggests that FABP-membrane interaction takes place through two different mechanisms. On one hand, some FABPs interact with membrane by a collisional mechanism, while other group interacts by aquous diffusion of ligand. However, recent bibliography suggests that diffusional mechanism also implies a direct contact with the membrane but without penetration of lipid bilayer. Proposed mechanisms have resulted in theoretical segregation of FABPs in collisional or diffusional groups. Several empirical researches have shown that despite of being structurally different, alpha helices have a critical rol in mechanism selection for both groups. Considering structural, ligand binding and ligand kinetic transfer differences, it has been proposed that collisional and diffusional FABPs may have different functions, contributing with lipid compartmentalization and lipid differential transport. Although both mechanisms have been and are still deeply studied, a detailed explanation of the relationship between structural differences and different mechanisms is still missing. Different computational biology techniques were used in this thesis in order to describe distinct aspects of FABP-membrane interaction. Employed techniques allowed structural analysis of FABP, the study of electrostatic as driving force of FABP-membrane interaction and the description of the system dynamic. Mammals FABP structural analysis shows that highly conserved charged residues are found in defined regions. Charge sign conservation in defined regions is equal for same mechanism FABPs. Electrostatics analysis pointed this very energy as driving force for the interaction. The relevance of the highly conserved charged amino acid for mechanism selection was confirmed. Reverse mutation of them caused inversion in the electrostatic interaction landscape. Molecular Dynamics results pointed to conserved residues as the first residues to contact lipid bilayer, in this way, structural results were confirmed. In summary, obtained results allowed the generation of a computational model that explains in detail the existence colisional and difusional mechanisms for mammals FABP.

Die akute Lungenarterienembolie stellt eine häufige Erkrankung dar, welche mit einer hohen Letalität einhergeht. Die Risikostratifizierung und Therapieplanung beruht derzeit primär auf klinischen Parametern, Biomarkerbestimmungen und den Ergebnissen der Echokardiographie. Fr¨¹here Untersuchungen zeigten einen möglichen Nutzen von Heart-type Fatty-Acid Binding Protein (H-FABP) als Prognosemarker. Hierbei wurden allerdings unselektierte Patientenkollektive verwendet. Der Nutzen von H-FABP in der Risikostratifizierung normotensiver Patienten mit einer akuten Lungenarterienembolie ist bislang nicht untersucht. H-FABP ist ein Enzym des Myokards, welches eine tragende Bedeutung im Fettstoffwechsel der Myozyten spielt. Es wird bei einer Schädigung des Herzmuskels ins Blutplasma freigesetzt und ist dort innerhalb kurzer Zeit als Marker einer Myokardischämie nachweisbar. Insgesamt wurde in dieser Studie die prognostische Aussagekraft von H-FABP bei 126 normotensiven Patienten mit einer akuten Lungenarterienembolie im Hinblick auf 30-Tages-Komplikationen sowie ihr Langzeit¨¹berleben untersucht. Innerhalb von 30 Tagen kam es bei 9 (7%) Patienten zu Komplikationen. In dieser Subpopulation zeigten sich signifikant (p<0,001) erhöhte H-FABP-Spiegel (median 11,2 ng/ml; IQR 8,0-36,8) im Vergleich zu der Patientengruppe, in der keine Komplikationen auftraten (median 3,4 ng/ml; IQR 82,1-4,9). 29 Patienten zeigten bei Aufnahme einen H-FABP-Wert oberhalb des mittels ROC-Analyse ermittelten cut-offs von 6 ng/ml. Hiervon entwickelten 8 (28%) Patienten Komplikationen. Von 97 Patienten mit einem normwertigen H-FABP-Wert kam es in einem Fall zu Komplikationen (negativer prädiktiver Wert 0,99; p<0,001). Damit lag bei einem erhöhten H-FABP-Spiegel ein 36,6-fach erhöhtes Komplikationsrisiko vor. Die etablierten Marker Troponin T und NT-proBNP waren hingegen nicht mit dem vermehrten Auftreten von Komplikationen assoziiert. Die Kombination von H-FABP mit einer Tachykardie erscheint ein äußerst n¨¹tzlicher und praktikabler Prognosemarker f¨¹r Komplikationen zu sein (OR 33,4; p<0,001). H-FABP zeigte sich außerdem als ein signifikanter Parameter f¨¹r ein verk¨¹rztes Langzeit¨¹berleben (HR 4,5; p<0,001). Die Ergebnisse dieser Studie deuten darauf hin, dass H-FABP ein n¨¹tzlicher Biomarker in der Risikostratifizierung hämodynamisch stabiler Patienten mit einer akuten Lungenarterienembolie ist.

The desert ant Cataglyphis fortis is one of the hyperthermophilic species of Cataglyphis. It lives in the Sahara desert and forages during the hottest hours of the day when it can get up to 70˚C in the sand. The body temperature of the ant during the foraging runs can reach a maximum of 55˚C. Since C.fortis is one of few eukaryotic hyperthermophilic species, its proteins probably have a high thermostability. Investigating the thermostability can give valuable information about the principles of protein folding and stability in hyperthermophiles.Fatty acid binding proteins (FABPs) have an important role in the cell taking up and transporting fatty acids and regulating metabolic and inflammatory pathways. FABPs have been extensively studied and structures from several species have been determined. The determined structures of all FABPs are very similar why thermostability studies of FABP from C.fortis are highly relevant.Fluorescence spectroscopy is an easy and fast method to measure intrinsic protein fluorescence. Tryptophans were genetically introduced into three different positions in FABP to be used as environmental sensitive probes. Complementing the measurement results with a model of the 3D structure of FABP from C.fortis gave additional information about the ligand binding.The (local) thermostability of the mutants can be detected by shift in wavelength maximum during temperature ramping experiments. All mutants are stabilised in the presence of fatty acids. The mutant with tryptophan positioned closest to the supposed ligand binding residues (Y11W) is most affected. The mutant with tryptophan situated farthest from the supposed binding residues (Y52W) shows a stabilisation of Tm less evident than for Y11W. Thus, the structural changes following fatty acid binding are more obvious in the environment close to the binding site.However, the third mutant C87W shows no significant stabilisation although positioned closer to the fatty acid binding site than Y52. This is probably due to the size difference between the original and introduced amino acid in the mutation. Since the high value of the starting λmax for C87W implies that C87W is quite exposed to the aqueous solvent, the residue is likely to not have subsumed in the protein tertiary structure.Further, the myristic acid stabilise the melting temperature of all the mutants while octanoic acid only has a local effect of Y11W increasing the cooperativity. This implies different binding properties and that myristic acid stabilise the entire protein while octanoic acid only has a local stabilisation effect around the ligand binding site.

Liver fatty acid binding protein has been reported to possess antioxidant properties in the liver. The aim of this study was to investigate the effect of this protein in a nonalcoholic fatty liver disease (NAFLD) cell culture model. Rat hepatoma cells were treated with an oleate:palmitate (2:1) mixture for either 1 and 2 days, or further treated with 500 µM clofibrate to induce L-FABP expression. Intracellular lipid accumulation was quantitated by Nile Red. Lipotoxicity was determined using the WST-1 assay. Dichlorofluorescein (DCF) was utilized to assess intracellular reactive oxidative species (ROS) level. Measurement of lipotoxicity showed statistical decreases in cell viability as lipid concentrations increased in a dose-dependent manner. NAFLD cell cultures showed characteristic cellular damage from increased ROS levels in fatty acid treated cells. All groups treated with clofibrate showed statistically increased intracellular L-FABP levels and reduced ROS levels. The results lead to the conclusion that clofibrate induces L-FABP expression and in this manner suppresses hepatocellular ROS generation.

Liver fatty acid binding protein has been reported to possess antioxidant properties in the liver. The aim of this study was to investigate the effect of this protein in a nonalcoholic fatty liver disease (NAFLD) cell culture model. Rat hepatoma cells were treated with an oleate:palmitate (2:1) mixture for either 1 and 2 days, or further treated with 500 µM clofibrate to induce L-FABP expression. Intracellular lipid accumulation was quantitated by Nile Red. Lipotoxicity was determined using the WST-1 assay. Dichlorofluorescein (DCF) was utilized to assess intracellular reactive oxidative species (ROS) level. Measurement of lipotoxicity showed statistical decreases in cell viability as lipid concentrations increased in a dose-dependent manner. NAFLD cell cultures showed characteristic cellular damage from increased ROS levels in fatty acid treated cells. All groups treated with clofibrate showed statistically increased intracellular L-FABP levels and reduced ROS levels. The results lead to the conclusion that clofibrate induces L-FABP expression and in this manner suppresses hepatocellular ROS generation.

L'intestin grêle à un rôle vital, il est impliqué dans de multiples fonctions : absorptive, endocrinienne, lymphoïde, barrière, et dans le métabolisme de l'arginine. L'exploration du grêle est difficile chez les patients graves hospitalisés en réanimation, alors même que ces patients sont à risque d'avoir une dysfonction intestinale aiguë du fait de la sensibilité du grêle à l'ischémie. La découverte et la validation de biomarqueurs entérocytaires, l'un de fonction, la citrullinémie, l'autre de lyse entérocytaire, l'intestinal fatty acid-binding protein (I-FABP), pourrait permettre de mieux évaluer l'intégrité et la fonctionnalité du grêle chez les patients graves. Les objectifs de cette thèse étaient de décrire la prévalence des anomalies entérocytaires chez des patients graves, de comprendre les mécanismes enjeu dans l'atteinte entérocytaire aigiie, d'évaluer la valeur pronostique de ces biomarqueurs, et d'évoquer des perspectives. Entre 2007 et 2013, nous avons mesuré la citrullinémie et/ou l'I-FABP chez des patients hospitalisés au CHU de Besançon ayant un risque théorique de souffrance entérocytaire : patients admis en réanimation, patients nécessitant un pontage aortocoronarien, patients nécessitant une anesthésie générale. Des anomalies de biomarqueurs entérocytaires étaient présentes chez plus de la moitié des patients hospitalisés en réanimation dès leur admission. Il s'agissait d'une citrullinémie basse et/ou d'une concentration plasmatique élevée d'I-FABP. La synthèse des résultats obtenus fait apparaître qu'une cytolyse entérocytaire précoce et courte est fréquente chez le patient admis en réanimation (élévation de la concentration plasmatique d'I-FABP), puis diminue avec la stabilisation clinique des patients. Ce pic initial d'I-FABP semble suivi d'une diminution de la citrullinémie au cours des 48 premières heures d'hospitalisation en réanimation. D'autre part, le sepsis, qu'il soit ou non d'origine digestive, est associé à une diminution de la citrullinémie, ce qui suggère une entéropathie septique indépendante du champ nosologique de la destruction entérocytaire ischémique. Le couple de biomarqueurs, l'un de lyse cellulaire (I-FABP), l'autre de fonction cellulaire (citrulline), semble permettre de caractériser des altérations entérocytaires aiguës du patient grave, en estimant les degrés de destruction ou de dysfonction entérocytaire, et leurs évolutions. Nos études ont mis en évidence une surmortalité chez les patients qui ont une citrullinémie basse et/ou une concentration plasmatique d'I-FABP élevée. La valeur pronostique de l'atteinte entérocytaire nécessite toutefois d'être confirmée par d'autre groupes. Si la compréhension des modifications des valeurs de ces biomarqueurs entérocytaires progresse, avec les limites d'interprétation de leurs variations, et si leur dosage devient plus rapide du fait d'une automatisation de la technique permettant un rendu rapide de résultat, ils pourraient devenir un outil de diagnostic et d'adaptation rapide de la prise en charge thérapeutique des patients graves. / The small bowel is a complex organ involved in numerous vital functions. The small bowel dysfunction is suspected to be the cornerstone of the development of multiple organ dysfunction syndrome in critically ill patients. The development of enterocyte biomarkers is promising for evaluating the small bowel dysfunction and damage in the most severe patients. Plasma citrulline concentration is a validated marker of small bowel function, reflecting the functional enterocyte mass. Plasma citrulline concentration is frequently decreased in critically ill patients, and a decreased plasma citrulline concentration at ICU admission is associated with elevated plasma CRP concentration, and with increased 28-day mortality. Intestinal fatty acid binding protein (I-FABP) is a small cytosolic protein that is released in extracellular space in case of enterocyte destruction. In critically ill patients plasma I-FABP concentration is increased in about one half of patients at ICU admission. Increased plasma I-FABP concentration is associated with shock state, and with elevated catecholamine dose. Both biomarkers are probably complementary and may help the clinician to identify patients presenting enterocyte damage. Further studies are needed to improve the interpretation of plasma citrulline concentration which is probably complex in critically ill patients.