Dissertations / Theses on the topic 'OGTT'

The regulation of glucose metabolism, in healthy subjects, is based on a complex control system which aims to maintain plasma glucose concentrations within a narrow range (70÷180 mg/dl). Insulin, a hormone secreted by pancreatic beta-cells, is fundamental in maintaining glucose homeostasis, by reducing liver glucose production, while promoting its utilization by the insulin-dependent organs. The inability of beta-cells to adequately secrete insulin creates metabolic disorders which can result in glucose intolerance and even diabetes mellitus. There are two different kinds of diabetes: type 1 diabetes (T1DM), characterized by a total inability of pancreatic beta-cells to secrete insulin, and type 2 diabetes (T2DM), in which, because of insulin resistance, tissues are unable to appropriately utilize glucose, and insulin secretion is unable to compensate for this defect. Given the increasing prevalence of diabetes, a complete understanding of all the mechanisms involved in the glucose regulation system is essential. The liver is a fundamental organ in glucose regulation, since it is also responsible for circulating insulin levels by extracting about 50% of insulin appearing in the portal circulation, with every passage through it. A quantitative estimation of hepatic insulin extraction (HE), both in basal and dynamic physiological conditions (such as after an oral glucose load) is therefore a key aspect for a systematic description of glucose metabolism. Since a direct measurement of HE is very invasive, requiring the insertion of catheters into the portal and hepatic veins, indirect methods employing mathematical models are used. Such models require measurement of plasma concentrations and knowledge of the kinetics of C-peptide, and insulin secretion and clearance. This is facilitated by the fact that insulin and C-peptide are secreted in a 1:1 ratio by the beta-cells, and that the liver extracts insulin, but not C-peptide. The first model available in the literature for assessing HE was developed by Toffolo et al. and describes HE during an insulin modified intravenous glucose tolerance test (IM-IVGTT); this model estimates the insulin secretion rate (ISR) and the insulin delivery rate (IDR) from C-peptide and insulin concentrations, respectively. HE is subsequently derived from these two fluxes. More recently, Campioni et al. proposed a model to estimate HE after meal ingestion. In this case HE is described as a piecewise linear function, with a fixed number of breakpoints, which are the model parameters to be estimated. The main limitation of this approach is that, although allowing a reconstruction of the HE profile, it does not provide a mechanistic relationship between the involved variables, and thus the resulting model parameters do not have an easy physiological interpretation. Moreover, model structure makes the parameter identification vulnerable to noise, since the HE profile may vary rapidly to fit fluctuations in peripheral insulin concentrations. The aim of this work is to overcome the disadvantages of the available HE description by proposing a new physiological model of insulin kinetics and extraction. The best model is selected from seven, including an increasing number of compartments and different mechanistic descriptions of HE, each taking into account the influence of one or more modifiers, such as plasma glucose and insulin concentrations. In fact, during an oral test, one observes that, while glucose and insulin concentrations rise, the HE time course decreases in the meantime. These models are tested against data of a frequently sampled mixed meal (21 plasma samples) measured in 204 healthy subjects. The best model was selected according to standard criteria (ability to describe the data, precision of parameter estimates, model parsimony). Such a model describes insulin kinetics with three compartments, and HE as a function of plasma glucose concentration. One of the peculiarities of this model is to provide an index of HE sensitivity to glucose (SGHE), besides total (HEtot) and basal (HEb) HE indexes, already adopted in the literature. Moreover, the new model performs well even in data sets with less frequent sampling (11 samples). The new model was then applied to three further databases, involving subjects with different degrees of glucose tolerance, studied with a standard mixed meal or the oral glucose tolerance test (OGTT). The first data set is composed of 62 prediabetic subjects (including healthy, glucose intolerant subjects, and subjects with impaired fasting glucose), who underwent a triple tracer mixed meal and an OGTT. The model was able to describe data during both the tests, and HE indexes are shown to correlate with the degree of dysfunction in glucose metabolism. The second data set consists of 11 healthy and 14 T2DM subjects, matched for age, weight and body mass index (BMI), who underwent a mixed meal test with the triple tracer technique. Also in this case, the new model predicts the data, and the estimated HE indexes (HEb, HEtot, SGHE) differ significantly between the two groups. The last database is composed of 14 subjects with T2DM who were treated with vildagliptin or placebo before the meal; moreover, at t = 300 min, 0.02 unit/kg insulin was administered intravenously (over a 5-min period), thus allowing a better estimation of insulin kinetics. In this case the model was used in two different ways: at first, analyzing all the available plasma samples, then, neglecting the insulin infusion and just considering the former part of the test. Interestingly, the model provided a good correlation among the HE parameters in these two different occasions. In summary, we have developed a model of insulin kinetics which contains a new physiological description of HE. This model allows a good prediction of the available data during meals and OGTT in all the spectrum of glucose tolerance (healthy, intolerant and T2DM), also providing a powerful new index of HE sensitivity to glucose.
La regolazione del metabolismo del glucosio, in soggetti sani, si basa su un complesso sistema di controllo, che mira a mantenerne la concentrazione plasmatica in un range limitato (70÷180 mg/dl). L'insulina, un ormone secreto dalle beta-cellule pancreatiche, ha un ruolo fondamentale nell'omeostasi del glucosio, riducendone la produzione epatica, e stimolandone l'utilizzazione da parte degli organi insulino-dipendenti. L'incapacitá da parte delle beta-cellule di secernere adeguatamente l'insulina puó creare problemi metabolici, che possono anche provocare uno stato di intolleranza al glucosio, o addirittura il diabete mellito. Esistono due diversi tipi di diabete: il diabete di tipo 1 (T1DM), caratterizzato da una totale impossibilitá di secernere insulina da parte delle beta-cellule pancreatiche, e il tipo 2 (T2DM), in cui, a causa dell'insulino-resistenza, i tessuti non riescono a utilizzare adeguatamente il glucosio, e la secrezione insulinica è insufficiente per compensare questo difetto. Data la crescente diffusione del diabete, comprendere tutti i meccanismi coinvolti nel sistema di regolazione del glucosio è molto importante. Il fegato è un organo fondamentale nella regolazione del glucosio, poichè è anche responsabile dei livelli di insulina plasmatica, estraendone circa il 50% dalla circolazione portale, ad ogni passaggio attraverso di esso. La quantificazione dell'estrazione insulinica epatica (HE), sia in condizioni basali che in condizioni dinamiche (come per esempio dopo un carico orale di glucosio), è quindi fondamentale per descrivere il metabolismo del glucosio. Dato che una misura diretta di HE è molto invasiva, richiedendo l'inserzione di cateteri nella vena porta e epatica, si preferisce utilizzare metodi indiretti, basati sui modelli matematici. Tali modelli richiedono misure delle concentrazioni plasmatiche, e la conoscenza della cinetica del C-peptide, della secrezione e della degradazione dell'insulina. È infatti noto che insulina e C-peptide sono secreti in maniera equimolare dalle beta-cellule pancreatiche, ma soltanto l'insulina viene poi estratta dal fegato. Il primo modello disponibile in letteratura per descrivere HE è stato sviluppato da Toffolo et al., e descrive HE durante un insulin-modified intravenous glucose tolerance test (IM-IVGTT); questo modello fornisce una stima della secrezione insulinica (ISR) e della velocitá di comparsa dell'insulina nel plasma (IDR), rispettivamente dalle concentrazioni di C-peptide e insulina. HE viene quindi calcolata da questi due flussi. Piú recentemente, Campioni et al. hanno proposto un modello di stima di HE durante pasto. In questo caso HE è descritta come una funzione lineare a tratti, con un numero prefissato di punti, che sono i parametri stimati dal modello. La limitazione principale di questo approccio è che, benchè il profilo di HE venga ricostruito, non è fornita una relazione meccanicistica tra le variabili coinvolte, e quindi i parametri del modello non hanno un'immediata interpretazione fisiologica. Inoltre, la struttura del modello rende l'identificazione parametrica sensibile al rumore, poichè il profilo di HE puó essere soggetto a rapide variazioni a seguito delle fluttuazioni della concentrazione di insula periferica. Lo scopo di questo lavoro è quindi di superare gli svantaggi della descrizione di HE attualmente disponibile, proponendo un nuovo modello fisiologico della cinetica e estrazione dell'insulina. Il modello migliore viene selezionato tra sette nuovi modelli, che includono un numero di compartimenti crescente, e diverse descrizioni fisiologiche di HE, ciascuna contenente l'influenza di uno o piú controlli, come le concentrazioni plasmatiche di glucosio e insulina. Infatti, durante un test orale è possibile osservare che, mentre le concentrazioni di glucosio e insulina salgono, il profilo temporale di HE decresce. Questi modelli sono stati testati in 204 soggetti sani studiati con un pasto misto e campionato frequentemente (21 campioni). Il modello migliore è quindi stato selezionato in base a criteri standard (abilitá di predizione dei dati, precisione delle stime parametriche, parsimonia). Tale modello risulta comprendere una descrizione della cinetica dell'insulina a tre compartimenti, dove HE è funzione della concentrazione di glucosio. Una delle peculiarietá del modello è la possibilitá di ottenere un indice di sensibilitá di HE al glucosio (SGHE), oltre agli usuali indici basale (HEb) e totale (HEtot) di HE, giá presenti in letteratura. Inoltre, il nuovo modello fornisce buone performance anche in dati raccolti con un campionamento standard, quindi meno frequente (11 campioni). Il modello selezionato è stato quindi utilizzato in altri tre diversi database, costituiti da soggetti con vari gradi di tolleranza al glucosio, studiati durante un pasto misto standard, o un test orale di tolleranza al glucosio (OGTT). Il primo data set impiegato è composto da 62 soggetti prediabetici (ovvero sani, intolleranti al glucosio, e soggetti con ridotta glicemia a digiuno), sottoposti sia a un pasto misto con triplo tracciante, sia a un OGTT. Il modello si è dimostrato in grado di descrivere i dati adeguatamente durante entrambi i test, e gli indici di HE mostrano una correlazione con il grado di disfunzione nel metabolismo del glucosio. Il secondo data set consiste di 11 soggetti sani e 14 T2DM, di simile etá, peso e indice di massa corporea (BMI), sottoposti a pasto misto con triplo tracciante. Anche in questo caso il nuovo modello è in grado di predire i dati, e gli indici di HE (HEb, HEtot, SGHE) risultano significativamente diversi nei due gruppi. L'ultimo database è costituito da 14 soggetti T2DM, trattati sia con vildagliptin che con placebo prima del pasto; inoltre in t = 300 min, sono state somministrate 0.02 unitá/kg di insulina per via endovenosa (in un periodo di 5 minuti), permettendo quindi una migliore stima della cinetica dell'insulina. In questo caso il modello è stato usato in due modi differenti: prima analizzando tutti i campioni plasmatici a disposizione, quindi, successivamente, trascurando l'infusione di insulina, e considerando solo la parte iniziale del test. Un risultato interessante riguarda il fatto che il modello fornisce una buona correlazione tra i parametri di HE, calcolati nelle due diverse identificazioni. Quindi, riassumendo, è stato sviluppato un modello della cinetica dell'insulina, contenente una nuova descrizione fisiologica di HE. Questo modello permette una buona predizione dei dati disponibili durante pasto e OGTT, in tutto lo spettro di tolleranza al glucosio (soggetti sani, intolleranti e T2DM), fornendo inoltre un nuovo indice di sensibilitá di HE al glucosio.

Résumé : L’aggravation de certaines caractéristiques cliniques des femmes enceintes (âge, poids) et l’augmentation de la prévalence du diabète gestationnel (DG) poussent à dépister le DG le plus tôt possible pour éviter chez la mère et le fœtus les complications à court et à long terme. Le dépistage du DG est recommandé à 24-28 semaines de grossesse, et le plus souvent un test de tolérance à 50g de glucose (TTG) est réalisé. Pour les femmes qui ont des facteurs de risque, ce test doit être effectué plus précocement, habituellement pendant le premier trimestre de la grossesse. Cette dernière recommandation est peu suivie, d’autant qu’il n’y a pas de consensus international sur le dépistage du DG pendant le premier trimestre de la grossesse. Objectifs. 1) Définir au premier trimestre de la grossesse la valeur de la glycémie du TTG qui prédit le diagnostic de DG à 24-28 semaines avec une sensibilité et une spécificité optimales à l’aide d’une courbe ROC. 2) Déterminer si la glycémie du TTG au premier trimestre est un facteur prédictif indépendant du DG. Méthodes. Étude prospective de cohorte. Les facteurs d'inclusion étaient : âge ≥ 18 ans et âge gestationnel entre 6 et 13 semaines après la dernière menstruation. Les TTG ont été effectués à la première visite prénatale. Une deuxième visite était programmée à 24-28 semaines pour faire une hyperglycémie provoquée par voie orale (HGPO) et donc un éventuel diagnostic de DG. Les critères utilisés pour ce diagnostic étaient ceux de l’Association américaine du diabète. Résultats. Les TTG ont été faits à 9,1±2,0 semaines et les HGPO à 26.5±1.1semaines chez 1180 femmes (28,2±4,4 ans, IMC : 25,2±5,5 kg/m[indice supérieur 2]). Un DG a été diagnostiqué chez 100 (8,4%) participantes. La valeur de glycémie du TTG à 5,6 mmol/L a prédit le DG avec une sensibilité de 84,1% et une spécificité de 62,3%, tandis que la valeur prédictive positive était de 0,121 et la valeur prédictive négative de 0,985. Cette valeur de 5,6 était indépendamment associée au DG (OR=2,806, IC 95%: 1,98 à 3,97, p <0,001). Comparé à d'autres facteurs de risque, le TTG était le plus puissant prédicteur indépendant du DG (OR=1,767, IC 95%: 1,52 à 2,05, p <0,001). Conclusions. Au premier trimestre, la valeur glycémie de 5.6 mmol/L du TTG prédit avec une bonne sensibilité et spécificité l’apparition d’un DG à 24-28 semaines. La glycémie du TTG au premier trimestre est le plus puissant prédicteur indépendant de DG.
Abstract : The changes in clinical characteristics of pregnant women and an increase in the prevalence of gestational diabetes mellitus (GDM) warrant the importance of screening as early as possible in order to possibly prevent short and long-term complications in both the mother and fetus. GDM screening is recommended at 24-28 weeks of pregnancy, using a 50g glucose challenge test (GCT) although women with multiple risk factors are expected to be assessed “early” in pregnancy, a recommendation poorly followed. Most importantly, there is no universal agreement currently in place for GDM screening, particularly during the first trimester of pregnancy. Objectives. 1) To define the cut-off value of GCT during the first trimester in order to predict GDM diagnosed at 24-28 weeks of gestation with optimal sensitivity and specificity using ROC curve. 2) To determine if GCT during the first trimester of pregnancy is an independent predictor of GDM diagnosed at 24-28 weeks gestation. Methods. This is a prospective cohort study. Women were recruited at their first prenatal visit. Inclusion factors were: age ≥ 18 years and gestational age between 6 and 13 weeks from their last menstrual period. GCT were performed at the first prenatal visit. The second visit was scheduled at 24-28 weeks for the diagnostic 75g oral glucose tolerance test (OGTT). GDM diagnosis was made in accordance with the American Diabetes Association guidelines. A variety of statistical analysis including multivariate logistic regression models and ROC curve were used to address the aims of the study. Results. Participants (n=1180, age: 28.2±4.4 years, BMI: 25.2±5.5 kg/m[superscript 2]) underwent GCT at 9.1±2.0 weeks and OGTT at 26.5±1.1 weeks of gestation. GDM was diagnosed in 100 (8.4%) women. The cut-off value of 5.6 mmol/L predicted GDM with 84.1% (75.4-92.7) sensitivity, 62.3% (59.5-65.1) specificity, while the positive predictive value was 0.121 (0.091-0.150) and the negative predictive value was 0.985 (0.975-0.994). This 5.6 value was independently associated with GDM (OR=2.806, 95% CI: 1.98-3.97, p<0.001). Compared to other risk factors, GCT was the strongest independent predictor of GDM (OR=1.767, 95% CI: 1.52-2.05, p<0.001). Conclusions. The cut-off value of 5.6 mmol/L has the optimal sensitivity and specificity for the GCT during the first trimester to predict GDM at 24-28 weeks of gestation according to ADA guidelines. GCT during the first trimester is the strongest independent predictor of GDM at 24-28 weeks of gestation.

INTRODUCTION/OBJECTIVES: Current screening guidelines for pre-diabetes and type 2 diabetes mellitus note that there are discrepancies in diagnosing the disease using the fasting plasma glucose test, oral glucose tolerance test, and HbA1c in high-risk populations. The objective of this study is to compare the effectiveness of screening methods for type 2 diabetes mellitus (T2DM) and pre-diabetes by race/ethnicity and gender. METHODS: Secondary analyses of the National Health and Nutrition Examination Survey (NHANES, 2005-2008) were performed using SPSS 19.0. Screening outcomes were assessed and compared for a sample of n=10,566, NHW, NHB, MA, and Multiracial/other men and women. Analyses included cross tabulations, ANOVA and partial correlations to establish disease prevalence, effectiveness of screenings, and statistical significance. RESULTS: It was found that the HbA1c test is comparable in precision, and is correlated with the FPG for racial and ethnic minorities. The specificities for detecting pre-diabetes using the HbA1c were higher (64-66%) for these groups than by using the standard, FPG screening method (42-49%). There were no strong, significant differences for screening effectiveness for men versus women. DISCUSSION: This study revealed that the HbA1c test might be an effective method for screening for pre-diabetes in racial and ethnic minorities instead of the FPG test alone. Screening in high-risk populations will help delay the onset of T2DM, with increased prevention during the pre-clinical phase.

Les phénomènes métaboliques postprandiaux sont primordiaux à la fois pour caractériser le statut glycémique (normal, prédiabète ou diabète, diagnostiqués au mieux par la charge orale de 75g en glucose (HGPO)) mais aussi en raison de modifications cardiovasculaires induites par la prise alimentaire. Un petit-déjeuner standardisé comprenant 75g de glucides (PDJ) pourrait être une alternative. Les holters glycémiques nous ont permis de montrer une grande concordance de l'amplitude/cinétique de la réponse métabolique (glycémies, index d’insulinorésistance, variabilité glycémique) à l’HGPO vs PDJ chez des sujets obèses sans diabète connu. Le PDJ offrait de bonnes performances diagnostiques. Nous l’avons également utilisé chez des patients obèses présentant une intolérance au glucose(étude ACCES) pour explorer les modifications métaboliques et cardiovasculaires (fonction endothéliale, microcirculation, système nerveux autonome, rigidité artérielle, fonction myocardique) avant et après PDJ selon que les patients avaient été randomisés pour un traitement de 12 semaines par saxagliptine, un inhibiteur de la dipeptidyl 4 (iDPP4), ou son placebo. Nous avons montré que ce traitement permettait la régression de l’intolérance au glucose pour 9 patients sur 10 dans le bras saxagliptine contre 4 sur 9 dans le bras placebo. Nous n’avons pas observé de modifications de nos paramètres cardiovasculaires d’intérêt sous iDPP4 vs placebo, à la fois à jeun et après le PDJ, après une seule prise et après les 12 semaines de traitement. Seule la diminution postprandiale de l’activité vagale était plus soutenue sous saxagliptine. Ces résultats sont en faveur de la sécurité cardiovasculaire de la saxagliptine. Au total, le PDJ standardisé apparaît être un test diagnostique de dysglycémie prometteur car de réalisation simple avec une bonne tolérance. Il peut également être utilisé pour explorer les modifications cardiovasculaires après un repas mixte, de façon plus physiologique qu’après HGPO
Postprandial metabolic changes are essential both to characterize glycemic status (normal, prediabetes or diabetes, best diagnosed by oral 75g glucose tolerance test of (OGTT)) but also because of cardiovascular changes induced by food intake. A standardized breakfast with 75g carbohydrates (SB) could be an alternative. The continuous glucose monitoring allowed us to show a great concordance of the amplitude / kinetics of the metabolic response (glycemia, insulin resistance indexes, glucose variability) after OGTT vs after SB in obese subjects without known diabetes. The SB also offered good diagnostic performance. We also used the SB to explore fasting and postprandial metabolic and cardiovascular changes (endothelial function, microcirculation, autonomic nervous system, arterial stiffness, myocardial function) in obese patients with impaired glucose tolerance (ACCES study), according to randomiziation to a 12-week treatment with Saxagliptin, a dipeptidyl 4 inhibitor (iDPP4), or its placebo. We showed that this treatment allowed the regression of glucose intolerance for 9 patients out of 10 in the saxagliptin arm against 4 out of 9 in the placebo arm. We did not observe any change in our cardiovascular parameters according to iDPP4 vs placebo, both at fasting and after the SB, after a single dose and after 12 weeks of treatment. Only the decrease in postprandial vagal activity was more sustained in the saxagliptin group.These results support the cardiovascular safety of saxagliptin.To conclude, the SB appears to be a promising diagnostic test for dysglycemia, as it is simple and well tolerated. It can also be used to explore cardiovascular changes after a mixed meal,with more physiological modifications than after OGTT

L’amélioration de l’espérance de vie des patients atteints de mucoviscidose est associée à l’apparition de comorbidités dont le diabète est la plus fréquente. L’objectif de ce travail est de mieux déterminer l’impact pronostique du diabète et des valeurs élevées des temps précoces du test d’hyperglycémie provoquée par voie orale (HGPO). Nous présentons et discutons quatre études sur l’évolution naturelle et l’impact pronostique des troubles du métabolisme glucidique associés à la mucoviscidose. Nous présentons ensuite deux études sur l’impact du diabète dans des situations spécifiques : la grossesse et la transplantation pulmonaire. Nous retenons les éléments suivants : les variations du statut glucidique intra-individuel sont très importantes au cours du temps, et nos résultats nuancent l’impact péjoratif du diabète décrit dans la littérature, et celui des valeurs élevées de glycémie et d’insulinémie à 1 heure du test HGPO. Les perspectives de recherche sont de poursuivre l’implémentation de la cohorte GLYCONE pour gagner en effectif et durée de suivi, d’évaluer l’association entre apports alimentaires et trouble du métabolisme glucidique, d’élaborer un processus de décision médicale partagée pour l’instauration d’une insulinothérapie pour les profils de patients stables, de déterminer la consommation de soins et les coûts de prise en charge des patients diabétiques, et d’évaluer les changements épidémiologiques induits par les modulateurs du CFTR sur la prévalence et ll’âge d’apparition du diabète et son pronostic
Life expectancy improvement for cystic fibrosis patients is associated with comorbidities with diabetes being the most common. The objective of this work is to better determine the prognosis impact of diabetes and high values of early times of the oral glucose tolerance test (OGTT). We present and discuss four studies on the natural evolution and prognosis impact of glucose metabolism disorders associated with cystic fibrosis. We then present two studies on the impact of diabetes in specific situations: pregnancy and lung transplantation. The following elements are important: changes in intra-individual glucose status are very important over time, and our results qualify the pejorative impact of diabetes described in the literature, and that of high blood glucose and insulin levels at one hour of the OGTT. The research perspectives are to continue the implementation of the GLYCONE cohort to increase the number of participants and the duration of follow-up, to evaluate the association between dietary intake and carbohydrate metabolism disorder, to develop a shared medical decision-making process for the introduction of insulin therapy for stable patient profiles, to determine the consumption of care and the costs of management of diabetic patients, and evaluate the epidemiological changes induced by CFTR modulators on the prevalence and the age of onset of diabetes and its prognosis

High intensity exercise is believed to yield greater results on health and human performance than moderate intensity exercise. Extensive research indicates that not only do high-intensity interval training (HIT) and sprint interval training (SIT) produce significant improvements in cardiovascular fitness and disease, they may be more effective at improving long-term metabolic function, including insulin sensitivity (Si), by producing more mitochondria. Moreover, compliance rates for HIT and SIT participation are reported to be the same or better than traditional moderate intensity exercise. Because lack of time is often cited as major hindrance to exercise participation, SIT is also seen as a time efficient option to improve health and performance. It does appear, however, that repeated sessions of SIT are needed before overall improvements can be measured. SIT protocols employing maximal 30 sec sprints with ~5 min rest [a 1:9 work-to-rest ratio (W:R)], have garnered much of the research focus, while those using minimal rest periods, like Tabata which uses 20 sec sprints and 10 sec rest (2:1 W:R), have been ignored. This may omit a possible SIT option that could influence acute and chronic adaptations. The role of inflammatory cytokines on Si remains an area of continued research. While endurance exercise is thought to create an overall anti-inflammatory environment that stimulates improvement in Si, SIT is often viewed as pro-inflammatory. However, few studies have provided significant insight into cytokine release following SIT, and none haveexplored its impact on Si. In addition, the impact of W:R on cytokine remains speculative at best. Therefore, the examination of the effect of different sprint protocols of similar total work (kJ) on performance, metabolic function, and inflammatory response may provide valuable insight into these adaptive processes.

The ogt gene encodes an O\(^6\)-alkylguanine DNA alkyltransferase, which is reported to repair DNA against methylation damage caused by reactive nitrogen species, which are generated during an anaerobic respiration in a presence of nitrate. This study examined transcription activation by NarL at the ogt promoter using biochemical techniques with various semi-synthetic ogt promoters. The ogt promoter has two crucial DNA sites for NarL dimers at positions -78.5 and -45.5 relative to the transcript start site. An interaction between NarL and αCTD was investigated using site-specific mutagenesis. Locations and orientations of NarL and RNA polymerase in the transcript initiation complex were also confirmed in this study. It was found that NarL, at position 45.5, is located on the different DNA helical face from αCTD, which binds to the minor groove immediately upstream the -35 element. This unrecognized promoter architecture allows residue 273 of αCTD to interact with residue 178 of NarL.

L'hépatite C est causée par le virus de l'hépatite C (HCV) qui est responsable de maladies chroniques du foie et l’une des principales causes de développement du carcinome hépatocellulaire (CHC). L'infection des hépatocytes humains par le HCV est un processus en plusieurs étapes impliquant des facteurs viraux et des facteurs de l'hôte. Les microARN (miR) sont de petits ARN non codants qui régulent l'expression des gènes au niveau post-transcriptionnel. En utilisant un criblage génomique de miR, nous avons identifié miR-501-3p et miR-619-3p comme modulateurs de l’assemblage et l’export du HCV. Nous avons découvert que ces miR régulent l'expression de l’OGT (UDP-N-acétylglucosamine-peptide N-acétylglucosaminyltransférase) et identifié l'OGT et la O-GlcNAcylation comme régulateurs de la morphogénèse et de l'infectiosité du HCV. De plus, l'expression de l'OGT dans les tissus hépatiques de patients infectés par le HCV était associée à la maladie hépatique et au cancer. En plus de son effet sur la morphogénèse du HCV, l'OGT peut donc jouer un rôle dans les maladies hépatiques induites par le HCV et l'hépatocarcinogenèse
Hepatitis C is caused by the hepatitis C virus (HCV) leading in most subjects to chronic liver infection resulting in chronic hepatitis and progressive liver disease and thereby to development of lethal complications, i.e. cirrhosis and hepatocellular carcinoma (HCC). Infection of human hepatocytes by HCV is a multistep process involving viral and host factors. microRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression. A functional high-throughput miRNA mimic screen identified miR-501-3p and miR-619-3p as novel modulators of HCV assembly/release. We discovered that these miRNAs regulate O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) protein expression and identified OGT and O-GlcNAcylation as regulators of HCV morphogenesis and infectivity. Furthermore, increased OGT expression in patient-derived liver tissue was associated with HCV liver disease and cancer. In addition to its effect on HCV morphogenesis, OGT may thus play a role in HCV-induced liver disease and hepatocarcinogenesis

Plants produce a vast array of secondary metabolites. The phenolic compounds flavonoids are metabolites ubiquitous among plants and are known to aid in processes such as plant reproduction, UV defense, pigmentation and development. In relation to human health, flavonoids have also been found to possess anti-inflammatory, anti-cancer, and anti-oxidant properties. Flavonoids ability to participate in so many interactions is due in part to their subclass variation and further chemical modification. One such modification is glucosylation, where a glucose molecule is added to the flavonoid substrate. The enzymes that catalyze these reactions are known as glucosyltransferases. Citrus paradisi contains a glucosyltransferase that is specific to the 3-O position of flavonols. To further understand the reactions it catalyzes, Cp3-O-GT structure was modeled against a anthocyanidin/flavonol 3 GT found in Vitis vinifera to identify candidate amino acids for mutations. Mutants were then created using site-directed mutagenesis, and one mutant, D344P, was constructed by an aspartate being replaced with a proline based off of the sequence comparison of the original enzymes. Biochemically characterizing the mutant D344P protein will determine whether the mutation has an effect on the regio and/or steriospecificity of Cp3-OGT. An initial screening assay has been performed using radioactive UDP- glucose as a sugar donor. Early results indicated that the mutant D344P has particular affinity for flavonols and for diosometin, a flavone. Kinetic assays are being performed to confirm these results. Studies of time course, enzyme concentration, HPLC product analysis, pH optimum and reaction kinetics will be performed to further complete D344P protein characterization.

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During the development of Grid middleware systems, researchers often employ simulation tools and techniques for validating new concepts and implementations. Simulation tools play a fundamental role on the development of Grid middleware systems since: (a) researchers often do not have access to huge Grid testbed environments, limiting the capacity for evaluating situations that demand high amount of resources; (b) it is difficult to explore in large scale application and resources scenarios involving several users in a repetitive and controlled way, due to the dynamic nature of Grid environments; (c) real Grid applications usually consume great amount of time, ranging from a few hours to even weeks. This work describes OGST, an object-oriented discrete event simulator whose main objective is to assist developers of opportunistic Grid middleware on validating new concepts and implementations. The preliminary motivation for OGST development was to provide a way for evaluating the behavior of scheduling algorithms commonly used on Grid environments under different execution environment conditions and the investigation of adaptive scheduling approaches. It was carefully designed to take into consideration the dynamics of opportunistic Grids, providing a set of features that hasten the development of simulations that takes into consideration the dynamism of the execution environment. The simulator was developed in the context of the InteGrade project, but was designed to allow the simulation of generic opportunistic Grids in order to be applied by other Grid middleware research projects.
Durante o desenvolvimento de sistemas de middleware de grade, pesquisadores freqüentemente empregam técnicas e ferramentas de simulação para valida- ção de novos conceitos e implementações. Ferramentas de simulação têm um papel fundamental no desenvolvimento de sistemas de middleware de grade uma vez que: (a) pesquisadores freqüentemente não têm acesso a grandes ambientes de grade para testes, limitando a capacidade para avaliar situações que demandam por uma grande quantidade de recursos; (b) é difícil explorar cenários com recursos e aplicações em larga escala envolvendo diversos usuários de forma repetitiva e controlada, devido à natureza dinâmica de ambientes de grade; (c) aplicações reais da grade geralmente consomem muito tempo, de poucas horas até mesmo a semanas. Este trabalho descreve o OGST, um simulador de eventos discretos orientado a objetos cujo principal objetivo é auxiliar desenvolvedores de sistemas de middleware de grade oportunista na validação de novos conceitos e implementações. A motivação preliminar para o desenvolvimento do OGST foi prover um caminho para avaliar o comportamento de algoritmos de escalonamento comumente usados em ambientes de grade sob diferentes condições do ambiente de execução e a investigação de abordagens de escalonamento adaptativo. Ele foi cuidadosamente projetado para levar em consideração a dinâmica de grades oportunistas, provendo um conjunto de funcionalidades que agilizam o desenvolvimento de simulações que consideram o dinamismo do ambiente de execução. O simulador foi desenvolvido no contexto do projeto InteGrade, mas foi projetado para permitir a simulação de grades oportunistas de uma maneira em geral com o propósito de ser aplicado a outros projetos de pesquisa envolvendo middleware de grades.

La O-GlcNAcylation est une modification post-traductionnelle réversible qui consiste en l'addition de N-acétylglucosamine (GlcNAc) sur des résidus de sérine et de thréonine de protéines cytoplasmiques, nucléaires et mitochondriales. Elle est contrôlée par deux enzymes : la O-GlcNAc transférase (OGT), qui ajoute le résidu de GlcNAc, et la O-GlcNAcase (OGA), qui l'hydrolyse. La O-GlcNAcylation a été décrite chez Toxoplasma gondii, parasite apicomplexe responsable de la toxoplasmose. D'autre part, les plantes expriment deux OGTs potentielles, SPINDLY (SPY) et SECRET AGENT (SEC), interférant avec divers processus biologiques. In fine, peu de choses avaient été décrites concernant la O-GlcNAcylation et l’OGT chez ces organismes: le but de ma thèse a ainsi consisté à approfondir nos connaissances en ce domaine en nous focalisant sur les parasites, T. gondii et P. falciparum, la plante supérieure A. thaliana et l'algue verte unicellulaire Chlamydomonas reinhardtii. En utilisant différentes approches biochimiques et protéomiques nous avons identifié tout un panel de protéines O-GlcNAcylées chez T. gondii et P. falciparum. En ce qui concerne les modèles végétaux, nous avons identifié une OGT potentielle chez C. reinhardtii mais avons été surpris de constater que les niveaux de O-GlcNAcylation chez cet organisme ainsi que chez la plante A. thaliana étaient très faibles. SPY a récemment été reclassée comme une O-Fucosyltransférase, perdant son statut d’OGT. Ces résultats remettent en question l’existence de la O-GlcNAcylation chez les organismes végétaux et questionnent sur l’apparition et la diversification des OGTs au cours de l’évolution des espèces
O-GlcNAcylation is a dynamic post-translational modification which consists in the addition of N-acetylglucosamine (GlcNAc) onto serine and threonine residues of proteins confined within the cytoplasm, the nucleus and the mitochondrion. O-GlcNAcylation is managed by two enzymes: the O-GlcNAc transferase (OGT) that adds the sugar donor UDP-GlcNAc, and the O-GlcNAcase (OGA) that hydrolyzes it. Occurrence of O-GlcNAcylation was described in Toxoplasma gondii, an apicomplexan parasite causing toxoplasmosis. In the other hand, plants express two distinct OGT, SPINDLY (SPY) and SECRET AGENT (SEC), capable to interfere with a variety of fundamental processes. In fine, relatively few things have been described regarding O-GlcNAcylation and OGT in these organisms: therefore, the aim of my thesis was to go further into our knowledges in this field by focusing on the parasites T. gondii and P. falciparum, the higher plant model Arabidopsis thaliana and the unicellular algae Chlamydomonas reinhardtii. By using a panel of biochemical and proteomics approaches we identified a set of O-GlcNAcylated proteins in T. gondii and P. falciparum, outlining for the very first time the O-GlcNAcome of these parasites. Concerning the plant models, we identified a putative OGT in C. reinhardtii but we were surprised to note that O-GlcNAcylation levels in this organism as for the plant A. thaliana were very low. Recently, it was revealed that SPY is in fact an O-Fucosyltransferase, losing its OGT status. The results question again the occurrence of O-GlcNAcylation in plants and about the apparition and the diversification of OGTs along species evolution

O-GlcNAcylation can be defined as an addition of O-GlcNAc to the Serine/Threonine residue of proteins by the enzymatic activity of O-GlcNAc transferase (OGT) enzyme. O-GlcNAcylation is considered a dynamic and reversible process as another enzyme called O-GlcNAcase (OGA) undoes the action of OGT. OGT being one of the only two enzymes modulating O-GlcNAcylation, has a pivotal role in a wide range of biological processes such as cell signalling and communication, cell division and invasion, transcription and translation of factors/co-factors. The roles played by O-GlcNAcylation and OGT in the pathology of metabolic disorders such as cancer, diabetes and neurodegenerative diseases have been documented in detail over the last few decades. However, its exact molecular mechanism is poorly understood due to the lack of specific tools to perturb its functionality within the biological systems. Therefore, attempts have been made to construct specific chemical tools in the form of OGT inhibitors but the lack of potency and selectivity has slowed down their progress. In the current thesis, focus was set on the construction of novel, specific and potent inhibitors of the targeted OGT enzyme with a view to gain a better understanding of its active role. In addition, an attempt was made to investigate the impact of OGT inhibition on the cytotoxic effects exerted by the anticancer agents. Overall, the outcomes of this work further enhanced our understanding of the intricacies involved in the development of selective and potent OGT inhibitors. Chapter-1 presents the introduction of the OGT enzyme and helps to understand the significance of studying O-GlcNAcylation and the OGT enzyme in addition to outlining the major aims of the thesis. This chapter contains literature review paper specifically focusing on the role of O-GlcNAcylation and OGT enzyme in the stabilization of various oncogenic factors (manuscript-1) Chapter-2 describes the specific details on the materials and methodology used in chapter 3 to 5. Chapter-3 discusses the design and development of novel inhibitors targeting the OGT enzyme using bisubstrate analogues as a template. This strategy employed the identification of suitable donor and acceptor substrates where a flexible aliphatic linker was introduced to replace the negatively charged pyrophosphate group. This was based on the hypothesis that such exchange could improve the cell permeability of inhibitors. Uridine and GlcNAc were selected as donor substrate as UDP-GlcNAc is acting as a donor substrate for OGT enzyme for O-GlcNAcylation. The results demonstrated that one compound from the series, compound-17 (uridine-O-peptide conjugate, manuscript-2) could inhibit the human OGT enzyme with an IC50 value of 29.34 μM. In-silico prediction for physicochemical properties showed that compound-17 has higher value of Log P than previously reported bisubstrate inhibitors (i.e. Goblin-1 and Thiogoblin-1) which means theoretically compound-17 has better cell permeability. Chapter-4 describes another series of bisubstrate inhibitors where uridine-mimetic scaffolds were selected as donor substrate. In this chapter, attempt was made to understand the impact of an exchange of modifiable amino acid (serine) with unmodifiable amino acid (alanine) as well as D-serine to evaluate chirality. One of the candidates from that series of compounds (compound-29, manuscript-3) was also found to have moderate inhibitory activity against OGT with an IC50 value of 134.40 μM. These preliminary findings laid a foundation for the better understanding of the nuances required within the basic scaffold of bisubstrate analogue inhibitors to achieve desired selectivity and potency. Chapter-5 discusses the exploration of a combinatorial approach involving an OGT inhibitor (OSMI-1) with existing chemotherapeutic agents (doxorubicin and docetaxel). OGlcNAcylation and OGT are indispensable for the signal transduction and activation of various oncogenic factors that are thought to be responsible for the reduced sensitivity of prostate cancer cells towards these anticancer agents (manuscript-4). Investigation of concomitant treatment of doxorubicin with an OGT inhibitor demonstrated synergistic effects in terms of cell death in prostate cancer cells (manuscript-5). Chapter-6 presents concluding remarks on the outcomes of the proposed work and future directions.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Pharmacy & Med Sci
Griffith Health
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Currently there is no curative therapy for Chronic Myelogenous Leukemia (CML), and patients must remain on the current prescribed treatment, tyrosine kinase inhibitors (TKI), indefinitely. Although many patients can survive in the chronic phase of the disease under TKI treatment, some patients do progress to the terminal blast crisis phase of the disease. Patients in this terminal phase do not respond to TKI treatment. We evaluated the therapeutic benefit of targeting the oncogene c-MYC in CML, using the CML cell line K562. This was achieved by inhibiting the enzyme O-linked β-N-acetylglucosamine Transferase (OGT), using two indirect inhibitors 2-deoxyglucose and Azaserine, and the direct inhibitor ST078925. Treatment with these inhibitors resulted in decreased half-life of c-MYC protein in K562, reduced c-MYC protein in K562 cells, and reduced K562 cell growth. Together these results suggest that targeting c-MYC through OGT may be a potential therapeutic option for patients with CML.

Si de nombreuses études soutiennent l’existence d’une relation étroite entre les désordres nutritionnels, les modifications épigénétiques et l’étiologie du cancer colorectal (CCR), les mécanismes sous-jacents restent à éclaircir. Les gènes suppresseurs de tumeurs de la famille UNC5H (UNC5A, B, C et D) qui codent des récepteurs membranaires contrôlant la balance survie/apoptose font partie des gènes fréquemment réprimés au cours de la carcinogenèse colique par des mécanismes épigénétiques encore peu compris. Dans le modèle murin de carcinogenèse colique AOM/DSS, nous avons montré que l’expression d’UNC5A, UNC5B et UNC5C était diminuée dans les tumeurs mais exclusivement chez les souris soumises à un régime riche en sucres (HCD) durant toute la durée de l’expérience, reliant ainsi la nutrition à leur perte d’expression dans le CCR. La O-GlcNAcylation est une modification post-traductionnelle ciblant des milliers de protéines nucléocytoplasmiques et mitochondriales intervenant dans divers processus cellulaires fondamentaux parmi lesquels la régulation épigénétique de l’expression génique et dont les niveaux sont augmentés au cours de la carcinogenèse colique. Les niveaux de O-GlcNAcylation dépendent étroitement du nucléotide sucre donneur de la réaction, l’UDP-GlcNAc, qui lui-même est au carrefour de plusieurs métabolismes définissant cette glycosylation comme un senseur nutritionnel. Dans ce contexte, nous avons émis l’hypothèse selon laquelle la O-GlcNAcylation puisse représenter un des relais moléculaires entre la nutrition et la répression des gènes de la famille UNC5H au cours de la carcinogenèse colique. Dans des cellules cancéreuses coliques humaines, par une combinaison d'approches incluant inhibitions pharmacologiques et interférence à l’ARN couplées à des analyses en RT-qPCR et à des tests d’activité promotrice, nous avons montré l’action conjointe de la O-GlcNAcylation et d’EZH2 (la sous-unité catalytique du complexe PRC2 responsable du dépôt de la marque épigénétique répressive H3K27Me3) dans la régulation de l’expression d’UNC5A. Plus précisément, des expériences de CUT&RUN nous ont permis de prouver que la O-GlcNAcylation d’EZH2 permet son recrutement sur le promoteur d’UNC5A afin de réprimer sa transcription. L’ensemble de nos résultats soutiennent donc l'hypothèse selon laquelle la O-GlcNAcylation pourrait représenter une nouvelle connexion entre la nutrition et la régulation épigénétique de gènes suppresseurs de tumeurs clés régissant la cancérisation de la muqueuse colique
Although many studies support a close relationship between nutritional disorders, epigenetic changes and the etiology of colorectal cancer (CRC), the underlying mechanisms remain to be elucidated. The UNC5H tumor suppressor genes (UNC5A, B, C and D) that code for membrane receptors controlling the survival/apoptosis balance are among the genes frequently repressed during colonic carcinogenesis by epigenetic mechanisms that are still poorly understood. In the AOM/DSS mouse model of colonic carcinogenesis, we showed that UNC5A, UNC5B and UNC5C expression was decreased in tumors but exclusively in mice subjected to a High Carbohydrate Diet (HCD) during all the time course of the experiment, thus linking nutrition to their repression in CRC. O-GlcNAcylation is a post-translational modification targeting thousands of nucleocytoplasmic and mitochondrial proteins involved in various fundamental cellular processes including epigenetic regulation of gene expression and whose levels are increased during colonic carcinogenesis. O-GlcNAcylation levels depend of UDP-GlcNAc, the sugar nucleotide donor of the reaction, which itself is at the crossroad of several metabolisms, thus defining this glycosylation as a nutritional sensor. In this context, we hypothesized that O-GlcNAcylation could be one of the molecular relays between nutrition and UNC5H genes repression during colonic carcinogenesis. In human colon cancer cells, by using a combination of pharmacological inhibitions and siRNA approaches coupled to RT-qPCR analyses and promoter activities studies, we showed that O-GlcNAcylation and EZH2 (the catalytic subunit of the PRC2 complex responsible for the deposition of the epigenetic repressive mark H3K27Me3) act jointly to repress UNC5A expression. More precisely, by CUT&RUN experiments, we demonstrated that O-GlcNAcylation of EZH2 allows its recruitment onto the UNC5A promoter to repress its transcription. To conclude, all these results confirm the hypothesis that O-GlcNAcylation could be a new connection between nutrition and epigenetic regulation of tumor suppressor genes governing the cancerization of the colonic mucosa

During my PhD course, I have been involved in studies aiming to identify novel properties of proteins having fundamental roles in the regulation of chromatin modifications and gene transcription. One of the two project was focused on the functional characterization of the products of the catalytic activity of Polycomb Repressive Complex 2 (PRC2). In spite of the wide knowledge about PRC2-dependent trimethylation of Lysine 27 of histone H3 (H3K27me3), the other forms of methylation on H3K27, namely mono (me1) and di-methylation (me2), are still poorly characterized. Using mouse embryonic stem cells (mESC) as model system, we were able to provide an extensive characterization of the functional properties of these methylation forms, highlighting their differential deposition along the genome, their fundamental role in the mechanisms of transcriptional regulation in mESC, and their potential implications during differentiation program. Our data demonstrated that while H3K27me1 was required for efficient transcription of genes and positively correlated with the deposition of H3K36me3, H3K27me2 was broadly deposited and protects the genome from aberrant firing of non specific cell type enhancers. My second project focused on the activity of the O-linked glycosyltransferase Ogt which is the only enzyme capable to catalyze O-linked GlcNAcylation within the cell. Sxc protein, which is the Drosophila orthologue of mammalian Ogt, is essential for Polycomb (PcG) function. Sxc null embryos showed lethal phenotypes at early developmental stages like those observed in PcG null embryos. Starting from this observation we found appealing to investigate whether Sxc function in Drosophila were conserved in mammals. In particular, we were interested in the discovery of possible role for Ogt in the context of PcG recruitment to chromatin and the mechanisms governing transcriptional regulation in mESC. With our study we have identified a novel partnership between Ogt and ten-eleven translocation (TET) proteins, which catalyze hydroxylation of methylated cytosine. We have shown that Tet1 protein recruits Ogt to target genes in proximity of transcription start sites (TSS) rich in cytosine-guanine dinucleotides (CpG). Tet1-Ogt co-localization at target genes correlated with low levels of cytosine modification, suggesting a role in the regulation of CpG island (CpGi) methylation.

The O-linked N-acetylglucosamine post-translational modification (O-GlcNAcylation) is the dynamic and reversible attachment of N-acetylglucosamine to serine and threonine residues of target proteins. It is abundant in metazoa, involving hundreds of proteins linked to a plethora of biological functions with implications in human diseases. The process is catalysed by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), that add and remove the sugar moiety, respectively. Ogt gene knock-out is embryonic lethal in a range of animal models, hampering the study of the biological role of O-GlcNAc. O-GlcNAcylation of nuclear and cytoplasmic proteins has been extensively studied, however little is known about the role of O-GlcNAc in mitochondria. A previous report suggested the presence of a mitochondrial OGT isoform (mOGT) in human cell lines in addition to the well-characterised nucleocytoplasmic one (ncOGT). Since this report more than one decade ago, this putative mOGT has not been studied further. Similarly, hundreds of O-GlcNAcylated nucleocytoplasmic proteins have been identified by high-throughput proteomic screens, whereas only a few mitochondrial proteins have been detected. Nevertheless, several studies suggest that altered O-GlcNAc signalling affects mitochondrial function and morphology, with potential clinical implications. The aim of this thesis work was to study and characterise the biological role of mOGT and determine the mitochondrial O-GlcNAc proteome. Firstly, the presence of mOGT in human cell lines and mouse tissues was investigated. Surprisingly, analysis of genomic sequences indicates that this isoform cannot be expressed at protein level in most of the species analysed, except human and some primates. In fact, the putative mOGT cDNA in most of the genomes analysed contains a stop codon that excludes the presence of such isoform. In addition, mOGT was not detected at protein level in a wide range of human cell lines. Knock-down experiments and Western blot analysis of all the predicted OGT isoforms suggested the expression of only a single OGT isoform. In agreement with this, overexpression of ncOGT in HEK 293 suspension cells led to increased O-GlcNAcylation of mitochondrial proteins, suggesting that ncOGT is necessary and sufficient for the generation of the mitochondrial O-GlcNAc proteome. These data point to a model where O-GlcNAc cycling of mitochondrial proteins occurs in the cytosol, followed by their import into mitochondria. Alternatively, ncOGT itself might be transported into mitochondria where it can take part to O-GlcNAc cycling inside the organelle. In parallel, some advance in determining the O-GlcNAc mitochondrial proteome has been undertaken. Different mitochondrial fractionation protocols, combined with O-GlcNAc enrichment methods have been explored in order to map novel glycosylation sites on mitochondrial proteins. A novel technique established in our research group, employing a bacterial OGA orthologue as a bait to trap O-GlcNAcylated proteins, has been applied to crude mitochondrial fractions allowing the identification of several hits, although site mapping has not been yet achieved. The second chapter describes the work that has been done to improve and optimise novel O-GlcNAc inhibitors previously designed in the laboratory, called goblins. The original objective was to make these molecules cell-permeable and possibly target them to mitochondria in order to inhibit mOGT. Several strategies were explored to deliver the compounds into living cells, including the use of transfection reagents and covalent linkage to linear cell-penetrant peptides. The above methods did not achieve cellular uptake, although recently designed cyclic cell-penetrant peptides, linked to fluorescein, were internalised by HeLa cells with immediate diffuse nucleocytoplasmic staining. These molecules will be linked to goblins aiming to use the inhibitors for cell biology studies. A different approach, based on the permeabilisation of Drosophila embryos, enabled the penetration of goblins into the organisms with consequent reduction of global O-GlcNAc levels. This method allowed the use of these novel bisubstrate inhibitors in vivo for the first time, with potential applications in studying the role of O-GlcNAc in Drosophila development and possibly for future therapeutic purposes after further development of the scaffold.

Background: La mielolesione determina la completa o l’incompleta alterazione delle funzioni sensomotorie sotto il livello neurologico lesionale, nello 0.1% della popolazione mondiale. Ciò provoca importanti limitazioni nelle autonomie quotidiane, compresa la funzione locomotoria: il suo recupero può essere perseguito tramite la riabilitazione fisioterapica convenzionale isolata (overground walking training – OGT) o associata a Robot-assisted gait training – RAGT (es: Lokomat ®). Obiettivo: Questa sintesi è orientata al confronto dell’efficacia del RAGT combinato alla fisioterapia convenzionale, rispetto all’efficacia esclusiva dell’OGT, nel recupero o miglioramento della locomozione in persone con lesione midollare incompleta di qualsiasi origine. Metodi: La Revisione Sistematica è stata redatta secondo la PRISMA statement 2020 checklist. Le banche dati indagate, da Marzo 2021 al 3 Ottobre 2021, sono state: PubMed, PEDro, Cochrane Central Register of Controlled Trials (Cochrane Library) e CINAHL database. Sono stati analizzati Randomized Controlled Trial di persone con mielolesione incompleta, trattate con RAGT e/o OGT. Il rischio di bias è stato valutato attraverso la Scala di PEDro. Risultati: Tra gli 84 RCT individuati, 4 sono risultati includibili nella sintesi, per un totale di 258 partecipanti. Le misure di esito analizzavano la funzione locomotoria attraverso la necessità di assistenza e la forza muscolare degli arti inferiori (scala WISCI-II e LEMS). È il trattamento robotico ad avere stimolato i maggiori miglioramenti nei quattro studi, che, però, non sempre erano statisticamente significativi. Conclusioni: Un protocollo riabilitativo che combina il RAGT alla fisioterapia convenzionale è più efficace, rispetto alla sola OGT, sul miglioramento del cammino in fase sub-acuta, ma sono necessarie ulteriori ricerche, data la ridotta numerosità degli articoli identificati e dei campioni considerati, oltre ad ulteriori valutazioni di fattibilità (setting e spesa).

La O-GlcNAc est une glycosylation dynamique, résidente du cytosol et du noyau, participant à la régulation de processus biologiques tels que le cycle cellulaire et l'embryogenèse. Nos travaux ont porté dans un premier temps sur le contrôle par la O-GlcNAc de la reprise méiotique de l'ovocyte deXenopus laevis, processus analogue à la transition G2/M du cycle cellulaire. Cette transition G2/M est caractérisée par l'activation simultanée du M-phase Promoting Factor, facteur universel d'entrée en phase M et de la voie MAPK-Erk2, et par une augmentation du niveau de O-GlcNAc. Nous avons démontré que cette augmentation de O-GlcNAc était primordiale pour la reprise méiotique ovocytaire puisque l'inhibition de l'OGT, l'enzyme transférant le résidu de GlcNAc, empêche la transition G2/M de l'ovocyte alors que sa surexpression accélère ce phénomène. Nous avons identifié 24 protéines dont le niveau de O-GlcNAc augmente au cours de la reprise méiotique dont des protéines du cytosquelette, la kinase erk2, la phosphatase PP2A, des enzymes de la glycolyse et des protéines ribosomales. Nous avons également entrepris l'étude des variations de O-GlcNAc, d'OGT et d'UDP-GlcNAc au cours de l'ovogenèse et de l'embryogenèse précoce chez Xenopus laevis et avons montré que la dynamique de la O-GlcNAc était complexe tout au long de ces deux processus. Notamment, nous avons observé une diminution drastique et transitoire de la O-GlcNAc au début de la gastrulation suggérant une implication de la glycosylation dans les phénomènes de migration cellulaire caractéristiques de cette étape du développement mettant en place les trois feuillets embryonnaires à l'origine de tous les tissus de l'adulte
O-GlcNAc is a dynamic and reversible post-translational modification found within the cytosol and the nucleus that take part in the regulation of many cellular processes among which cell cycle and embryogenesis. First, our works have focused on the study of O-GlcNAc implication in the control of Xenopus laevis oocyte meiotic resumption, a process analogous to the G2/M transition of the cell cycle. This G2/M transition is characterized by the simultaneous activation of the M-phase Promoting Factor, the universal regulator of the M-Phase entry and of the MAPK-Erk2 pathway but also by a sudden increase in the oocyte O-GlcNAc content. We have demonstrated that this O-GlcNAc increase was essential for meiotic resumption since the inhibition of OGT, the enzyme transferring the O-GlcNAc, prevents the oocyte G2/M transition whereas OGT overexpression accelerates this process. We identified 24 proteins that O-GlcNAc modification increases during meiotic resumption among which cytoskeletal proteins, the kinase erk2, the phosphatase PP2A, several glycolysis enzymes and sorne ribosomal proteins. Second, we have undertaken the study of O-GlcNAc, OGT and UDP-GlcNAc variations during the oogenesis and the early development of Xenopus laevis and we showed that the O-GlcNAc dynamism is intricate from the Xenopus oogenesis to embryogenesis. ln particular, we observed a drastic and transitory O-GlcNAc decrease at the onset of gastrulation, suggesting a role for O-GlcNAc in the regulation of cell migration characteristic of this stage of development since it permits the generation of the three germ layers, precursors ofthe whole adult tissues

La O-GlcNAcylation (O-N-acétylglucosaminylation) est une MPT (modification post-traductionnelle) dynamique et réversible catalysée par un unique couple d’enzymes antagonistes : l’OGT (O-GlcNAc transférase) et l’OGA (O GlcNAcase). Elle est considérée comme un véritable senseur nutritionnel et régule un grand nombre de mécanismes cellulaires fondamentaux. En ciblant des oncoprotéines et des suppresseurs de tumeur, sa dérégulation est associée à la cancérogenèse et la progression tumorale. En revanche, son rôle dans la réponse aux thérapies anti-cancéreuses est très peu étudié. Il a été néanmoins montré récemment que l’hyper-O-GlcNAcylation impacte la réponse de certains cancers à des drogues telles que le tamoxifène, le cisplatine, le bortézomib et le 5-FU (5-fluorouracile). Le 5-FU est la chimiothérapie de référence du CCR (cancer colorectal) et la TS (Thymidylate Synthase) sa cible principale. La surexpression de la TS est un biomarqueur de résistance au 5-FU utilisé en clinique. La TS a été montrée comme étant O-GlcNAcylée mais le rôle de cette MPT n’a pas été élucidé. Il nous est donc paru intéressant d’analyser le « cross-talk » entre O-GlcNAcylation et réponse au 5-FU dans le CCR dans l’hypothèse que la O-GlcNAcylation pourrait impacter la sensibilité au 5-FU en régulant sa cible TS. Un modèle murin in vivo de CCR humains et des cellules coliques non cancéreuses et cancéreuses ont été utilisés pour analyser l’effet du 5-FU sur la O-GlcNAcylation globale des protéines et réciproquement l’impact de la O-GlcNAcylation sur le niveau et l’activité de la TS, et la réponse au 5-FU. Nos données in vitro corroborent nos résultats in vivo et soutiennent que le 5-FU diminue la O-GlcNAcylation globale et que, réciproquement, la O-GlcNAcylation augmente le niveau de TS et sensibilise le CCR au 5-FU. Nous avons déchiffré le mécanisme moléculaire sous-jacent mettant en lumière le rôle de la O-GlcNAcylation dans la stabilisation de la TS et sa protection contre la dégradation protéasomale. Deux sites de O-GlcNAcylation de la TS ont été identifiés : la Thr251 à l’interface de dimérisation de l’enzyme et la Thr306 dans la séquence dégron carboxy-terminale connue pour contrôler sa dégradation. Ensemble nos résultats proposent une nouvelle stratégie thérapeutique combinant le 5-FU à un inhibiteur de l’OGA afin d’améliorer la réponse du CCR à la chimiothérapie à base de 5-FU
O-GlcNAcylation (O-N-acetylglucosaminylation) is a dynamic and reversible PTM (post-translational modification) controlled by a couple of unique antagonist enzymes : OGT (O-GlcNAc transferase) and OGA (O-GlcNAcase). O GlcNAcylation is considered as a nutritional sensor and regulates a plethora of fundamental cellular mechanisms. By targeting oncoproteins and tumor suppressors, dysregulation of O-GlcNAcylation is associated with carcinogenesis and tumor progression. However, its role in the anti-cancer therapy response has been poorly investigated. Recently, hyper O-GlcNAcylation has been shown to impact the response of some cancers to drugs such as tamoxifen, cisplatin, bortezomib and 5-FU (5-fluorouracil). 5-FU is the CRC (colorectal cancer) gold standard chemotherapy and TS (Thymidylate synthase) is its main target. Overexpression of TS is a biomarker of 5-FU resistance already used clinically. TS has been shown to be O-GlcNAcylated but the role of this PTM has not been elucidated. We therefore analyzed the « cross-talk » between O-GlcNAcylation and 5-FU response based on the hypothesis that O-GlcNAcylation impacts the sensitivity to 5-FU by regulating TS. In vivo mouse model of human CRC and colon non-cancerous and cancerous cells were used to analyze the effect of 5 FU on total O-GlcNAcylation and, reciprocally, the impact of O-GlcNAcylation on TS level/activity and 5-FU response. Our in vitro data corroborate our in vivo results and support that 5-FU decreases O-GlcNAcylation and, reciprocally, that O-GlcNAcylation increases TS level and sensitizes CRC to 5-FU. We deciphered the underlying molecular mechanism which highlights the role of O-GlcNAcylation towards TS stability and protection against proteasomal degradation. Two TS O-GlcNAcylated sites have been identified: at Thr251 within the dimerization interface and at Thr306 within the carboxy-terminal degron sequence known to control TS degradation. Together, our results propose a new therapeutic approach combining 5-FU-based therapy with an OGA inhibitor to improve the CRC drug response

Le differenti strategie terapeutiche utilizzate nella gestione dell’acromegalia, possono avere un diverso impatto sull’assetto glicometabolico. Abbiamo pertanto studiato, utilizzando il monitoraggio glicemico continuo (CGM), 41 pazienti acromegalici (20 M/21 F), (56±13.9 anni), a target per la patologia di base da almeno due anni, omogenei per BMI, durata media di malattia, latenza diagnostica media. I pazienti sono stati suddivisi in base al tipo di terapia: 15 analoghi della somatostatina di I (SSA), 4 analogo di II generazione/Pasireotide (PAS), 10 Pegvisomant (PEG), 12 operati con successo. I pazienti sono stati sottoposti a: OGTT (75 gr di glucosio), CGM (72 ore), polisonnografia (PSG), monitoraggio pressorio delle 24 h, ecocardiografia con doppler e colordoppler. RISULTATI 14/41 diagnosi di IFG (8 SSA, 2 PEG, 4 senza terapia); 9/41 diagnosi di IGT (4 SSA, 3 PAS, 2 senza terapia); 4/41 diagnosi di diabete mellito (3 SSA, 1 PEG). • La glicemia media dell’OGTT significativamente più alta rispetto alla glicemia media CGM in toto (p꞊0.0001) e nei sottogruppi di terapia (SSA vs PEG) (p꞊0.0019); • glicemia media OGTT significativamente più alta nel gruppo di pazienti SSA, sia rispetto ai pazienti Peg (p=0,047) sia rispetto alla sola chirurgia (p=0,037); • correlazione diretta picco CGM (p꞊0.0021); • correlazione diretta tra media OGTT e AUC CGM per iperglicemia (p꞊0.0016); • correlazione diretta tra AUC CGM per iperglicemia (p꞊0.05). Non sono emerse differenze statisticamente significative tra presenza, severità di OSAS, tipo di terapia, profilo glico-metabolico DISCUSSIONE I pazienti trattati con SSA presentano un’alterata risposta in termini di secrezione insulinica ad un input particolarmente consistente, quale il carico orale di glucosio. L’analogo della somatostatina infatti, ha un duplice effetto sul metabolismo glicidico: da un lato riducendo i valori del GH riduce l’insulino resistenza e la produzione di glucosio, dall’altro esercita un’azione inibitoria sia verso la secrezione di insulina che di glucagone, compromettendo il controllo glicemico. I dati ottenuti dal nostro studio ci lascerebbero quindi ipotizzare che i valori glicemici a digiuno si mantengano stabili per il miglioramento della sensibilità insulinica, che ne va a controbilanciare la ridotta secrezione. Abbiamo escluso la presenza di ipoglicemie a digiuno, ipotizzata nei pazienti trattati con SSA per un eventuale deficit di controregolazione. Una problematica aperta è quella relativa ai pazienti critici, resistenti a terapia con analoghi classici e pegvisomant. Questi, pur traendo beneficio in termini di controllo biochimico di malattia acromegalica dall’utilizzo di pasireotide, presentano un deterioramento del quadro glicometabolico. L’utilizzo del CGMS potrebbe rappresentare un valido ausilio nella scelta terapeutica dell’aspetto glicometabolico e del suo monitoraggio.

碩士
國立臺灣大學
公共衛生學系
85
ABSTRACT The prevalence and incidence of diabetes mellitus were studied from a cohort WHO had a voluntary health examination in a medical center during 1990 to 1992.A total of 2116 persons( 771 males and 558 females ) were enrolled in our study, and were divided into three groups :diabetes mellitus ( DM );impaired glucose tolerance (IGT); and normal group, by WHO criteria for DM classification.The crude prevalence of DM was 14.5% in this chort (males 15.2%, females13.5%, respectively). The age-adjusted prevalence of DM was 10.8% (males 11.9%, females 9.1%, respectively). There were 184 newly diagnosed DM cases and only 123 of them had a past history of DM. The crude prevalence of IGT was 23% (males 22.3%, females 23.2%, respectively). The age-adjusted prevalence of IGT was 21.4% (males 21.7%, females 20.5%, respectively). The follow-up study of IGT group and normal group was conducted in 1996-1997, which were about 6 years later from the initial work-up. The accumulative incidence of DM for IGT group was 29.4% (males 30.8%, females27.8%, respectively). The accumulative incidence of DM for normal group was 9.3% (males 11.3%, females6.3%, respectively). The risk ratio of IGT group to develop DM(NIDDM) was 3.46(95% C.I.:2.46-5.13). The risk ratio of IGT group to develop DM was 3.37 (95% C.I.: 2.13-5.33) for males, and 3.58 (95% C.I.:3.19-10.37) for females. The univariate and multivariate analyses revealed that there were significant associations between the incidence of DM and risk factors including blood sugar (fasting, 2 hr-postprandial, and OGTT), Hba1c, systolic blood pressure, uric acid ( for females) ,age, obesity, and nativity ( for females).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1991.
Vita. Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves: 396-410).

博士
元智大學
資訊工程學系
107
While occurring enzymatically in biological systems, O-linked glycosylation affects protein folding, localization and trafficking, protein solubility, antigenicity, biological activity, as well as cell-cell interactions on membrane proteins. Catalytic enzymes involve glycotransferases, sugar-transferring enzymes and glycosidases which trim specific monosaccharides (sugars) from precursors to form intermediate structures. Due to the difficulty of experimental identification, a web server OGTSite has been proposed to computationally identify O-GlcNAcylation sites. In addition, due to the very important roles of glycotransferases by recognizing specific protein substrate and catalyzing the attachment of glycan to the target protein, the investigation of the glycotransferases regulations and glycosylated substrate proteins is emerging as a hot topic. However, there is a lack of methods proposed and tools designed to explore the regulatory networks of glycotransferases for glycosylated proteins. Thus, we are motivated to develop a new method to explore the regulatory networks of glycotransferases for glycosylated proteins. In this dissertation, we focus on the integration of glycosylation site databases, identification of glycotransferase-specific glycosylation sites, and systematic discovery of glycotransferase-substrate network in protein glycosylation. We incorporate computational model with protein associations (protein-protein interactions, functional associations, and gene expression profile of OGT, substrates and OGT-interacting proteins in liver hepatocellular carcinoma (LIHC)) to identify the catalytic glycotransferases for each glycoprotein with experimental glycosylated sites. With the highly predictive performance of glycosylation sites, a better understanding of relationships between glycotransferases and substrates will be facilitated and engineered to analyze the therapeutic usefulness. The identified glycotransferase-substrate interactions are used to comprehensively construct the intracellular glycosylation network starting from receptor glycotransferases, with the information of protein-protein interactions and gene expression profile of OGT, substrates and OGT-interacting proteins in LIHC. We present the clustering analysis for OGT-interacting proteins in Liver. The OGT were involved in regulation of insulin receptor signaling pathway and in response to insulin process. The biological processes investigation revealed that the second group of proteins were involved in positive regulation of transcription from RNA polymerase II promoter,which has the same function as OGT. KEGG pathways of the second group of proteins is biased toward the Insulin resistance. Insulin resistance is a condition where cells become resistant to the effects of insulin. It is often found in people with health disorders, including obesity, type 2 diabetes mellitus, non-alcoholic fatty liver disease, and cardiovascular diseases. High glucose and insulin levels may promote liver tumor growth. A case study has demonstrated that the proposed method could be a feasible means of conducting preliminary analyses of protein O-GlcNAcylation based on clustering analysis for OGT-interacting proteins in liver hepatocellular carcinoma (LIHC) .

Les modifications post-traductionnelles telles que la phosphorylation, l’OGlcNAcylation et l’ubiquitination jouent des rôles critiques dans la coordination des fonctions protéiques et par conséquent influencent grandement de nombreux processus cellulaires. Il est à noter que ces modifications sont hautement dynamiques et finement regulées. Par exemple, l’ubiquitination peut être réversible via l’action des déubiquitinases comme le suppresseur de tumeurs BAP1. Parmis les gènes codant pour les déubiquitinases, BAP1 est la plus souvent mutée dans le cancer. Des études récentes ont démontré l’importance des dynamiques de modifications post-traductionnelles dans la régulation du complexe BAP1. En plus, BAP1 forme un complexe multi-protéiques contenant plusieurs régulateurs transcriptionnels comme la protéine polycomb OGT et les facteurs de transcription FOXK1 et FOXK2. OGT est une enzyme unique qui catalyze l’ajout d’un groupement O-GlcNAc sur ses substrats afin d’en moduler l’activité enzymatique, les interactions protéines-protéines et leur localisation cellulaire. Cette modification est aussi liée au métabolisme puisque son substrat donneur, l’UDP-GlcNAc, est dérivé de la voie biosynthétique des hexosamines. Parallèlement, FOXK1/2 ont aussi été démontrés comme étant critiques à des processus métaboliques telles que la myogenèse et l’autophagie. Lors de nos études, nous avons identifié FOXK1 comme un nouveau substrat d’OGT. De plus, les niveaux d’O-GlcNAcylation de FOXK1 fluctuent lors de l’entrée/sortie du cycle cellulaire. En outre, nous avons identifié l’importance de FOXK1 dans l’adipogenèse et observé que l’interaction FOXK1/BAP1 est affectée par le métabolisme cellulaire. En résumé, nos études ont révélé l’importance d’OGT dans la régulation de certaines composantes du complexe BAP1, ce qui aidera à la compréhension de l’effet suppresseur de tumeur de BAP1 ainsi que son mécanisme d'action dans différents processus tel que le remodelage de la chromatine.
Post-translational modifications such as phosphorylation, O-GlcNAcylation and ubiquitination play critical roles in coordinating protein function and are therefore involved in diverse cellular processes. Of relevance here, ubiquitination may be removed by deubiquitinases such as the tumour suppressor BAP1, which represents the most mutated deubiquitinase gene in the human genome. Recent studies have revealed that important and dynamic post-translational modifications regulate several functions of the BAP1 complex. Indeed, BAP1 has been shown to form a multi-protein complex with several transcriptional regulators including the polycomb group protein OGT and the transcription factors FOXK1 and FOXK2. OGT is a unique enzyme that catalyzes the addition of an O-GlcNAc moiety to target proteins, which impacts protein function including enzymatic activity, protein-protein interactions and subcellular localization. This modification is also highly linked to cellular metabolism, as the donor substrate for the reaction, UDP-GlcNAc, is derived from the hexosamine biosynthesis pathway. Similarly, FOXK1 and FOXK2 have been shown to be implicated in metabolic processes such as myogenesis and autophagy. During our studies, we identified FOXK1 but not FOXK2 as a novel substrate of OGT. Further, we found that this OGlcNAcylation is modulated during the entry/exit of cell cycle. We also found that FOXK1 is critical for adipogenesis and that the interaction between FOXK1/BAP1 is compromised during nutrient starvation. Thus, our studies have revealed that OGT selectively modulates and regulates components of the BAP1 complex which may impact different cellular processes, notably chromatin remodelling and could help understanding how BAP1 acts as a tumor suppressor.

L’O-GlcNAcylation est une modification post-traductionnelle qui consiste en l’ajout covalent du N-acetylglucosamine au groupement hydroxyle des sérines et thréonines des protéines nucléaires et cytoplasmiques. Ce type de glycosylation atypique est régulé de manière très dynamique par l’action de l’O-GlcNAc transférase (OGT) et de l’O-GlcNAcase (OGA) qui catalysent et hydrolysent cette modification respectivement. Aujourd’hui, OGT émerge comme un régulateur transcriptionnel et senseur critique du métabolisme où les protéines ciblées par l’O-GlcNAcylation couvrent la presque totalité des voies de signalisation cellulaire. Récemment, des études ont aussi proposé qu’OGT soit impliquée dans la régulation épigénétique par l’O-GlcNAcylation des histones. Dans le but de caractériser le rôle fonctionnel d’OGT dans la régulation épigénétique, nous avons revisité le concept d’O-GlcNAcylation des histones et, de manière surprenante, n’avons pu confirmer cette observation. En fait, nos données indiquent que les outils disponibles pour détecter l’O-GlcNAcylation des histones génèrent des artéfacts. De ce fait, nos travaux supportent plutôt un modèle où la régulation épigénétique médiée par OGT se fait par l’O-GlcNAcylation de régulateurs transcriptionnels recrutés à la chromatine. Parmi ceux-ci, OGT s’associe au complexe suppresseur de tumeurs BAP1. En étudiant le rôle d’OGT dans ce complexe, nous avons identifié le facteur de transcription FOXK1 comme un nouveau substrat d’OGT et démontrons qu’il est régulé par O-GlcNAcylation durant la prolifération cellulaire. Enfin, nous démontrons que FOXK1 est aussi requis pour l’adipogenèse. Ensemble, nos travaux suggèrent un rôle important d’OGT dans la régulation du complexe BAP1.
O-GlcNAcylation is a post-translational modification which consists in the covalent addition of an N-acetylglucosamine sugar to the hydroxyl group of serine and threonine residues of nuclear and cytoplasmic substrates. This atypical glycosylation is regulated in a very dynamic manner through the action of the O-GlcNAc transferase (OGT) and the O-GlcNAcase (OGA) that catalyze and hydrolyze this modification respectively. OGT has emerged as a critical transcriptional regulator and sensor of metabolism whereby proteins targeted by O-GlcNAcylation cover several cell signaling pathways. Recently, studies have also suggested that OGT may be involved in epigenetic regulation through the O-GlcNAcylation of histones. For the purpose of characterizing the functional role of OGT in epigenetic regulation, our group revisited the concept of histone O-GlcNAcylation and surprisingly, our work could not confirm this observation. In fact, our data indicate that the available tools for histone O-GlcNAcylation detection generate artifacts. Consequently, our work rather supports a model whereby OGT-mediated epigenetic regulation is indirectly achieved through O-GlcNAcylation of chromatin-associated transcriptional regulators. Among these, OGT strongly associates with the BAP1 tumor suppressor complex. Thus, by focusing on the role of OGT in this complex, we identified the transcription factor FOXK1 as a novel substrate of OGT and demonstrate that it is regulated throught O-GlcNAcylation during cell proliferation. Finally, we demonstrate that FOXK1 is also required for adipogenesis. Taken together, these data suggest an important role of OGT in regulating the BAP1 complex.

L’ubiquitination, une modification post-traductionnelle importante pour le contrôle de nombreux processus cellulaires, est une réaction réversible. La réaction inverse, nommée déubiquitination est catalysée par les déubiquitinases (DUB). Nous nous sommes intéressés dans nos travaux à étudier l’ubiquitination de l’histone H2A (H2Aub), au niveau des résidus lysines 118 et 119 (K118/K119), une marque épigénétique impliquée dans la régulation de la prolifération cellulaire et la réparation de l’ADN. Le régulateur transcriptionnel BAP1, une déubiquitinase nucléaire, a été initialement identifié pour sa capacité à promouvoir la fonction suppressive de tumeurs de BRCA1. BAP1 forme un complexe multi-protéique avec plusieurs facteurs transcriptionnels et sa fonction principale est la déubiquitination de H2Aub. Plusieurs études ont démontré que BAP1 est un gène suppresseur de tumeurs majeur et qu’il est largement muté et inactivé dans une multitude de cancers. En effet, BAP1 émerge comme étant la DUB la plus mutée au niveau des cancers. Cependant, le ou les mécanismes d’action et de régulation du complexe BAP1 restent très peu connus. Dans cette étude nous nous sommes intéressés à la caractérisation moléculaire et fonctionnelle des partenaires protéiques de BAP1. De manière significative nous avons caractérisé un mécanisme unique de régulation entre deux composants majeurs du complexe BAP1 à savoir, HCF-1 et OGT. En effet, nous avons démontré que HCF-1 est requis pour maintenir le niveau protéique de OGT et que cette dernière est indispensable pour la maturation protéolytique de HCF-1 en promouvant son clivage par O-GlcNAcylation, une signalisation cellulaire nécessaire au bon fonctionnement de HCF-1. Également, nous avons découvert un nouveau mécanisme de régulation de BAP1 par l’ubiquitine ligase atypique UBE2O. En effet, UBE2O agit comme un régulateur négatif de BAP1 puisque l’ubiquitination de ce dernier induit sa séquestration dans le cytoplasme et l’inhibition de sa fonction suppressive de tumeurs. D’autre part nous nous sommes penchés sur la caractérisation de l’association de BAP1 avec deux facteurs de la famille des protéines Polycombes nommés ASXL1 et ASXL2 (ASXL1/2). Nous avons investigué le rôle de BAP1/ASXL1/2, particulièrement dans les mécanismes de déubiquitination et suppression de tumeurs. Nous avons démontré que BAP1 interagit directement iii via son domaine C-terminale avec le même domaine ASXM de ASXL1/2 formant ainsi deux complexes mutuellement exclusifs indispensables pour induire l’activité déubiquitinase de BAP1. De manière significative, ASXM s’associe avec BAP1 pour créer un nouveau domaine composite de liaison à l’ubiquitine. Ces interactions BAP1/ASXL1/2 régulent la progression harmonieuse du cycle cellulaire. De plus, la surexpression de BAP1 et de ASXL2 au niveau des fibroblastes induit la sénescence de manière dépendante de leurs interactions. D’autre part, nous avons identifié des mutations de cancers au niveau de BAP1 le rendant incapable de lier ASXL1/2, d’exercer sa fonction d’autodéubiquitination et de ce fait d’agir comme suppresseur de tumeurs. Ainsi nous avons révélé un lien étroit entre le gène suppresseur de tumeurs BAP1, son activité déubiquitinase et le contrôle de la prolifération cellulaire.
The reverse reaction of ubiquitination, a crucial post-translational modification, is catalyzed by deubiquitinases (DUBs). BAP1 is an ubiquitously expressed nuclear DUB that recently emerged as an important tumor suppressor highly mutated and inactivated in an increasing number of cancers of diverse origins. Both somatic and germline mutations with loss of heterozygosity were observed in tumors, making BAP1 the most mutated DUB in human malignancies. We previously reported that BAP1 is a component of a large multi-protein complex that includes several transcription regulators. The Drosophila homologue of BAP1, Calypso, forms the Polycomb-repressive DUB (PR-DUB) complex with Additional Sex Comb, ASX. This complex catalyzes the deubiquitination of histone H2A, an essential chromatin modification that regulates gene expression. Despite the ever increasing number of findings describing the occurrence of BAP1 mutations in cancers, few studies investigated the mechanisms of action of this DUB as a tumor suppressor. Therefore, the biological function and the mechanism of action and regulation of BAP1 remains largely uncharacterized. In the work described in this thesis, we investigated the roles of BAP1 partners in modulating its catalytic activity and tumor suppressor function. More specifically we discovered a unique mechanism of regulation between two major components of BAP1 complexes, namely HCF-1 and OGT. Indeed, HCF-1 is important for the maintenance of the cellular levels of OGT. OGT, in turn, is required for the proper proteolytic maturation of HCF-1 by promoting its O-GlcNAcylation. This signaling event is required for HCF-1 function as a cell cycle regulator. On the other hand, we deciphered an intricate mechanism of regulation of BAP1 by the atypical E2/E3 ligase, UBE2O. UBE2O, promote the multi-monoubiquitination of BAP1 on its NLS mediating its cytoplasmic sequestration and thus inhibition of its tumor suppressor function. Another aspect of modulation of BAP1 H2Aub catalysis is provided by the association of BAP1 with ASXL1 and ASXL2 (ASXL1/ASXL2), two orthologs of ASX. We investigated the role of BAP1/ASXL1/2, particularly in the mechanisms of deubiquitination and tumor suppression. We have demonstrated that BAP1 interacts directly via its C-terminal domain with the ASXM domain of ASXL1/2, thus forming two mutually exclusive complexes. Significantly, ASXM promote, through assembly with BAP1, the generation of a composite ubiquitin binding domain (CUBI), indispensable for inducing the deubiquitinase activity of BAP1 towards H2Aub. The interactions between BAP1 and ASXL1/2 regulate cell cycle progression. In addition, overexpression of BAP1 or ASXL2 in fibroblasts induces senescence in CTD- and ASXM-dependent manner. We also identified cancer-derived mutation of BAP1 that selectively abolish its interaction with ASXL1 and ASXL2 as well as its H2A deubiquitinase activity. Significantly, this mutant suppressed senescence induced by BAP1 overexpression. Thus we provided a link between the tumor suppressor BAP1, its deubiquitinase activity and the control of cell proliferation.

Tumor-induced immunosuppression can occur by multiple mechanisms, each posing a significant obstacle to immunotherapy. Evidence presented in this dissertation suggests that aberrant cytokine signaling, as a result of altered metabolism of Chronic Lymphocytic Leukemia (CLL) cells, confers a selective advantage for tumor survival and growth. Cells from CLL patients with aggressive disease (as indicated by high-risk cytogenetics) were found to exhibit prolongation in Interferon (IFN)-induced STAT3 phosphorylation, and increased levels of reactive oxygen species (ROS) in these cells reflected these signaling processes. Changes in the relative balance of phospho-STAT3 and phospho-STAT1 levels, in response to combinations of IL-2 + Toll-like receptor (TLR)-7 agonist + phorbol esters, as well as IFN, were associated with the immunosuppressive and immunogenic states of CLL cells. In addition, immunosuppressive leukemic cells were found to express high levels of proteins with O-linked N-acetylglucosamine (O-GlcNAc) modifications, due to increased metabolic activity through the Hexosamine Biosynthetic Pathway (HBP), which caused impaired intracellular signaling responses and affected disease progression. A conclusion of the studies presented here is that the intrinsic immunosuppressive properties of leukemic cells may be overcome by agents such as Resveratrol that target metabolic pathways of these cells.