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Chemotherapy and radiotherapy can induce permanent infertility of the treated patient. For prepubertal boys, no option is currently available for the restauration of fertility after anti-cancer treatments. This represents a major concern that moved several centers all over Europe and North America to develop fertility preservation programs that though experimental, are met with high rates of acceptance by the patients and their parents. As no mature gametes can be found in the testicle before puberty, the only available option is the preservation of spermatogonia, diploid germ cells that are precursors of spermatozoa. Retrieval of immature testicular tissue (ITT) through a small biopsy is a safe and efficient method to preserve germ cells before exposure to gonadotoxic treatments. Amongst the several alternatives currently under-study for fertility restoration, transplantation of frozen-thawed ITT fragments is the most promising option. This procedure has not been performed yet on human subjects, although the xeno-transplantation of such fragments to the nude mice provided a valuable model to study. However, every report on human ITT xeno-transplants described an arrest of spermatogenesis at the pachytene spermatocytes stage and an important germ cells loss. After multiple reports of failed attempts at improving the transplantation procedure through in vivo administration of antioxidants or hormonal supplementation and in vitro culture with supporting molecules before grafting, application of tissue engineering led to promising results in autografts of mice testicular tissue. Indeed, administration of nanoparticles delivering Vascular endothelial growth factor (VEGF) and encapsulation in an alginate matrix improved respectively short-term vascularization and spermatogonial survival. The purpose of our project was to obtain a further improvement of testicular tissue transplantation technique applying tissue engineering. In the first part of the experience, we compare, (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2022

Ovarian tissue cryopreservation and further transplantation has proved its effectiveness in restoring fertility after cancer, with over 200 live births worldwide to date. However, it is limited by massive ischemic damage that occurs shortly after transplantation, causing a significant decline in the ovarian reserve. This project aimed to develop a novel strategy using adipose tissue-derived stem cells (ASCs) for ovarian tissue transplantation, after our preliminary results revealed their impact on hypoxia and revascularization. ASCs are able to protect the ovarian reserve from hypoxic damage, thereby reducing follicle loss. Moreover, their secretome, rich in growth factors, is able to both enhance graft revascularization and reperfusion, and mitigate the primordial follicle burn-out due to abnormal activation soon after transplantation. The present work proved the effecicacy of ASC use in ovarian tissue transplantation, which is now one step closer to clinical application., (MED - Sciences médicales) -- UCL, 2022

In vitro differentiation of human induced pluripotent stem cells (iPSCs) into beta cells represents an important cell source for diabetes research. Here, we fully characterized iPSC-derived beta cell function in vitro and in vivo in humanized mice. Using a 7-stage protocol, human iPSCs were differentiated into islet-like aggregates with a yield of insulin-positive beta cells comparable to that of human islets. The last three stages of differentiation were conducted with two different 3D culture systems, rotating suspension or static microwells. In the latter, homogeneously small-sized islet-like aggregates were obtained, while in rotating suspension size was heterogeneous and aggregates often clumped. In vitro function was assessed by glucose-stimulated insulin secretion, NAD(P)H and calcium fluctuations. Stage 7 aggregates slightly increased insulin release in response to glucose in vitro. Aggregates were transplanted under the kidney capsule of NOD-SCID mice to allow for further in vivo beta cell maturation. In transplanted mice, grafts showed glucose-responsiveness and maintained normoglycemia after streptozotocin injection. In situ kidney perfusion assays showed modulation of human insulin secretion in response to different secretagogues. In conclusion, iPSCs differentiated with equal efficiency into beta cells in microwells compared to rotating suspension, but the former had a higher experimental success rate. In vitro differentiation generated aggregates lacking fully mature beta cell function. In vivo, beta cells acquired the functional characteristics typical of human islets. With this technology an unlimited supply of islet-like organoids can be generated from human iPSCs that will be instrumental to study beta cell biology and dysfunction in diabetes.

Aims/hypothesis: The rapid remission of type 2 diabetes by a diet very low in energy correlates with a marked improvement in glucose-stimulated insulin secretion (GSIS), emphasising the role of beta cell dysfunction in the early stages of the disease. In search of novel mechanisms of beta cell dysfunction after long-term exposure to mild to severe glucotoxic conditions, we extensively characterised the alterations in insulin secretion and upstream coupling events in human islets cultured for 1–3 weeks at ~5, 8, 10 or 20 mmol/l glucose and subsequently stimulated by an acute stepwise increase in glucose concentration. Methods: Human islets from 49 non-diabetic donors (ND-islets) and six type 2 diabetic donors (T2D-islets) were obtained from five isolation centres. After shipment, the islets were precultured for 3–7 days in RPMI medium containing ~5 mmol/l glucose and 10% (vol/vol) heat-inactivated FBS with selective islet picking at each medium renewal. Islets were then cultured for 1–3 weeks in RPMI containing ~5, 8, 10 or 20 mmol/l glucose before measurement of insulin secretion during culture, islet insulin and DNA content, beta cell apoptosis and cytosolic and mitochondrial glutathione redox state, and assessment of dynamic insulin secretion and upstream coupling events during acute stepwise stimulation with glucose [NAD(P)H autofluorescence, ATP/(ATP+ADP) ratio, electrical activity, cytosolic Ca2+ concentration ([Ca2+]c)]. Results: Culture of ND-islets for 1–3 weeks at 8, 10 or 20 vs 5 mmol/l glucose did not significantly increase beta cell apoptosis or oxidative stress but decreased insulin content in a concentration-dependent manner and increased beta cell sensitivity to subsequent acute stimulation with glucose. Islet glucose responsiveness was higher after culture at 8 or 10 vs 5 mmol/l glucose and markedly reduced after culture at 20 vs 5 mmol/l glucose. In addition, the [Ca2+]c and insulin secretion responses to acute stepwise stimulation with glucose were

In vitro differentiation of human induced pluripotent stem cells (iPSCs) into beta cells represents an important cell source for diabetes research. Here, we fully characterized iPSC-derived beta cell function in vitro and in vivo in humanized mice. Using a 7-stage protocol, human iPSCs were differentiated into islet-like aggregates with a yield of insulin-positive beta cells comparable to that of human islets. The last three stages of differentiation were conducted with two different 3D culture systems, rotating suspension or static microwells. In the latter, homogeneously small-sized islet-like aggregates were obtained, while in rotating suspension size was heterogeneous and aggregates often clumped. In vitro function was assessed by glucose-stimulated insulin secretion, NAD(P)H and calcium fluctuations. Stage 7 aggregates slightly increased insulin release in response to glucose in vitro .Aggregates were transplanted under the kidney capsule of NOD-SCID mice to allow for further in vivo beta cell maturation. In transplanted mice, grafts showed glucose-responsiveness and maintained normoglycemia after streptozotocin injection. In situ kidney perfusion assays showed modulation of human insulin secretion in response to different secretagogues. In conclusion, iPSCs differentiated with equal efficiency into beta cells in microwells compared to rotating suspension, but the former had a higher experimental success rate. In vitro differentiation generated aggregates lacking fully mature beta cell function. In vivo ,beta cells acquired the functional characteristics typical of human islets. With this technology an unlimited supply of islet-like organoids can be generated from human iPSCs that will be instrumental to study beta cell biology and dysfunction in diabetes., SCOPUS: ar.j, info:eu-repo/semantics/published

A growing body of evidence indicates that mitochondria play a key role in many disorders as well as in cancer progression and response to treatment. Next to being the main cellular energy generator through respiration, mitochondria are also the major producer of superoxide and other downstream reactive oxygen species in the cell. In this thesis, we aimed to develop an integrated electron paramagnetic resonance (EPR) toolbox enabling the assessment of mitochondrial (dys)function by measuring the oxygen consumption rate and superoxide production in a same cellular or mitochondrial preparation. This EPR toolbox was used for the benefit of two different projects: discovering potential radiosensitizers in anticancer therapy (like statins) and assessing the impact of agents such as Boscalid and Bixafen, which are succinate dehydrogenase inhibitors (SDHI), on the mitochondrial function of human cells., (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2021

Metallothioneins (MTs) are low molecular weight, cysteine-rich, metal-binding proteins whose precise biological roles have not been fully characterized. Existing evidence implicated MTs in heavy metal detoxification, metal ion homeostasis and antioxidant defense. MTs were thus categorized as protective effectors that contribute to cellular homeostasis and survival. This view has, however, been challenged by emerging evidence in different medical fields revealing novel pathophysiological roles of MTs, including inflammatory bowel disease, neurodegenerative disorders, carcinogenesis and diabetes. In the present focused review, we discuss the evidence for the role of MTs in pancreatic beta-cell biology and insulin secretion. We highlight the pattern of specific isoforms of MT gene expression in rodents and human beta-cells. We then discuss the mechanisms involved in the regulation of MTs in islets under physiological and pathological conditions, particularly type 2 diabetes, and analyze the evidence revealing adaptive and negative roles of MTs in beta-cells and the potential mechanisms involved. Finally, we underscore the unsettled questions in the field and propose some future research directions.

The mechanisms underpinning beta cell compensation for obesity-associated insulin resistance and beta cell failure in type 2 diabetes remain poorly understood. We used a large-scale strategy to determine the time-dependent transcriptomic changes in islets of diabetes-prone db/db and diabetes-resistant ob/ob mice at 6 and 16 weeks of age. Differentially expressed genes were subjected to cluster, gene ontology, pathway and gene set enrichment analyses. A distinctive gene expression pattern was observed in 16 wk db/db islets in comparison to the other groups with alterations in transcriptional regulators of islet cell identity, upregulation of glucose/lipid metabolism and various stress response genes, and downregulation of specific amino acid transport and metabolism genes. In contrast, ob/ob islets displayed a coordinated downregulation of metabolic and stress response genes at 6 weeks of age, suggestive of a preemptive reconfiguration in these islets to lower the threshold of metabolic activation in response to increased insulin demand thereby preserving beta cell function and preventing cellular stress. In addition, amino acid transport and metabolism genes were upregulated in ob/ob islets, suggesting an important role of glutamate metabolism in beta cell compensation. Gene set enrichment analysis of differentially expressed genes identified enrichment of binding motifs for transcription factors, FOXO4, NFATC1 and MAZ. siRNA-mediated knockdown of these genes in MIN6 cells altered cell death, insulin secretion and stress gene expression. In conclusion, these data revealed novel gene regulatory networks involved in beta cell compensation and failure. Preemptive metabolic reconfiguration in diabetes-resistant islets may dampen metabolic activation and cellular stress during obesity.

Aims/hypothesis: Nicotinamide nucleotide transhydrogenase (NNT) is involved in mitochondrial NADPH production and its spontaneous inactivating mutation (NntTr [Tr, truncated]) is usually considered to be the main cause of the lower glucose tolerance of C57BL/6J vs C57BL/6N mice. However, the impact of this mutation on glucose tolerance remains disputed. Here, we singled out the impact of NntTr from that of other genetic variants between C57BL/6J and C57BL/6N mice on mitochondrial glutathione redox state (EGSH), glucose-stimulated insulin secretion (GSIS) and glucose tolerance. Methods: Male and female N5BL/6J mice that express wild-type Nnt (NntWT) or NntTr (N5-WT and N5-Tr mice) on the C57BL/6J genetic background were obtained by crossing N5BL/6J NntWT/Tr heterozygous mice. C57BL/6J and C57BL/6N mice were from Janvier Labs. The Nnt genotype was confirmed by PCR and the genetic background by whole genome sequencing of one mouse of each type. Glucose tolerance was assessed by IPGTT, ITT and fasting/refeeding tests. Stimulus-secretion coupling events and GSIS were measured in isolated pancreatic islets. Cytosolic and mitochondrial EGSH were measured using the fluorescent redox probe GRX1-roGFP2 (glutaredoxin 1 fused to redox-sensitive enhanced GFP). Results: The Nnt genotype and genetic background of each type of mouse were confirmed. As reported previously in C57BL/6N vs C57BL/6J islets, the glucose regulation of mitochondrial (but not cytosolic) EGSH and of NAD(P)H autofluorescence was markedly improved in N5-WT vs N5-Tr islets, confirming the role of NNT in mitochondrial redox regulation. However, ex vivo GSIS was only 1.2-1.4-times higher in N5-WT vs N5-Tr islets, while it was 2.4-times larger in C57BL/6N vs N5-WT islets, questioning the role of NNT in GSIS. In vivo, the ITT results did not differ between N5-WT and N5-Tr or C57BL/6N mice. However, the glucose excursion during an IPGTT was only 15-20% lower in female N5-WT mice than in N5-Tr and C57BL/6J mice and r

Pathological remodeling of the myocardium has long been known to involve oxidant signaling, but strategies using systemic antioxidants have generally failed to prevent it. We sought to identify key regulators of oxidant-mediated cardiac hypertrophy amenable to targeted pharmacological therapy. Specific isoforms of the aquaporin water channels have been implicated in oxidant sensing, but their role in heart muscle is unknown. RNA sequencing from human cardiac myocytes revealed that the archetypal AQP1 is a major isoform. AQP1 expression correlates with the severity of hypertrophic remodeling in patients with aortic stenosis. The AQP1 channel was detected at the plasma membrane of human and mouse cardiac myocytes from hypertrophic hearts, where it colocalized with NADPH oxidase-2 and caveolin-3. We show that hydrogen peroxide (H 2 O 2 ), produced extracellularly, is necessary for the hypertrophic response of isolated cardiac myocytes and that AQP1 facilitates the transmembrane transport of H 2 O 2 through its water pore, resulting in activation of oxidant-sensitive kinases in cardiac myocytes. Structural analysis of the amino acid residues lining the water pore of AQP1 supports its permeation by H 2 O 2 .Deletion of Aqp1 or selective blockade of the AQP1 intrasubunit pore inhibited H 2 O 2 transport in mouse and human cells and rescued the myocyte hypertrophy in human induced pluripotent stem cell–derived engineered heart muscle. Treatment of mice with a clinically approved AQP1 inhibitor, Bacopaside, attenuated cardiac hypertrophy. We conclude that cardiac hypertrophy is mediated by the transmembrane transport of H 2 O 2 by the water channel AQP1 and that inhibitors of AQP1 represent new possibilities for treating hypertrophic cardiomyopathies., info:eu-repo/semantics/published

Friedreich ataxia is an autosomal recessive neurodegenerative disease associated with a high diabetes prevalence. No treatment is available to prevent or delay disease progression. Friedreich ataxia is caused by intronic GAA trinucleotide repeat expansions in the frataxin-encoding FXN gene that reduce frataxin expression, impair iron-sulfur cluster biogenesis, cause oxidative stress, and result in mitochondrial dysfunction and apoptosis. Here we examined the metabolic, neuroprotective and frataxin-inducing effects of glucagon-like-peptide 1 (GLP-1) analogs in in vivo and in vitro models and in Friedreich ataxia patients. The GLP-1 analog exenatide improved glucose homeostasis of frataxin-deficient mice through enhanced insulin content and secretion in pancreatic β-cells. Exenatide induced frataxin and iron-sulfur cluster-containing proteins in β-cells and brain, and was protective to sensory neurons in dorsal root ganglia. GLP-1 analogs also induced frataxin expression, reduced oxidative stress and improved mitochondrial function in Friedreich ataxia patients' induced pluripotent stem cell-derived β-cells and sensory neurons. The frataxin-inducing effect of exenatide was confirmed in a pilot trial in Friedreich ataxia patients, showing modest frataxin induction in platelets over a 5-week treatment course. Taken together, GLP-1 analogs improve mitochondrial function in frataxin-deficient cells and induce frataxin expression. Our findings identify incretin receptors as a therapeutic target in Friedreich ataxia., info:eu-repo/semantics/published

AMP-activated protein kinase (AMPK) is a key player in the control of cellular energy homeostasis. AMPK activation is associated with beneficial effects to counteract the metabolic syndrome, and AMPK is now recognized as a potential drug target for the treatment of type 2 diabetes. However, research findings have been controversial. The aim of the thesis was to understand whether genetic deletion of AMP-metabolizing enzymes would be a viable approach to achieve AMPK activation and whether enzyme inhibition could be a pharmacological strategy for the treatment of type 2 diabetes. The first part of the thesis focused on the effects of cytosolic 5`-nucleotidase 1A (NT5C1A) and of cytosolic 5`-nucleotidase II (NT5C2) deletion in incubated skeletal muscles. The second part focused on whether NT5C2 deletion could improve glycaemic control in mice fed normal and high fat diet in comparison to wild-type mice., La protéine kinase activée par l'AMP (AMPK) est impliquée dans le contrôle de l’homéostasie énergétique cellulaire. Son activation a des effets bénéfiques pour contrecarrer le syndrome métabolique et l'AMPK est reconnue comme cible potentielle pour le traitement du diabète de type 2. Cependant, les résultats actuels de la recherche font débat. Cette thèse visait à évaluer si la délétion génétique des enzymes métabolisant l’AMP constitue une approche valable pour activer l’AMPK et si l’inhibition de cette dernière pouvait servir de stratégie pharmacologique pour le traitement du diabète de type 2. La première partie de l’étude s’est concentrée sur les effets liés aux délétions des 5`-nucleotidase 1A (NT5C1A) et 5`-nucleotidase II (NT5C2) cytosoliques dans les muscles squelettiques incubés. La seconde a investigué si la délétion de NT5C2 améliore le contrôle de la glycémie chez des souris soumises à un régime normal ou riche en graisse, en comparaison à des souris de type sauvage., (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2019

Aims/hypothesis: The mechanisms responsible for beta cell compensation in obesity and for beta cell failure in type 2 diabetes are poorly defined. The mRNA levels of several metallothionein (MT) genes are upregulated in islets from individuals with type 2 diabetes, but their role in beta cells is not clear. Here we examined: 1) the temporal changes of islet Mt1 and Mt2 gene expression in mouse models of beta cell compensation and failure; and 2) the role of Mt1 and Mt2 in beta cell function and glucose homeostasis in mice. Methods: Mt1 and Mt2 expression was assessed in islets from: (1) control lean (chow diet-fed) and diet-induced obese (high-fat diet-fed for 6 weeks) mice; (2) mouse models of prediabetes (6-week-old db/db mice) and diabetes (16-week-old db/db mice) and age-matched db/+ (control) mice; and (3) obese non-diabetic ob/ob mice (16-week-old) and age-matched ob/+ (control) mice. MT1E, MT1X and MT2A expression was assessed in islets from humans with and without type 2 diabetes. Mt1-Mt2 double-knockout (KO) mice, transgenic mice overexpressing Mt1 under the control of its natural promoter (Tg-Mt1) and corresponding control mice were also studied. In MIN6 cells, MT1 and MT2 were inhibited by small interfering RNAs. mRNA levels were assessed by real-time RT-PCR, plasma insulin and islet MT levels by ELISA, glucose tolerance by i.p. glucose tolerance tests and fasting 1 h refeeding tests, insulin tolerance by i.p. insulin tolerance tests, insulin secretion by RIA, cytosolic free Ca2+ concentration with Fura-2 leakage resistant (Fura-2 LR), cytosolic free Zn2+ concentration with Fluozin-3, and NAD(P)H by autofluorescence. Results: Mt1 and Mt2 mRNA levels were reduced in islets of murine models of beta cell compensation, whereas they were increased in diabetic db/db mice. In humans, MT1X mRNA levels were significantly upregulated in islets from individuals with type 2 diabetes in comparison with non-diabetic donors, while MT1E and MT2A mRNA levels were unchange

Photodynamic therapy (PDT) is a medical treatment aimed at destroying pathological tissues including (pre-)cancerous lesions using photosensitizers (PS). Exposure of these compounds to light (often from a laser) induces local cytotoxicity following the photochemical production of singlet oxygen and reactive oxygen species. The induction of immunogenic cell death by PDT has recently suggested its potential use in cancer immunotherapy. OR141 is the lead compound of a family of benzophenazine-type PS discovered at UCLouvain. Here, we explored the direct anticancer effects of OR141 and its ability to stimulate the immune system, including through the optimization of a dendritic cell-based antitumor vaccine. This research has demonstrated the possibility of using white light to activate this PS, considerably broadening its spectrum of use. Our work also revealed the value of a submaximal dose of PS to promote the host's immune response and the existence of an optimal timing of PDT delivery when combined with radiotherapy., La thérapie photodynamique (PDT) est un traitement médical visant à détruire des tissus pathologiques dont des lésions (pré-)cancéreuses à l’aide de photosensibilisateurs (PS). L’exposition à la lumière (souvent d’un laser) de ces composés induit une cytotoxicité locale suite à la production photochimique d’oxygène singulet et d’espèces réactives de l’oxygène. L’induction d’une mort cellulaire de type immunogénique par la PDT a récemment suggéré son utilisation potentielle dans l’immunothérapie du cancer. OR141 est le chef de file d’une famille de PS découverts à l’UCLouvain. Nous avons exploré les effets anticancéreux directs d’OR141 et sa capacité à stimuler le système immunitaire, ce compris via l’optimisation d’un vaccin antitumoral à base de cellules dendritiques. Ces recherches ont démontré la possibilité d’utiliser une lumière blanche pour activer ce PS, élargissant considérablement son spectre d’utilisation. Notre travail a également révélé l’intérêt d’une dose submaximale de PS pour favoriser la réponse immunitaire de l’hôte ainsi que l’existence d’un timing optimal de délivrance de la PDT lorsque combinée à la radiothérapie., (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2019