The central nervous system (CNS) melanocortin signaling pathway plays a critical role in the regulation of metabolism. However, the regulatory effects of CNS melanocortin signaling on hepatic lipid metabolism and fatty liver disease have not been well established. Although the activity of the CNS melanocortin system is regulated by metabolic signals, the mechanism for this regulation is not fully understood. Variants of the FTO (fat mass and obesity-associated) gene are associated with obesity and FTO is expressed in the hypothalamic neurons including proopiomelanocortin (POMC) neurons. Therefore, it is hypothesized that hypothalamic FTO plays a role in the regulation of metabolism by mediating the effect of metabolic signals on hypothalamic melanocortinergic neurons, and that impairments in this regulation may cause metabolic impairments including obesity and fatty liver disease. Intracerebroventricular (i.c.v.) treatment with SHU9119, a melanocortin antagonist, increased hepatic lipid accumulation and the expression of genes encoding lipogenic enzymes in lean mice. Conversely, i.c.v. treatment with MTII, a melanocortin agonist, reduced the expression of hepatic lipogenic genes in association with reduction in body weight in ob/ob mice, a mouse model of fatty liver disease. Immunohistochemical analysis demonstrated that Fto is co-expressed in both POMC and agouti-related protein (AgRP) neurons in the mouse hypothalamus. Fto mRNA and protein expression was reduced by fasting and increased by glucose treatment in nutritionally important hypothalamic nuclei. Fasting-induced reduction in hypothalamic Fto expression was observed in both lean wild-type and obese ob/ob mice, while the stimulatory effect of glucose on hypothalamic Fto expression was absent in ob/ob mice. These findings support the hypothesis that central melanocortin signaling regulates hepatic lipid metabolism in part by regulating de novo lipogenesis. Impairments in the central melanocortin signaling lead to the development of hepatic steatosis, while enhanced melanocortin signaling may be beneficial in reversing abnormal hepatic lipid metabolism in fatty liver disease (Poritsanos et al., 2008). These findings also support the hypothesis that Fto is expressed in the hypothalamic melanocortinergic neurons and is regulated by metabolic signals involving changes in CNS glucose availability and/or glucose action. Impairments in this regulation may cause metabolic impairments including obesity and fatty liver disease.

In the last three decades carbon nanomaterials such as fullerenes, carbon nanotubes and graphene have attracted significant attention from the scientific community due to their unique electronic, optical, thermal, mechanical and chemical properties. Among them carbon nanotubes and graphene have been used in numerous applications for future nanoelectronics, biochemical sensors and energy harvesting technologies due to their unique properties including exceptionally high electronic conductivity and mechanical strength. Carbon nanotubes are cylindrical structures and considered to be large mesoscopic molecules with high aspect ratios. Graphene is a single atomic layer of crystalline graphite and prepared by stripping layers off the graphite using Scotch tape. Apart from this scotch tape method, chemical ex-foliation and reduction of graphite oxide produces large amounts of reduced graphene oxide which has similar properties as graphene. This thesis reports on the biosensors made of reduced graphene oxide and single walled carbon nanotubes based on their electronic properties. We also demonstrate the changes in electronic properties of single walled carbon nanotubes due to interactions with dendrimer molecules. Finally, the yielding and flow behaviour of graphene oxide nematic gel are discussed. Chapter 1 gives a general introduction about the preparation and characterization along with the electronic properties of the systems studied in this thesis, namely graphene oxide, reduced graphene oxide and single walled carbon nanotubes. We have also discussed about the experimental techniques such as Raman, UV-visibe and infrared spectroscopy, atomic force and scanning tunneling microscopy and different types of rheometers used in this thesis work. In Chapter 2, we discuss top-gated field effect transistor characteristics of the devices made of reduced graphene oxide monolayer by dielectrophoresis. Raman spectrum of RGO flakes shows a single 2D band at 2687 cm 1, characteristic of a single layer graphene. The two probe current - voltage measurements of RGO flakes, deposited in between the patterned electrodes using a.c. dielectrophoresis show ohmic behavior with a resistance of 37kΩ. The temperature dependence of the resistance (R) of RGO measured between temperatures 305K to 393K yields the temperature coefficient of resistance of -9.5 10 4/K. Ambipolar nature of graphene flakes is observed upto a doping level of 6 1012/cm2 and carrier mobility of 50cm2/V-sec. The source - drain current characteristics shows a tendency of current saturation at high source - drain voltage which is analyzed quantitatively by a diffusive transport model. In Chapter 3, We demonstrate the detection of glucose molecules by using reduced graphene oxide (RGO) and aminophenylboronic acid (APBA) complex with detection limit of 5 nM. APBA functionalized RGO (APBA-RGO) flakes, prepared by stirring the aqueous GO suspension in the presence of APBA molecules at 100◦C, were used as conducting channel in our field effect transistor (FET) devices. The APBA-RGO complex formation was confirmed by atomic force microscopy (AFM), x - ray photoelectron, Raman and UV-visible spectroscopic studies. Detection of glucose molecules was carried out by monitoring the changes in electrical conductance of the APBA-RGO flake in the FET device. FET devices made of non-covelently functionalized APBA-RGO complex (nc-APBA-RGO) exhibited enhanced sensitivity over the devices made of covalently functionalized APBA-RGO complex (c-APBA-RGO). Change in normalized conductance in the FET devices made of nc-APBA-RGO flakes ( 85%) is 4 times more than that of in the devices made of c-APBA-RGO flakes in response to aqueous glucose solution with different concentrations. Specificity of APBA-RGO complex to glucose was proved from the observation of negligible change in electrical conductance of the FET devices made of nc-APBA-RGO complex after exposure to 10 mM lactose solution. Chapter 4 reports unipolar resistive switching in ultrathin films of chemically produced graphene (reduced graphene oxide) and multiwalled carbon nanotubes. The two - terminal devices with yield > 99% are made at room temperature by forming continuous films of graphene of thickness 20 nm on indium tin oxide coated glass electrode, followed by metal (Au or Al) deposition on the lm. These memory devices are non - volatile, rewritable with ON/OFF ratios up to 105 and switching times up to 10 s. The devices made of MWNT films are rewritable with ON/OFF ratios up to 400. The resistive switching mechanism is proposed to be nanogap formation. In the first part of Chapter 5, we study the interactions between SWNT and PETIM dendrimer by measuring the quenching of inherent fluorescence of the dendrimer. Also, the dendrimer - nanotube binding results in the increased electrical resistance of the hole-doped SWNT due to charge transfer interaction between the dendrimer and the nanotube. This charge transfer interaction was further corroborated by observing a shift in frequency of the tangential Raman modes of SWNT. Experimental studies were supplemented by all atom molecular dynamics simulations to provide a microscopic picture of the dendrimer - nanotube complex. The complexation was achieved through charge - transfer and hydrophobic interactions, aided by multitude of oxygen, nitrogen and n-propyl moieties of the dendrimer. We also studied the effect of acidic and neutral pH conditions on the binding affinities. In the second part, we show that SWNT decorated with sugar functionalized PETIM dendrimer is a very sensitive platform to quantitatively detect carbohydrate recognizing proteins, namely, lectins. The changes in electrical conductivity of SWNT in field effect transistor device due to carbohydrate - protein interactions forms the basis of this study. The mannose sugar attached PETIM dendrimers undergo charge - transfer interactions with the SWNT. The changes in the conductance of the dendritic sugar functionalized SWNT after addition of lectins in varying concentrations were found to follow the Langmuir type isotherm, giving the concanavalin A (Con A) - mannose affinity constant to be 8.5 106 M-1. The increase in the device conductance observed after adding 10 nM of Con A is same as after adding 20 µM of a non - specific lectin peanut agglutinin, showing the high specificity of the Con A - mannose interactions. The specificity of sugar-lectin interactions was characterized further by observing significant shifts in Raman modes of the SWNT. Chapter 6 reports the metal to semiconductor transition in metallic single-wall carbon nanotubes (SWNT) due to the wrapping of mannose attached poly (propyl ether imine) dendrimer (DM) molecule. Scanning tunneling spectroscopic (STS) measurements and ionic liquid top gated field effect transistor (FET) characteristics of the nanotube-dendrimer complex gives a band gap of 0.42eV, close to the E11 energy gap between the first van Hove singularities of 1.7nm diameter semiconducting nanotubes. The absence of Breit-Wigner-Fano (BWF) component in G band in the Raman spectrum of the nanotube-dendrimer complex corroborates the semiconductor nature of the tubes after wrapping with the dendrimer molecules. Dendrimer molecule breaks the symmetry in metallic SWNT by wrapping around it through the charge transfer interactions. In the first part of Chapter 7, we demonstrate a rigidity percolation transition and the onset of yield stress in a dilute aqueous dispersion of graphene oxide platelets (aspect ratio 5000) above a critical volume fraction of 3.75x10-4 with a percolation exponent of 2.4 ± 0.1.The viscoelastic moduli of the gel at rest measured as a function of time indicates the absence of structural evolution of the 3D percolated network of disks. However, a shear-induced aging giving rise to a compact jammed state and shear rejuvenation indicating a homogenous flow is observed when a steady shear stress (σ ) is imposed in creep experiments. We construct a shear diagram (σ vs volume fraction ϕ) and the critical stress above which shear rejuvenation occurs is identified as the yield stress σ y of the gel. The minimum steady state shear rate ƴm obtained from creep experiments agrees well with the end of the plateau region in a controlled shear rate flow curve, indicating a shear localization below ƴm. A steady state shear banding in the plateau region of the flow curve observed in particle velocimetry measurements in a couette geometry confirms that the dilute suspensions of GO platelets form a thixotropic yield stress fluid (TYSF). In the second part, we report that the creep experiments on a nematic liquid crystalline suspension of Graphene Oxide platelets which was established recently as a TYSF exhibit two characteristic timescales Tc and Tf marking the onset of yielding, and a final steady state of flow respectively. We show that both Tc and Tf exhibit a power law dependence on the applied stress σ which can be linked to the steady state flow behaviour of a TYSF. The smooth transition from Andrade creep to the onset of flow with ƴ~ t 0.7 at a critical strain ƴc for different applied stresses, is well captured by the master curve for the creep compliance, obtained through a simple scaling of the creep times with either Tc or Tf . We propose that the absence of diverging timescales for onset of flow as σ→ yield stress σy from above, is a characteristic feature of TYSF. The thesis concludes with a summary of the main results and a brief account of the scope of future work described in Chapter 8.

L’homéostasie énergétique se définit comme le maintien de l’équilibre entre les apports et les dépenses d’énergie. La régulation nerveuse de cet équilibre est principalement assurée par l’hypothalamus. Il existe dans cette structure des neurones spécialisés dont l’activité électrique est modifiée par des signaux nerveux, métaboliques et hormonaux.Nous avons travaillé sur la détection du glucose dans cette structure, qui permet l’élaboration d’une réponse adaptée en termes de prise alimentaire et de contrôle du métabolisme. Lors de cette détection, l’utilisation du glucose conduit à la formation d’Espèces Actives de l’Oxygène d’origine mitochondriale (mEAOs) par la chaîne respiratoire mitochondriale (CRM), constituant une signalisation redox indispensable aux réponses physiologiques. De récentes études in vitro (cultures de myoblastes, hépatocytes) ont par ailleurs mis en évidence le rôle de la dynamique mitochondriale, qui contrôle la morphologie des mitochondries par des mécanismes de fission et de fusion, sur la production de mEAOs induite par une hyperglycémie. Cette dernière déclenche la fission des mitochondries de façon concomitante à la production de mEAOs. En revanche, le blocage de la fission empêche la production de mEAOs lors de l’hyperglycémie dans ces cultures. Ces études suggéraient donc que la fission soit déclenchée par l’hyperglycémie et permette alors la production de mEAOs. Mon projet de thèse a consisté à déterminer l’implication de la dynamique mitochondriale dans la signalisation mEAOs lors de la détection hypothalamique du glucose. Nos résultats nous ont permis de mettre en évidence, dans un premier temps, un adressage de la protéine de fission DRP1 à la mitochondrie dans l’hypothalamus lors d’une hyperglycémie cérébrale, évènement nécessaire au déclenchement de la fragmentation des mitochondries. Cette fragmentation est confirmée en imagerie où l’analyse morphologique montre des mitochondries plus petites, plus sphériques et moins allongées que celles des témoins. Dans un deuxième temps, nous avons déterminé l’implication de cette fission mitochondriale dans la détection hypothalamique du glucose. Son importance a pu être évaluée en bloquant la fission des mitochondries par l’inhibition de l’expression de la protéine de fission DRP1 spécifiquement dans le VMH, par interférence ARN. Cette stratégie nous a permis d’obtenir une inhibition de l’expression de DRP1 de près de 80%, 72h après l’injection. Cette inhibition est localisée au VMH et a pour conséquence une élongation des mitochondries qui présente un réseau mitochondrial plus filamenteux. L’étude du phénotype des animaux a mis en évidence une hyperphagie associée à l’inhibition de la fission mitochondriale dans le VMH. Cette hyperphagie n’entraine cependant aucune modification du poids corporel. Ceci suggère une augmentation des dépenses énergétiques chez ces animaux. De plus, ils présentent une perte de sensibilité hypothalamique au glucose qui conduit à un défaut du contrôle nerveux de la sécrétion d’insuline, ainsi qu’à une perte de l’effet satiétogène du glucose lors d’un test de réalimentation. Nous montrons que cette perte de sensibilité au glucose est due à un défaut de production hypothalamique des mEAOs en réponse au glucose, production qui est nécessaire à la signalisation responsable des réponses effectrices. Ce défaut de production de mEAOs est associé à un dysfonctionnement de la CRM. L’ensemble de ce travail permet donc de montrer pour la première fois, in vivo, que la fission mitochondriale est indispensable à la production hypothalamique de mEAOs lors d’une hyperglycémie cérébrale. Cette production est nécessaire au déclenchement du contrôle nerveux permettant d’une part la sécrétion d’insuline et d’autre part le rassasiement induit par le glucose intra-hypothalamique. / Energetic homeostasis results in the balance between energy intake and expenditure. The hypothalamus plays an important role in the regulation of both energetic metabolism and food intake in sensing hormonal and metabolic signals. For instance, changes in hypothalamic glucose level modulate food intake and insulin secretion. We have previously found that 1) increased hypothalamic glucose level triggers production of mitochondrial reactive oxygen species (mROS) from the electron transport chain; 2) hypothalamic mROS production is involved in glucose homeostasis and food intake control. The molecular mechanisms involved in glucose-induced hypothalamic mROS production are still unknown. Mitochondrial dynamics control mitochondrial morphology through fission or fusion mechanisms. Recent in vitro studies have shown that mitochondrial fission is involved in glucose-induced myoblasts and hepatocytes mROS production. The main hypothesis of my thesis was that mitochondrial dynamics were involved in 1) hypothalamic glucose-induced mROS signaling and 2) hypothalamic glucose sensitivity.We first showed in vivo that increased hypothalamic glucose level in response to an intracarotid glucose injection induces recruitment of the mitochondrial fission protein DRP1 at the mitochondria and triggers mitochondrial fragmentation. The second part of my work was to determine whether mitochondrial fission is involved in hypothalamic glucose sensitivity. Therefore, we inhibited DRP1 expression in the ventromedial hypothalamus (VMH) by siRNA injection. 72h post siDRP1 injection, VMH DRP1 expression was decreased by 80%. At this time, we found that increased hypothalamic glucose level failed to increase hypothalamic mROS production. In addition, intracarotid glucose injection-induced insulin secretion was decreased. Finally, VMH glucose injection-induced food intake inhibition was attenuated in siDRP1 treated animals. In a last set of experiments, we found ex vivo by oxygraphy that hypothalamic mROS production is associated with electron transport chain dysfunction. Altogether, our work shows for the first time that mitochondrial fission is involved in mROS dependent hypothalamic glucose sensitivity. Furthermore, this work demonstrates that mitochondrial fission plays a critical role in the regulation of glucose homeostasis and food intake.

L’hypothalamus participe au contrôle de l’homéostasie énergétique en détectant les signaux circulants tels que le glucose. L’hypothalamus médiobasal (MBH) en particulier, est capable de détecter l’hyperglycémie afin d’initier des réponses physiologiques adaptées, comme par exemple la sécrétion d’insuline via le système nerveux autonome (par un contrôle vagal). Notre équipe a récemment montré que la détection du glucose nécessite la production d’espèces actives de l’oxygène d’origine mitochondriale (mROS), fortement dépendante de la dynamique mitochondriale (fusion et fission). Récemment, l’étude de modèles génétiques ont permis de faire un lien entre ces évènements dynamiques dans le MBH et le développement de pathologies métaboliques. L’objectif de ma thèse a été tout d’abord été de mettre en place un modèle expérimental présentant uniquement une altération de la détection hypothalamique du glucose induite par l’exposition à un régime gras enrichi en saccharose (HFHS) chez le rat. Après avoir caractérisé ce modèle, nos objectifs ont été de déterminer si l’exposition à ce régime hypercalorique avait un impact sur la dynamique mitochondriale ainsi que la signalisation mROS, via la fonction respiratoire de la mitochondrie dans l’hypothalamus. Nous avons finallement réversé quelques acteurs métaboliques dérégulés, potentiellement impliqués dans la dynamique mitochondriale, dans le but de réverser le phénotype observé chez les rats HFHS. Nos résultats montrent qu’après 3 semaines d’exposition au régime HFHS, les rats ont un poids corporel normal malgré l’augmentation de leur masse grasse, comparés aux rats contrôles. Les rats HFHS présentent aussi une intolérance au glucose et une augmentation de la glycémie basale sans modification de leur insulinémie. La sécrétion d’insuline en réponse à la détection hypothalamique du glucose, mesurée après une injection intra-carotidienne de glucose en direction du cerveau qui induit une hyperglycémie uniquement cérébrale, a été fortement diminuée. Cependant, la capacité sécrétoire des îlots pancréatiques est normale chez les rats HFHS. Ces défauts sont associés à une diminution de la production de ROS dans le MBH en réponse au glucose, sans modification du status redox. L’efficacité de la respiration mitochondriale hypothalamique a été mesurée par oxygraphie, et les résultats montrent une déficience de la respiration mitochondriale chez les rats HFHS. La translocation de la protéine de fission DRP1 à la mitochondrie est diminuée en réponse au glucose, suggérant une diminution de la fission mitochondriale. L’augmentation de l’activation de l’AMPK dans l’hypothalamus n’est pas responsable de l’altération de la détection hypothalamique du glucose car sa réversion avec une injection intracérébroventriculaire (ICV) de composé C, n’a pas permis de restaurer la sécrétion d’insuline en réponse à l’hyperglycémie cérébrale. De même, une injection ICV de leptine induisant l’activation de STAT3 n’a pas permis de restaurer la sécrétion d’insuline en réponse à l’hyperglycémie cérébrale. Enfin, la diminution de l’activation d’AKT suggère une résistance centrale à l’insuline. Ces résultats démontrent pour la première fois que l’altération hypothalamique de la signalisation ROS, de la fission et de la respiration mitochondriale, sont présent chez les rats exposés pendant 3 semaines à un régime HFHS. Ces défauts précoces hypothalamiques pourraient ainsi participer à un défaut primaire du contrôle de la sécrétion d’insuline, et finallement, à l’installation d’un phénotype diabétique. / The hypothalamus participates in the control of energy homeostasis by detecting circulating nutrients, such as glucose. The mediobasal hypothalamus (MBH), in particular, senses hyperglycemia and initiates physiological responses, e.g., insulin secretion via the autonomous (vagal) nervous system. We have recently demonstrated that glucose sensing requires mitochondrial reactive oxygen species (mROS) signaling heavily dependant on mitochondrial fusion and fission (dynamics). Recently, genetic models have associated some of these dynamics within the MBH to their obesogenic susceptibility. The aims of my thesis were first to establish a model that only presents a hypothalamic glucose sensing defect induced by a high fat high sucrose (HFHS) feeding in rats. After caracterizing this model, our objectives were to determine whether modulating the diet affects mitochondrial dynamics, and thus, mROS signaling, through the mitochondrial respiratory function in the hypothalamus. We finally reversed some dysregulated metabolic signalings potentially involved in mitochondrial dynamics in order to reverse the phenotype observed in HFHS fed rats. Our results demonstrate that after 3 weeks of HFHS feeding, rats had a normal body weight despite an increase in the fat mass compared to control rats. HFHS fed rats displayed also a glucose intolerance, increased fasting glycemia but no modification of fasting insulinemia. Hypothalamic glucose sensing induced insulin secretion, measured after an intra-carotid glucose injection towards the brain that only increases brain glycemia without alteration in peripheral glycemia, was drastically decreased. However, glucose stimulated insulin secretion in isolated islets was not different compared to controls. These defects correlate with a decrease of MBH ROS production in response to glucose, with no modification in the redox status. Efficiency of hypothalamic mitochondrial respiration was evaluated using oxygraphy, and results showed mitochondrial respiratory deficiencies in HFHS fed rats. The fission protein DRP1 exhibited decreased mitochondrial translocation in the MBH in response to glucose, suggesting decreased mitochondrial fission. The increase of AMPK activation in the hypothalamus was not responsible for the alteration of hypothalamic glucose sensing since its reversal with an intracerebroventricular (ICV) injection of compound C failed to restore brain hyperglycemia induced insulin secretion. Likewise, an ICV injection of leptin that induced STAT3 activation also failed to restore brain hyperglycemia induced insulin secretion. Finally, the decrease in AKT activation suggested a central insulin resistance. These results demonstrate for the first time that hypothalamic alteration of mitochondrial ROS signaling, fission and respiration were present in rats exposed to a 3 weeks HFHS diet. Such hypothalamic glucose sensing defects are early events preceding those in islets. These early but drastic hypothalamic modifications could participate in a primary nervous defect of the control of insulin secretion, and finally, the etablishment of a diabetic phenotype.

The accurate and rapid detection of clinically relevant analytes at the point-of-care (POC) is a crucial element for the increase in our quality of life. There are multiple detection techniques for sensing a target analyte in biological samples. However, electrochemical sensors excel because of their versatility, accuracy and sensitivity. Among the many challenges in the fabrication of electrochemistry-based POC sensors, the miniaturization of the working electrodes is one of the most difficult to overcome. Decreasing the size of the sensors will result in less electroactive surface area (ESA) and, therefore, lower sensitivity. Thus, the design of miniaturized electrodes with high ESA is desired in this research field. The methodology developed in our laboratory to accomplish this goal is based on the fabrication of microstructured gold electrodes (MSEs) by depositing, via sputtering, a gold thin-film onto a pre-stressed polystyrene substrate masked with adhesive vinyl stencils and thermally shrinking the substrate at high temperatures (135-160 °C). In my thesis work, I developed cost-effective sensitive electrochemical platforms using only bench-top approaches. First, the ESA and, thus, the sensitivity of the MSEs were enhanced by using a simple and rapid nano-roughening approach. The ESA of MSEs was increased 4x by applying high voltage pulsing in sulfuric acid. The resulting electrodes possessed high anti-fouling capabilities and excellent response toward the enzyme-free detection of glucose with a limit of detection (LOD) of 0.62 mM in the presence of bovine serum albumin (BSA) and ascorbic acid. Furthermore, the fabrication cost of the MSEs electrodes was decreased by 5x by replacing the sputtering deposition step with a cost-effective solution-based electroless deposition technique. In this case, the PS substrates were coated with a polydopamine adhesion layer and noble metal films (copper, silver and gold) were subsequently plated. Not only the cost of the gold electrode was substantially reduced but, due to the intrinsic roughness of the surface, the MSEs electrodes obtained via electroless deposition showed a higher ESA than those made via sputtering. Furthermore, the developed electroless method was extended for the fabrication of paper-based sensing devices. The sensing versatility of these surfaces was demonstrated by electrochemically detecting mercury with a 0.27 ppb LOD and by sensing thiophenol via surface-enhanced Raman scattering (SERS). The MSEs electrodes fabricated via electroless deposition were subjected to the nano-roughening technique to generate affordable and high ESA electrodes. These platforms were used to design enzyme-based biosensors to accurately detect glucose and urea in complex samples. Glucose was detected in four different types of wine, with matrix interference measured below 10%, and in human serum, with a measured concentration that was not statistically different from that obtained from commercially available biosensors. Urea was detected in human urine and plasma with matrix interferences measured to be below 8% in both cases. We envision that the fabrication techniques developed in this thesis will rapidly grow in the scientific community for the prompt and accurate design of POC electrochemical devices. / Thesis / Doctor of Philosophy (PhD)

Trypanosoma brucei, the causative agent of African trypanosomiasis, and its relatives Trypanosoma cruzi and several Leishmania species belong to a class of protozoa called kinetoplastids that cause a significant health burden in tropical and semitropical countries across the world. While an improved therapy was recently approved for African trypanosomiasis, the therapies available to treat infections caused by T. cruzi and Leishmania spp. remain relatively poor. Improving our understanding of T. brucei metabolism can inform on metabolism of its relatives. The purpose of the research presented in this dissertation was to develop novel tools and methods to study metabolism in T. brucei with the ultimate aim to improve treatments of all kinetoplastid diseases. We developed a novel tool to study glycosomal pH in the bloodstream form of T. brucei. Using this tool, we discovered that this life stage regulates glycosomal pH differently than the procyclic form, or insect-dwelling stage, and only uses sodium/proton transporters to regulate glycosomal pH. I pioneered a thermal proteome profiling method in this parasite to discover drug targets and their effects on cell pathways. Using this method, I found that other proteins may be involved in glycosomal pH regulation, including PEX11 and a vacuolar ATPase. This method also illuminated several important pathways influenced by glycosomal pH regulation, including glycosome proliferation, vesicle trafficking, protein glycosylation, and amino acid transport. Metabolic studies in kinetoplastid parasites are currently hampered by the lack of available chemical probes. We developed a novel flow cytometry-based high-throughput drug screening assay to discover chemical probes of T. brucei glycolysis. This method combines the advantages of phenotypic (or cell-based) screens with the advantage of targeted (purified protein) screens by multiplexing biosensors that measure multiple glycolytic metabolites simultaneously, such as glucose, ATP, and glycosomal pH. The complementary information gained is then used to distinguish the part of glycolysis identified inhibitors target. We validated the method using the well characterized glycolytic and alternative oxidase inhibitors 2-deoxyglucose and salicylhydroxamic acid respectively. We demonstrated the screening assay with a pilot screen of 14,976 compounds with decent hit rates for each sensor (0.2-0.4%). About 64% of rescreened hits repeated activity in at least one sensor. We demonstrated one compound with micromolar activity against two biosensors. In summary, we developed and demonstrated a novel screening method that can discover glycolytic chemical probes to better study metabolism in this and related parasites. There are few methods to study enzyme kinetics in the live-cell environment. I developed a kinetic flow cytometry assay that can measure enzyme and transporter activity using fluorescent biosensors. I demonstrated this by measuring glucose transport kinetics and alternative oxidase inhibition kinetics, with the measured kinetic parameters similar to those previously reported. We plan to expand on this method to measure transport kinetics in the glycosome and other organelles which has not been done before. We previously performed a drug screen to identify inhibitors that decrease intracellular glucose in T. brucei. I have performed preliminary work identifying the glucose transporter THT1 as one of the targets of optimized glucose inhibitors using the previously mentioned thermal proteome profiling method. We expect this finding will improve our ability to move these compounds from hit to lead in the drug discovery pipeline. Together, I have developed several flow cytometry and proteomics methods to better study metabolism in T. brucei. These tools are beginning to be used in related parasites. We expect the discoveries made using these tools will improve our ability to treat these neglected tropical diseases.

Le foie est un organe indispensable dans le contrôle de l'homéostasie énergétique du corps humain. En particulier, le métabolisme hépatique est crucial pour l'homéostasie glucidique et lipidique. Les voies cataboliques et anaboliques sont en équilibre constant et régulées de façon synergique en fonction de la disponibilité en nutriments et de la demande en énergie. La perturbation de cet équilibre, notamment en cas d'obésité, peut conduire à l'accumulation intra-hépatique de lipides, qui est une des causes principales de la survenue de l'insulino-résistance hépatique (IRH), conduisant à l'hyperglycémie chronique et au diabète de type 2 (DT2). La cellule eucaryote est une structure hautement compartimentée, et à ce titre la compartimentalisation des processus cataboliques et anaboliques est une part intégrante de la gestion des voies métaboliques. Dans cet ensemble, la mitochondrie est un organite clef, qui abrite l'oxydation des lipides, le cycle de l'acide citrique (CAC) et la respiration cellulaire. De cette manière, la fonction mitochondriale est un élément crucial dans le maintien de l'état énergétique et d'oxydation-réduction de la cellule dans une gamme physiologique, ainsi que dans la régulation de l'activité du métabolisme du glucose et des lipides pour l'homéostasie du corps entier. La fonction mitochondriale est directement régulée par son interaction avec le réticulum endoplasmique (RE) via des zones de proximité entre les organites appelées Mitochondria-Associated-Endoplasmic-Reticulum-Membranes ou MAM. Dans ce contexte, j'ai participé au cours de mon travail de thèse à une étude qui a montré l'importance des interactions mitochondrie-RE dans la signalisation de l'insuline et mise en lumière la perturbation des MAM comme acteur principal dans l'IRH. De plus, j'ai étudié la régulation des MAM dans le contexte physiologique de la transition nutritionnelle dans le foie sain et insulino-résistant (IR) / The liver is an essential organ in the control of energetic homeostasis of the human body. Particularly, hepatic metabolism is crucial for glucose and lipid homeostasis. Catabolism and anabolism of both substrates are in constant equilibrium and synergically regulated in regard of nutrient availability and energetic demand. Disruption of this equilibrium, especially in the case of obesity, can lead to hepatic accumulation of lipids, which is a major cause of hepatic insulin resistance (HIR) leading to chronic hyperglycaemia and type 2 diabetes (T2D). The eukaryotic cell is a highly compartmented structure, and in this respect compartmentation of anabolic and catabolic processes is an integral part of managing metabolic pathways together. In this context, the mitochondrion is a key organelle, housing oxidation of lipids, the tricarboxylic acid (TCA) cycle and cellular respiration. In this way, mitochondrial function is a crucial element in maintaining energetic and reductionoxidation state of the cell within physiological ranges, as well in regulating the proper activity of glucose and lipid metabolism for the all body homeostasis. Mitochondrial function is directly regulated by its interaction with the endoplasmic reticulum (ER) via proximity points between the organelles called Mitochondria-Associated-ER-Membranes (MAM). In this context I have participated during my Ph.D. in a work that has shown the importance of mitochondria-ER interactions in insulin signalling and highlighted MAM disruption as a main actor in HIR. Furthermore, I have studied the regulation of MAM in the physiological context of nutritional transition in the healthy and insulin resistant (IR) liver. Particularly, we have shown that MAM disruption induces impaired insulin signalling, while their reinforcement protects against its appearance and restore insulin sensitivity in lipid-induced IR condition. Moreover, we have pointed out a consistent decrease of MAM quantity in the IR liver of ob/ob, high-fat high-sucrose diet (HFHSD) and Cyclophilin D - knock-out (CypD-KO) mice

L'hypothalamus est fortement impliqué dans la régulation nerveuse de l'homéostasie énergétique. Il existe dans cette structure des neurones spécialisés (gluco-sensibles) qui détectent notamment l’hyperglycémie puis déclenchent des réponses adaptées comme le maintien de la glycémie, en stimulant la sécrétion d’insuline ou encore le rassasiement. Les astrocytes sont suspectés de participer à la détection neuronale du glucose. Dans l’ensemble du cerveau, il existe un couplage métabolique entre astrocytes et neurones. Le lactate, issu de la métabolisation du glucose par les astrocytes, est transporté par les neurones par des transporteurs aux monocarboxylates (MCTs). De plus, il a récemment été montré que les jonctions gap (GJ), à l’origine de la formation de réseaux au sein des astrocytes sont indispensables au passage du glucose de la circulation sanguine vers les neurones en activité. Ces GJ astrocytaires sont formées majoritairement de connexines 43 et 30 (Cxs).Mon travail de thèse s’est orienté suivant deux axes, qui ont visé à étudier le rôle des astrocytes dans la détection hypothalamique du glucose et du lactate. Dans un premier temps, nous avons montré que le lactate, comme le glucose, est détecté au niveau central et induit une sécrétion d’insuline. Dans un modèle de rat hyperglycémique pendant 48h (qui présente aussi une hyperlactatémie), nous avons montré que la détection du glucose et du lactate est altérée. Ces modifications ne sont pas dues à une variation de l’expression protéique des MCTs astrocytaires ou neuronale de l’hypothalamus.Dans un deuxième temps, nous nous sommes intéressés au rôle des Cxs astrocytaires. La Cx43 est très exprimée autour des micro-vaisseaux sanguins de l’hypothalamus médio-basal (MBH), un site présentant de nombreux neurones gluco-sensibles. L’expression de la Cx30 est plus diffuse dans cette structure. Nous montrons également que l’expression protéique des Cxs astrocytaires varie très rapidement suite à des modifications du statut métabolique (jeûne, réalimentation, hyperglycémie). Afin d’évaluer l’implication de la Cx43 astrocytaire (majoritaire) dans la détection hypothalamique du glucose, nous avons inhibé son expression dans le MBH, in vivo, en injectant des siRNA permettant d’inhiber la synthèse de cette protéine. L’inhibition de la Cx43 (30% à 72h) induit une diminution de la prise alimentaire sans modification du poids, de la glycémie et de l’insulinémie comparée aux témoins. Suite à l’injection carotidienne de glucose (censée mimer une hyperglycémie), la sécrétion d’insuline est fortement inhibée chez les animaux siCx43. De même, l’effet satiétogène du glucose semble inhibé chez ces animaux lors de la réalimentation après un jeûne.Ces résultats montrent pour la première fois, de façon intégrée, l’importance des connexines, et probablement des réseaux astrocytaires, lors de la détection hypothalamique du glucose. Ces nouvelles données renforcent l’importance du rôle métabolique des astrocytes lors de fonctions neuronales précises / The hypothalamus plays a pivotal role in the nervous control of glucose homeostasis. This area contains gluco-sensitive neurons. Some of them detect increases in glucose levels and regulate glucose homeostasis by stimulating insulin secretion or inhibiting food intake. It is widely accepted that astrocytes are metabolically coupled to neurons. Lactate, resulting from the metabolism of glucose by astrocytes, is transported via the monocarboxylate transporters (MCTs). In addition, gap junctions (GJ), that form networks within astrocytes, are essential to transfer glucose from the bloodstream to the active neurons. These astroglial GJ mainly consist of connexins 43 and 30 (Cxs).The aims of my thesis are twofold: first, to show that an intracarotid lactate injection toward the brain, as for glucose, triggers insulin secretion and, second, to investigate the role of astroglial Cxs.Our results demonstrate that lactate and glucose sensing are altered in 48h hyperglycemic rats (accompanied by high blood lactate level). These alterations are not due to changes in protein expression of astroglial or neuronal MCTs in the hypothalamus. We then show that Cx43 is highly expressed in astrocytic end-feet enwraping blood vessels, in medio-basal hypothalamus (MBH) where many gluco-sensitive neurons are present. The Cx30 expression is more diffuse in this structure. We also show that the protein expression of astroglial Cxs varies very rapidly due to changes in metabolic status (fasting, refeeding and hyperglycemia). To evaluate the involvement of astroglial Cx43 (the major isoform) in the hypothalamic glucose sensing, we silenced its expression in the MBH in vivo by injecting specific siRNA. A 30% diminution in protein levels (after 72h) induced a decrease in food intake without changes in weight, blood glucose and insulin levels compared to vehicle treated animals. The central response to glucose is drastically inhibited in terms of insulin secretion in siCx43 animals. Similarly, an intracarotid injection of glucose towards the brain does not reduce refeeding in siRNA treated animals.These results demonstrate for the first time in vivo, the importance of connexins and astroglial networks in hypothalamic glucose sensing mechanism. These new data reinforce the importance of the metabolic role of astrocytes in specific neuronal functions

L’hyperglycémie est détectée et intégrée au niveau de l’hypothalamus médio-basal (MBH) qui inhibe la prise alimentaire et déclenche la sécrétion d’insuline. Le MBH renferme des neurones spécialisés gluco-sensibles (GS) qui détectent directement ou indirectement des variations de la concentration extracellulaire en glucose. Dans une première étude, nous suggérons que la détection indirecte du glucose par les neurones GS hypothalamiques repose sur la libération d’endozépines par les astrocytes, un gliotransmetteur connu pour inhiber la prise alimentaire en réponse à l’hyperglycémie. Nous travaux montrent que les endozépines activent spécifiquement les neurones à pro-opiomélanocortine (POMC) du MBH pour générer leur effet anorexigène. Dans une seconde étude, nous montrons que la détection directe de l’hyperglycémie implique les neurones hypothalamiques dits « high gluco-excited » (HGE). Grâce à des approches pharmacologiques et génétiques, nous mettons en évidence que les canaux redox sensibles Transient Receptor Potential Canonical 3 et 4 (TRPC3/4) sont fondamentaux pour la détection du glucose par les neurones HGE in vitro, la stimulation de la sécrétion d’insuline et la diminution de la prise alimentaire en réponse à l’hyperglycémie cérébrale in vivo. De plus, nos travaux démontrent que les canaux TRPC3 du MBH jouent un rôle clef dans le contrôle de l’homéostasie énergétique. Les travaux de cette thèse permettent de mettre en évidence deux nouveaux mécanismes de détection hypothalamique de l’hyperglycémie : l’un reposant sur l’implication des canaux TRPC3/4 dans les neurones HGE et l’autre proposant les endozépines astrocytaires comme relai du signal « glucose » aux neurones POMC. / Hyperglycemia is detected and integrated by the mediobasal hypothalamus (MBH) which, in turn, inhibits food intake and triggers insulin secretion. The MBH houses specialized glucose-sensitive (GS) neurons, which directly or indirectly modulate their electrical activity in response to changes in glucose level. In a first study, we hypothesized that indirect detection of glucose by MBH GS neurons involves the secretion of endozepine by astrocytes, a gliotransmitter known to inhibit food intake in response to hyperglycemia. The present work shows that endozepines selectively activate anorexigenic MBH pro-opiomelanotortine (POMC) neurons. In the second study, we show that the direct detection of increased glucose level involves hypothalamic glucose-excited (HGE) neurons. Using pharmacological and genetic approaches, we demonstrate that the redox-sensitive Transient Receptor Potential Canonical 3 et 4 (TRPC3/4) channels are involved in MBH HGE response to glucose in vitro and increased insulin secretion and decreased food intake in response to cerebral hyperglycemia in vivo. We also obtained evidences that MBH TRPC3 channel is a critical new player for energy homeostasis. This thesis work identifies two new mechanisms involved in hypothalamic detection of hyperglycemia: the first based on the involvement of TRPC3/4 channels in HGE neurons and the second highlighting the astroglial endozepines as a relay of the “glucose” signal to POMC neurons.

This thesis, written by Ingrid Öberg Månsson at KTH Royal Institute of Technology and entitled “Electroanalytical devices with fluidic control using textile materials and methods”, presents experimental studies on the development of textile based electronic devices and biosensors. One of the reasons why this is of interest is the growing demand for integrated smart products for wearable health monitoring or energy harvesting. To enable such products, new interdisciplinary fields arise combining traditional textile technology and electronics. Textile based devices have garnered much interest in recent years due to their innate ability to incorporate function directly into, for example, clothing or bandages by textile processes such as weaving, knitting or stitching. However, many modifications of yarns required for such applications are not available on an industrial scale. The major objective of this work has been to study how to achieve the performance necessary to create electronic textile devices by either coating yarns with conductive material or using commercially available conductive yarns that are functionalized to create sensing elements. Further, liquid transport within textile materials has been studied to be able to control the contact area between electrolyte and electrodes in electrochemical devices such as sensors and transistors. Yarns with specially designed cross-sections, traditionally used in sportswear to wick sweat away from the body and enhance evaporation, was used to transport electrolyte liquids to come in contact with yarn electrodes. The defined area of the junction where the fluidic yarn meets the conductive yarn was shown to increase stability of the measurements and the reproducibility between devices. The results presented in the two publications of this thesis as well as additional results presented in the thesis itself show the promising potential of using textile materials to integrate electronic and electrochemical functionality in our everyday life. This is shown by using basic textile materials and processing techniques to fabricate complex devices for various application areas such as sensors and diagnostics as well as electrical and energy harvesting components. / Denna avhandling, skriven av Ingrid Öberg Månsson vid Kungliga Tekniska Högskolan och titulerad ”Elektroanalytiska sensorer med vätskekontroll integrerad genom användande av textila material och metoder”, presenterar experimentella studier inom utvecklingen av textilbaserade elektroniska komponenter och biosensorer. Detta är av intresse på grund av den ökade efterfrågan på integrerade smarta produkter som till exempel bärbara sensorer för hälsoövervakning eller för att samla upp och konvertera energi till elektricitet. För att möjliggöra denna typ av produkter föds nya interdisciplinära fält där traditionell textilteknologi och elektronik möts. Textilbaserade enheter har väckt stort intresse under de senaste åren på grund av den naturliga förmågan att integrera funktion i till exempel kläder eller förband genom textila tillverkningsprocesser som väveri, stickning eller sömnad. Många modifikationer hos garner som krävs för att möjliggöra sådana tillämpningar är dock inte tillgängliga i större skala. Därför har det huvudsakliga syftet med denna studie varit att undersöka hur man kan uppnå den prestanda som krävs för att tillverka elektroniska textila komponenter, antingen genom att belägga garner med elektroniskt ledande material eller genom att använda kommersiellt tillgängliga ledande garner som sedan modifieras kemiskt för att skapa sensorer. Utöver detta har vätsketransport inom textila material studerats för att kunna styra och kontrollera kontaktytan mellan elektrolyt och elektroder i elektrokemiska enheter så som sensorer och transistorer. Garner med speciella tvärsnitt, som traditionellt använts i sportkläder för att transportera svett bort från kroppen och underlätta avdunstning, har använts för att transportera elektrolytvätska till elektroder av garn. Den definierade kontaktytan där det vätsketransporterade garnet korsar elektrodgarnet har visats öka stabiliteten av mätningen och reproducerbarheten mellan mätenheter. Resultaten som presenteras i de två artiklar som denna avhandling bygger på samt i avhandlingen själv visar på lovande potential för användandet av textila material för att integrera elektronisk och elektrokemisk funktionalitet i våra vardagsliv. Detta har uppnåtts genom att använda grundläggande textila material och tillverkningsprocesser för att tillverka komplexa enheter för olika tillämpningsområden så som sensorer för diagnostik samt elektroniska komponenter. / <p>QC 2020-08-21</p>