Hyperglycemia in critically ill patients was considered for many years an adaptive response to stress conditions being present in both patients with and without previous history of diabetes. Hyperglycemia is caused mainly by peripheral insulin resistance induced by the factors acting counteracting insulin signalling at the postreceptor level. Furthermore, hyperglycemia itself can then increase serum levels of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-6 (Il-6) and interleukin-8 (Il- 8) and others. On the contrary, peripheral insulin resistance induced by pro- inflammatory cytokines may further potentiate hyperglycemia. White adipose tissue represents in addition to its energy storage function also a very active endocrine active organ. In addition to regulation of a number of metabolic processes it also significantly modulates the inflammatory response. In critically ill patients, adipose tissue changes its morphology, i.e. the adipocytes are shrinking and adipose tissue is abundantly infiltrated by macrophages. Paradoxically, overweight and obese critically ill patients have lower mortality than underweight, lean and morbidly obese subjects. In our studies, we selected population of the patients undergoing elective major cardiac surgery with extracorporeal...

Indiana University-Purdue University Indianapolis (IUPUI) / FGF23 is the central regulator for phosphate homeostasis. Both FGF23 and phosphate dysregulation are highly related with the progression of chronic kidney disease (CKD), which is a global health problem. In previous studies, FGF23 was found to be produced in bone and targeting the kidneys to regulate phosphate reabsorption and excretion. In the FGF23 signaling axis, it binds a receptor complex (αKlotho and FGFRs) in the distal convoluted tubules (DCT) and causes its biological effects in the proximal tubules (PT). The mechanism of how the signals passing on from DCT to PT is not clear. In my research, experiments were focused on the FGF23 signaling pathway within the kidney to study the communication steps between tubular cells. HBEGF treatment was given to FGF23 signaling impaired mouse models resulting in significant change of genes regulated by FGF23, indicating that HBEGF was important in the FGF23 signaling axis. Then high quality rabbit anti-mouse HBEGF antibodies were made to better study HBEGF activity in vivo and in vitro. A new cell model was characterized to test FGF23 effects on HBEGF signaling using Western blots and immunofluorescence. Lastly, the location of HBEGF activity was examined in the kidney in vivo. Immunostaining suggested that HBEGF activated the mitogen activated protein kinase (MAPK) pathway. This mapping may provide important information for the molecular relationships between FGF23 and HBEGF.

Indiana University-Purdue University Indianapolis (IUPUI) / Photoreceptors are a class of sensory neuronal cells that are deleteriously affected in many disorders and injuries of the visual system. Significant injury or loss of these cells often results in a partial or complete loss of vision. While previous studies have determined many necessary components of the gene regulatory network governing the establishment, development, and maintenance of these cells, the necessary and sufficient profile and timecourse of gene expression and/or silencing has yet to be elucidated. Arduous protocols do exist to derive photoreceptors in vitro utilizing pluripotent stem cells, but only recently have been able to yield cells that are disease- and/or patient-specific. The discovery that mammalian somatic cells can be directly reprogrammed to another terminally-differentiated cell phenotype has inspired an explosion of research demonstrating the successful genetic reprogramming of one cell type to another, a process which is typically both more timely and efficient than those used to derive the same cells from pluripotent stem cell sources. Therefore, the emphasis of this study was to establish a novel system to be used to determine a minimal transcriptional network capable of directly reprogramming mouse embryonic fibroblasts (MEFs) to rod photoreceptors. The tools, assays, and experimental design chosen and established herein were designed and characterized to facilitate this determination, and preliminary data demonstrated the utility of this approach for accomplishing this aim.

For any drug candidate to be approved by the U.S. Food and Drug Administration, it must meet strict standards for safety and efficacy. While the field of nuclear medicine is over 100 years old, traditional methods such as external beams or systematic administration have rarely met these standards or have limited application. Ligand-targeted therapy and diagnostics, or “theranostics,” has emerged in the past several decades as an exciting field that offers new possibilities to design drugs that are both safe and effective. When applied to nuclear medicine, the field of ligand-targeted radioactive theranostics is younger still, with many critical lessons being discovered and applied currently. This dissertation outlines the necessary principles of radioactive theranostic drug design, then demonstrates the application of several more recent techniques to improve both the efficacy and safety of radioactive theranostics targeting two high priority oncological targets: fibroblast activation protein alpha and folate receptor.

Spinal Muscular Atrophy with Progressive Myoclonic Epilepsy (SMA-PME), Farber Lipogranulomatosis (FL), and a rare variant form of Neonatal Progeroid Syndrome (NPS) are three monogenetic rare disorders caused by pathogenic variation in genes encoding lipid modifying proteins. FL and SMA-PME are caused by loss of function mutations in ASAH1, encoding the acid ceramidase (aCDase) enzyme. It is not, however, known how aCDase deficiency can produce either the isolated neurological symptoms of SMA-PME or the predominantly systemic symptoms of FL. Further, a recently identified variant form of NPS has been attributed to variants in ANO6, encoding a dual function calcium-activated chloride channel and glycerophosphoserine (GPS) scramblase. Here, it is not known how ANO6 mutation causes the premature aging phenotype that defines NPS. To address these questions, I sought to elucidate pathogenic changes in lipid metabolism that associate clinical phenotype. I show here that the different patient mutations in ANO6 cause a non-physiological gain of channel function and either a loss or gain of scramblase function depending on the variant expressed. Both variants, however, alter GPS metabolic homeostasis suggesting a common mechanism of action. To provide in vivo insight, I characterized a novel mouse model based on our NPS patient genetics, showing extremely low penetrance of disease symptoms in terms of live births yet confirming that affected animals show impaired GPS metabolism in affected organs. Next, I characterized the clinical presentation of six new patients with SMA-PME and identified distinct sphingolipid metabolic fingerprints in FL and SMA-PME cells. I show that FL is defined by a hypometabolic sphingolipid phenotype with cellular and molecular features of a classic lysosomal storage disorder. By contrast, SMA-PME has a hypermetabolic sphingolipid phenotype with features of non-classic lysosomal trafficking disorders. To provide clinical insight, I assessed the potential of enzyme replacement therapy, demonstrating a rescue of sphingolipid metabolism in SMA-PME patient cells. Together, this thesis identified changes in the cellular and tissue lipid profiles of patients with ANO6-NPS, SMA-PME, or FL, elucidating some of the lipid-centric pathomechanisms of these diseases.

Annually, about 80,000 people die in the United Kingdom due to myocardial infarction, congestive heart failure, stroke, or from other diseases related to blood vessels. The current gold standard treatment for replacing the damaged blood vessel is by autograft procedure, during which the internal mammary artery (IMA) graft or saphenous vein graft (SVG) are usually employed. However, some limitations are associated with this type of treatment, such as lack of donor site and post-surgery problems that could negatively affect the patient’s health. Therefore, this present work aims to fabricate a synthetic blood vessel that mimics the natural arteries and to be used as an alternative method for blood vessel replacement. Polymeric materials intended to be used for this purpose must possess several characteristics including: (1) Polymers must be biocompatible; (2) Biodegradable with adequate degradation rate; (3) Must maintain its structural integrity throughout intended use; (4) Must have ideal mechanical properties; and (5) Must encourage and enhance the proliferation of the cells. The feasibility of using synthetic biodegradable polymers such as poly (ε- caprolactone) (PCL) and poly (lactide-co-glycolic acid) (PLGA) for fabricating tubular vascular grafts was extensively investigated in this work. Many fundamental experiments were performed to develop porous tissue- engineered polymeric membranes for vascular graft purposes through electrospinning technique to achieve the main aim. Electrospinning was selected since the scaffolds produced by this method usually resemble structural morphology similar to the extracellular matrix (ECM). Hence, four 6mm in diameter tubular shape vascular grafts PCL only, PLGA only, coaxial (core-PCL and shell-PLGA), and bilayer (inner layer-PCL and outer layer-PLGA) was designed and fabricated successfully. The structure and properties of each scaffold membrane were observed by scanning electron microscopy (SEM), and these scaffolds were fully characterized by Fourier-transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), thermogravimetric analysis (TGA), water contact angle measurements, mechanical tensile test, as well as cell culture studies were carried out by seeding human umbilical vein cells (HUVEC) and human vascular Fibroblast cells (HVF). Moreover, all polymeric grafts underwent degradation process, and the change in their morphological structure properties was studied over 12 weeks at room temperature. All scaffolds were also exposed to a controlled temperature of 37°C for four weeks, in phosphate-buffered saline solution (pH, 7.3). It was found that all scaffolds displayed exceptional fibre structure and excellent degradability with adequate steady weight-loss confirming the suitability of the fabricated scaffolds for tissue engineering applications. The coaxial and bilayer scaffolds degraded at a much slower (and steadier) rate than the singular PCL and PLGA tubular scaffolds. Coaxial grafts fabricated via coaxial needle showed an increase in their fibre diameter and pore size volume than other membranes, but also showed to have significant tensile strength, elongation at fracture, and Young’s modulus. To conclude, all scaffolds have demonstrated to be reliable to adhere and proliferate HUVEC, and HVF cells, but these cells were attracted to the PLGA membrane more than other fabricated membranes.

No description available.

La forme canadienne-française du syndrome de Leigh (LSFC) est une maladie métabolique associée à une déficience en cytochrome oxydase (COX) et caractérisée par des crises d’acidose lactique, menant à une mort prématurée. Les mécanismes qui sous-tendent l’induction des crises restent inconnus et il n’existe aucune thérapie efficace pour les prévenir. Cette étude vise à caractériser l'effet de facteurs métaboliques périphériques potentiellement altérés chez les patients LSFC sur la mort de lignées cellulaires issues de ces patients et de témoins puis, à identifier des agents thérapeutiques pouvant la prévenir. Nous postulons que (i) ces facteurs métaboliques induiront une mort prématurée des cellules de patients et que (ii) les interventions susceptibles de la prévenir pallieront les conséquences de la déficience en COX, soit la diminution des taux d’adénosine triphosphate (ATP) et l’augmentation du stress oxydant, du nicotinamide adénine dinucléotide (NADH) et des lipides toxiques. Un criblage de 8 facteurs sanguins et 10 agents thérapeutiques a été réalisé. Les paramètres mesurés incluent la nécrose, l’apoptose, l’ATP et l’activité de la COX. Les fibroblastes LSFC sont plus susceptibles à la mort par nécrose (39±6%) induite par du palmitate plus lactate, un effet associé à des niveaux d’ATP diminués (53±8%). La mort cellulaire est réduite de moitié par l’ajout combiné d’agents ciblant le NADH, l’ATP et les lipides toxiques, alors que l’ajout d’antioxydants l’augmente. Ainsi, un excès de nutriments pourrait induire la mort prématurée des cellules LSFC et, pour atténuer cette mort, il serait important de combiner plusieurs interventions ciblant différents mécanismes. / Leigh syndrome French-Canadian variant (LSFC) is a metabolic disease associated with cytochrome c oxidase (COX) deficiency and characterized by episodes of lactic acidosis, referred to as “crisis”, leading to death at an early age. The mechanisms underlying a crisis and its cellular consequences remain elusive, and there is no effective therapy. The aim of this study was to characterize the effect of peripheral metabolic factors that are potentially altered in patients with LSFC on their cells death and to identify therapeutic agents able to prevent them using cell-lineage from LSFC patients and controls. The hypothesis are that (i) these metabolic factors can induce premature death in patient cells, and (ii) interventions that could rescue these cells may target potential consequences of COX deficiency, namely low adenosine triphosphate (ATP), high nicotinamide adenine dinucleotide (NADH) and toxic lipids, as well as oxidative stress. A screening of 8 blood factors and 10 therapeutic agents was conducted in fibroblasts. Parameter measured included cell death by necrosis and apoptosis, as well as ATP level and COX activity. LSFC fibroblasts were more susceptible to necrosis (39±6%) induced by high palmitate plus lactate and this was associated with a lower ATP (53±8%). Cell death decreased 2-fold with combined interventions, which presumably act on NADH, ATP, and the accumulation of toxic lipids, but increased with antioxidants. Collectively, our results emphasize the importance of nutrient overload as a factor eliciting premature cell death in LSFC cells and of combining interventions acting through various mechanisms for cell death rescue.

La sclérose systémique (ScS) est une maladie auto-immune d’origine inconnue qui est caractérisée par des atteintes vasculaires, des dérèglements cellulaire et immunitaire. La majorité des patients atteints de ScS possède des auto-anticorps dirigés contre des protéines nucléaires. Ces auto-anticorps sont associés à des manifestations cliniques spécifiques favorisant la classification et le diagnostic de la ScS. Les anti-ADN topoisomérase I (antitopo) sont l’un des principaux auto-anticorps retrouvés dans la ScS. Ils sont associés à la forme la plus grave de la maladie, soit la forme diffuse. Celle-ci se caractérise par une importante fibrose progressant vers une atteinte viscérale. La fibrose résulte d’une production excessive et dérégulée de matrice extracellulaire par les fibroblastes. Bien que les anti-topo soient associés à un très mauvais pronostic et qu’ils corrèlent avec l’activité et la sévérité de la maladie, leur rôle dans la pathogenèse de la ScS n’est pas élucidé. Toutefois, depuis que certains auto-antigènes ont démontré des fonctions additionnelles lorsque retrouvés dans le milieu extracellulaire, leur contribution suscite un intérêt marqué. En effet, ces auto-antigènes, dits bifonctionnels, influencent la physiologie de certaines cellules en se liant à leur surface. Ainsi, la détermination du rôle de ces autoantigènes ouvre la voie pour l’exploration du rôle potentiellement pathogène de leurs autoanticorps. Tout d’abord, nous avons démontré que l’auto-antigène topo, ciblée par les antitopo, pouvait influencer la physiologie du fibroblaste suite à l’activation de voies de signalisations intracellulaires stimulant la migration cellulaire. Nos résultats suggèrent fortement que la topo stimule le fibroblaste suite à son interaction avec le CCR7, un récepteur de chimiokine, présent à sa surface. Nous avons également démontré que la topo utilisait les protéoglycans à chaînes d’héparanes sulfates (HSPG) à titre de corécepteurs. Il avait été démontré que la topo liée à la surface des fibroblastes entraînait le recrutement d’anti-topo, l’adhésion et l’activation monocytaires. Nous avons ici démontré que la présence d’anticorps anti-topo entraîne l’amplification de la liaison de la topo au niveau des HSPG. De ce fait, le complexe immun à la surface des fibroblastes pourrait contribuer à l’initiation d’une cascade inflammatoire propice au développement d’une fibrose, caractéristique de la ScS. En dernier lieu, nos résultats nous ont permis de suggérer l’utilisation de l’héparine et des héparines de bas poids moléculaires comme approche thérapeutique pour la ScS puisqu’elles permettent autant de prévenir la liaison du complexe immun topo/anti-topo au niveau des HSPG que de le dissocier une fois lié. En résumé, notre étude soutient d’abord le rôle actif de l’auto-antigène dans la physiologie des fibroblastes mais également le rôle pathogène des anti-topo en présence de la topo dans la ScS. Finalement, les résultats de notre étude permettent de proposer une approche thérapeutique potentielle pour inhiber le développement d’une cascade inflammatoire et pro-fibrotique. / Systemic sclerosis (SSc) is an autoimmune disease of unknown etiology characterized by vascular damage, cellular and immunological disorders. The vast majority of patient sera are characterized by the presence of autoantibodies directed against nuclear proteins. The autoantibodies are associated with specific clinical manifestations and thus useful for diagnostic and classification of the disease. One of the major autoantibody groups are the anti-DNA topoisomerase I (anti-topo). They are associated with the diffuse form of the disease which is characterized by extensive cutaneous and visceral fibrosis. Increased extracellular matrix synthesis and deposition by fibroblast result in the development of fibrosis. Although anti-topo are associated with the worst form of the disease, correlated with the activity and the severity of SSc, their exact role in the pathogenesis of SSc is controversial and still unravelled. On the other hand, there is now strong evidence for active contribution of autoantigens, targeted by autoantibodies, in autoimmune diseases. Indeed, numerous cells have been shown to be influenced by the interaction of autoantigens with their cognate receptors present on their surface. These autoantigens display cytokine-like effects toward their target cell and are called bifunctional autoantigen. Hence, determination of the exact role of these autoantigens and characterization of their interaction with their target cell may open up research perspectives for the elucidation of the potential pathogenic role of their autoantibodies. In our study, we demonstrated that topo activates intracellular signaling pathways leading to the stimulation of fibroblast migration. We undertook experiments to characterize the interaction of the autoantigen topo with fibroblasts responsible of these cellular effects. Our results strongly suggest a direct interaction of topo with CCR7, a chemokine receptor, present on the surface of fibroblasts. Heparan sulfate proteoglycans (HSPG), abundantly present on fibroblast surfaces, were found to act as coreceptors for topo binding. Previous work has demonstrated that once bound to fibroblast surfaces, topo recruits anti-topo autoantibodies, which subsequently lead to adhesion and activation of monocytes. Here, we demonstrated that anti-topo autoantibodies from SSc sera lead to the amplification of topo binding to HSPG on fibroblast surfaces. The binding of topo/anti-topo IC could mediate the initiation and maintenance of an inflammatory cascade and further fibrosis development. Hence, perturbing the binding of topo/anti-topo immune complexes to HSPG became an interesting therapeutic approach. Heparin and low molecular weight heparins were found to prevent the binding of topo and topo/anti-topo immune complexes to the fibroblast surfaces. Moreover, topo/anti-topo immune complexes could be dissociated from fibroblast surfaces by these molecules. Hence, the prevention of topo/anti-topo immune complexes binding to HS chains could result in the absence of the inflammatory cascade initiation. Overall, our results support an active role for topo as a bifunctional autoantigen toward fibroblasts and a pathogenic role for anti-topo autoantibodies in SSc. Finally, a potential therapeutic approach is proposed which could target inflammatory and fibrotic development characteristic of SSc.

INTRODUÇÃO: O hiperparatireoidismo secundário (HPTS) é uma complicação da doença renal crônica que compromete a integridade do esqueleto. Pacientes com HPS submetidos à paratireoidectomia (PTX) passam de uma condição de níveis séricos de paratormônio (PTH) muito elevados para outra, onde esses níveis hormonais caem drasticamente. Os efeitos da PTX no tecido ósseo são mal compreendidos, especialmente no que se refere às proteínas expressas por osteócitos, como o fator de crescimento de fibroblastos 23 (FGF23), dentin matrix protein 1 (DMP-1), fosfoglicoproteína de matriz extracelular (MEPE), esclerostina, Fator nuclear Kappa beta ligante (RANKL) e osteoprotegerina (OPG), que regulam a remodelação e a mineralização óssea. OBJETIVOS: Caracterizar a expressão óssea dessas proteínas por imuno-histoquímica e estabelecer relações com os dados da histomorfometria do tecido ósseo em pacientes com HPS, antes e após a PTX. MÉTODOS: Estudamos biópsias ósseas obtidas de um banco de biópsias de 23 pacientes com DRC e HPTS, que foram realizadas antes e 12 meses após a PTX. RESULTADOS: A avaliação dos parâmetros histomorfométricos demonstrou uma melhora da microarquitetura óssea, porém com um maior retardo em sua mineralização após a PTX. A análise da expressão das proteínas osteocíticas revelou um aumento significativo na expressão da esclerostina e da OPG e uma diminuição da relação RANKL/OPG após a PTX, sugerindo a participação dessas proteínas na melhora das lesões ósseas decorrentes do HPTS. Observamos um aumento significativo na expressão da OPG no grupo de pacientes que evoluiu com defeito de mineralização somente após a cirurgia, sugerindo a participação dessa proteína no retardo de mineralização óssea desses pacientes. A expressão das proteínas osteocíticas que participam da formação e mineralização óssea apresentou correlação com parâmetros envolvidos na remodelação óssea. CONCLUSÕES: Mudanças significativas na expressão óssea de proteínas osteocíticas que podem potencialmente regular a remodelação e a mineralização óssea foram observadas após a PTX / INTRODUCTION: Secondary hyperparathyroidism (SHPT) is a complication of chronic kidney disease that compromises skeletal integrity. Patients with SHPT undergoing parathyroidectomy (PTX) go from a very high serum parathyroid hormone (PTH) condition to another, where these hormonal levels dramatically fall. The effects of PTX on bone tissue are poorly understood, especially as regards proteins expressed by osteocytes, such as fibroblast growth factor 23 (FGF23), dentin matrix protein 1 (DMP-1), extracellular matrix phosphoglycoprotein (MEPE), sclerostin, Kappa beta ligand nuclear factor (RANKL) and osteoprotegerin (OPG), which regulate bone remodeling and mineralization. OBJECTIVES: Characterize bone expression of these proteins by immunohistochemistry and establish relations with bone tissue histomorphometry data in SHPT patients, before and after PTX. METHODS: We studied bone biopsies obtained from a biopsy database of 23 patients with CKD and SHPT, which were performed before PTX and 12 months after PTX. RESULTS: Evaluation of histomorphometric parameters showed improvement of bone microarchitecture, but with longer delay in mineralization after PTX. Analysis of osteocyte protein expression revealed significant increase in sclerostin and OPG expression and decrease in RANKL/OPG ratio after PTX, suggesting participation of these proteins in improvement of bone lesions due to SHPT. We observed significant increase in OPG expression in the group of patients who evolved with mineralization defect only after surgery, suggesting participation of this protein in bone mineralization delay of these patients. Expression of osteocyte proteins that participate in bone formation and mineralization correlated with parameters involved in bone remodeling. CONCLUSIONS: Significant changes in bone expression of osteocyte proteins that can potentially regulate bone remodeling and mineralization were observed after PTX