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Corrélations génotype/phénotype dans la maladie de Charcot-Marie-Tooth : l'exemple des mutations du gène INF2 / Genotype-phenotype correlations in Charcot-Marie-Tooth disease : The example of mutations of the INF2Mathis, Stéphane 04 December 2014 (has links)
La maladie de Charcot-Marie-Tooth (CMT) est une pathologie neurologique affectant le système nerveux périphérique. Bien que décrite à la fin du XIXème siècle, la découverte d’une anomalie génétique n’a été identifiée chez ces patients que dans les années 1990 (duplication du gène PMP22). Depuis, de nombreux gènes ont été incriminés, et leur nombre ne cesse d’augmenter. Ainsi, cette multitude de gènes nous incite à rechercher des corrélations phénotype-génotype qui permettent d’orienter au mieux le diagnostic et la prise en charge de ces patients. Comme nous le montrons au travers de nos travaux, il est possible de s’appuyer sur des données cliniques, biologiques, électrophysiologiques (voire radiologiques) et histo-pathologiques (biopsie de nerf) pour orienter la recherche d’anomalies génétiques. Pour illustrer ceci, nous nous sommes appuyés sur l’exemple des mutations du gène INF2, gène récemment associé à la maladie de Charcot-Marie-Tooth. Dans ce cas précis, l’atteinte rénale, le profil électrophysiologique (forme « intermédiaire » de CMT) et surtout les données histo-pathologiques (la biopsie de nerf permettant de retrouver la présence d’expansions schwanniennes caractéristiques) sont évocatrices de la présence d’une anomalie portée par ce gène. D’autres exemples de corrélations génotype-phénotype sont apportés au travers d’observations. / Charcot-Marie-Tooth disease (CMT) is a neurological disorder of the peripheral nervous system. Even if it was described in the end of the nineteenth century, the first genetic abnormality (PMP22 duplication) was found only in the end of the twentieth century. Several other genes were found to be associated with this disease. This important number of potential genes leads us to find genotype-phenotype correlations in order to better and earlier diagnose these patients. As we can show it in our work, it is possible to use biological, electrophysiological (sometimes radiological) and pathological (nerve biopsy) in order to direct the genetic analysis towards the incriminated gene. To illustrate this, we have particularly study the INF2 gene, a gene recently associated with CMT. In this example, clinical (CMT phenotype and renal failure), electrophysiological (intermediate form of CMT), and pathological (supernumerary extensions of Schwann cells cytoplasm) features call to mind mutations in the INF2 gene. Other examples of genotype-phenotype correlations associated with various genes are reported in this manuscript.
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Myopathy and peripheral neuropathy associated with the 3243A>G mutation in mitochondrial DNAKärppä, M. (Mikko) 19 March 2004 (has links)
Abstract
Neurological features are common in mitochondrial diseases because tissues depending upon oxidative phosphorylation bear the brunt of the pathogenesis. The 3243A>G mutation in the MTTL1 gene in mitochondrial DNA is regarded as the most frequent mitchondrial point mutation and classically presents with mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS). Myopathy and peripheral neuropathy have been documented in patients with mitochondrial diseases, but not properly characterised in patients with the 3243A>G mutation. We have previously determined the prevalence of patients with this mutation in a defined population in northern Finland.
The clinical spectrum and molecular aspects of myopathy and peripheral neuropathy are analysed here in a population-based cohort of patients with 3243A>G. Fifty patients were examined neurologically in order to define the frequency of myopathy and its histological, ultrastructural and clinical features. The frequency and phenotypic variability of peripheral neuropathy were determined in 32 patients and muscle computed tomography findings recorded in 24 patients. Finally, variations in mutation heteroplasmy were analysed in 10 patients using single muscle fibre PCR analysis.
The frequency of peripheral neuropathy was 22% (95% confidence interval (CI), 9–40%) and that of clinical myopathy 50% (95% CI, 36–64%). Moderate limb weakness was the most common myopathic feature, but mild weakness and external ophthalmoplegia were also present. CT scans revealed myopathic changes in 54% of the patients (95% CI, 33–76%), most frequently in the pelvic muscles. The incidence of myopathy was highest in the fifth decade of life, and higher age and male gender increased the risk of neuropathy. Muscle histology was abnormal in 72% of the cases examined (95% CI, 55–86%). The presence of intramitochondrial crystals and COX-negative fibres and variations in the size and shape of mitochondria were more common in the muscle of myopathic patients. Single muscle fibre analysis pointed to a correlation between the mutation load in ragged red fibres and in adjacent histologically normal fibres, and the proportion of 3243A>G in histologically normal muscle fibres showed a pattern compatible with random genetic drift.
The results indicate that myopathy and peripheral neuropathy are common in patients with the 3243A>G and that myopathy is highly variable in presentation. Segregation of 3243A>G in individual muscle fibres showed a complex process with random and non-random elements.
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The Biochemical Characterization of Human Histidyl-tRNA Synthetase and Disease Associated VariantsAbbott, Jamie Alyson 01 January 2017 (has links)
Human histidyl-tRNA synthetase (HARS) is an aminoacyl-tRNA synthetase (AARS) that catalyzes the attachment of the amino acid histidine to histidyl-tRNA (tRNAHis) in a two-step reaction that is essential for protein translation. Currently, two human diseases, Usher Syndrome IIIB (USH3B) and an inherited peripheral neuropathy, Charcot Marie Tooth Syndrome (CMT), have been linked genetically to single point mutations in the HARS gene. The recessive HARS USH3B mutation encodes an Y454S substitution localized at the interface between the anticodon-binding domain and the catalytic domain of the opposing subunit. Patients with Usher Syndrome IIIB lose their sight and hearing during their second decade of life, and clinicians have observed that the onset of deafness and blindness may be episodic and correlate with febrile illness. Furthermore, some young USH3B patients present with a fatal form of acute respiratory distress. In addition to the single HARS mutation linked to Usher Syndrome, eight other mutations in the HARS gene are associated with CMT, an inherited peripheral neuropathy. Peripheral neuropathies are associated with progressive and length-dependent damage of the motor and sensory neurons that transmit information to the spinal cord. The age of onset and phenotypic severity of CMT linked to HARS is highly variable. When expressed in a yeast model system, the HARS variants are dominantly lethal, and confer defects in axonal guidance and locomotor deficiencies when expressed in C.elegans. Here, the biochemical characterization of the HARS USH3B and three peripheral neuropathy variants are described. The approaches included enzyme kinetic analysis with purified HARS enzymes to monitor catalytic deficiencies, differential scanning fluorimetry (DSF) to evaluate structural instability, and cellular models to detect physiological effects of axonal outgrowth by CMT variants. The results suggest that Usher Syndrome IIIB is unlikely to be a consequence of a simple loss of aminoacylation function, while HARS-linked peripheral neuropathy variants all share common catalytic defects in aminoacylation. The HARS system represents a notable example in which two different complex human diseases arise from distinct mutations in the same parent gene. By understanding the biochemical basis of these inherited mutations and their link to Usher Syndrome and CMT, it may be possible to develop mechanism-based therapies to improve the quality of life of patients afflicted with them.
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Flimmerverschmelzungsfrequenz bei Normalpersonen, AMD und Optikopathien / Critical Flicker-fusion Frequency in healthy probands, patients with AMD and optic neuropathyMeyer-Rüsenberg, Hans Helge 25 September 2017 (has links)
No description available.
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The pathogenesis of diabetic neuropathy : a proteomic, metabolomic and electrophysiological investigationFreeman, Oliver January 2015 (has links)
Diabetes mellitus affects more than 382 million people worldwide and an estimated 30-50% of patients develop some form of neuropathy. Patients typically present with sensory symptoms including hypersensitivity/pain and/or loss of somatosensation. In diabetic neuropathy, the longest nerves of the peripheral nervous system (PNS) show the worst pathology and symptoms are typically felt in the distal extremities. The cause of this apparent length-dependent pathology remains unknown. Through comprehensive integration of untargeted proteomic and metabolomic analyses of the PNS in the streptozotocin rat model of diabetes, we showed that bioenergetic pathways were more dysfunctional in the distal sciatic nerve (SN) than the lumbar 4/5 dorsal root ganglia (DRG) and cranial trigeminal ganglia (TG). Whilst glucose levels increased in all tissues in diabetes, there was extensive upregulation of proteins involved in mitochondrial oxidative phosphorylation in the distal SN compared to healthy age/weight-matched controls which was not evident in the proximal DRG or TG. There were significant changes in lipid metabolites in the SN, a phenomenon which is less apparent in the DRG and not evident in the TG. We investigated the therapeutic potential of copper chelation with triethylenetetramine to reverse such changes and whilst copper chelation prevented nerve conduction velocity deficits, it did not alter aberrant nerve metabolism. To further understand the functional deficits in diabetic neuropathy, we performed in vivo microelectrode recordings from the TG and the thalamic ventral posteromedial (VPM) nucleus in control and diabetic rats in response to precise whisker stimulation. Recordings from the TG showed that the tuning of the primary afferents to graded stimuli is preserved in diabetes. Furthermore, we found that neurons within the VPM showed increased spontaneous activity in diabetes, but maintained tuning to graded whisker stimulation in their evoked firing rate. Thus, the cranial TG appear to be relatively unaffected by diabetes at a biochemical or physiological level, but diabetes may lead to pathophysiological changes within the thalamus which could alter somatosensory processing. Despite a global metabolic insult in diabetes, the molecular consequences are not consistent throughout the nervous system. We show that metabolic dysfunction occurs specifically in regions known to be more affected in neuropathy. Due to such a focal dysfunction, aberrant oxidative phosphorylation in the sciatic nerve may be a key driver to the distal pathogenesis of diabetic neuropathy.
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Membranous core domain of Complex I and mitochondrial disease modelingKervinen, M. (Marko) 30 May 2006 (has links)
Abstract
Human mitochondria contain a circular genome called mitochondrial DNA (mtDNA). It encodes subunits of the respiratory chain enzymes involved in energy conservation in oxidative phosphorylation and the necessary RNA needed for their expression. Errors in these genes have been shown to cause diseases, called mitochondrial diseases, which mainly affect tissues with high energy-demand, such as brain, heart, and skeletal muscle, or to lead to the production of harmful by-products in the form of reactive oxygen species (ROS) during cellular respiration. ROS damage lipids, proteins, and DNA, especially mtDNA. Accumulation of mtDNA mutations has also been associated with aging.
Mitochondrial complex I is located in the inner mitochondrial membrane and catalyzes NADH-ubiquinone oxidoreduction coupled to the translocation of four protons from the inside of the mitochondrion to the intermembranous space. Bacteria contain a homologous but simpler enzyme, NDH-1, with the same catalytic mechanism and which is therefore considered the catalytical core of mitochondrial complex I. Seven of the conserved membranous subunits in complex I are encoded in the mtDNA and are targets for mutations causing mitochondrial diseases, like MELAS syndrome or Leber hereditary optic neuropathy (LHON).
We used Paracoccus denitrificans and Escherichia coli NDH-1 enzymes to reveal the role of selected conserved charged residues and MELAS or LHON amino acid substitutions in enzyme catalysis. The growth phenotypes and NDH-1-dependent activities in mutant bacterial membranes were characterized, in addition to the sensitivity to selected complex I inhibitors. In order to enable ROS production measurements in the bacterial model of human mitochondrial diseases, we evaluated the reliability of two superoxide detecting probes, lucigenin and coelenterazine.
Elimination of the acidic residue in ND1 (position E228) previously found to cause MELAS, was found detrimental for NDH-1 assembly and activity. Also, elimination of the acidic residue at position E36 in ND4L resulted in an inactive enzyme. ND1-E216A, ND4L-E72Q and -E36Q/I39D/A69D/E72Q substitutions decreased NDH-1 activity somewhat (normal activity in the last mutant), but displayed a negative growth phenotype under NDH-1 dependent conditions, suggestive of impaired energy conservation in these mutants. ND1-Y229, whose substitution causes MELAS, charged residues in loop five of ND1, and ND1-E157, whose substitution causes LHON, were also found important for the enzyme activity.
Coelenterazine was found a reliable probe for quantitative superoxide production measurement in mitochondrial or bacterial membranes, and its sensitivity is not affected by the reduction level of the respiratory chain. Therefore, coelenterazine is suitable for quantitative superoxide production measurements.
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Etude des canaux ioniques TREK 1 et HCN dans la neuropathie chimio-induite à l'oxaliplatine. / Involvement of ionic channels, molecular targets for the development of new analgesics, in the treatment of the neuropathy induced by oxaliplatin-chemotherapy.Poupon, Laura 18 September 2015 (has links)
L’oxaliplatine, anticancéreux utilisé en première intention dans le traitement du cancercolorectal, engendre des neuropathies qui se caractérisent par des dommages du systèmenerveux périphériques responsables de l’apparition de troubles sensitifs douloureux dès lapremière cure ainsi que de troubles moteurs mais également des comorbidités qui s’installentavec la répétition des cures. Les traitements actuellement disponibles sont peu efficaces pourtraiter ces neuropathies qui sont à l’origine d’une altération de la qualité de vie des patients etpeuvent conduire à une diminution des doses de chimiothérapie utilisées voire même à l’arrêtdu traitement, compromettant ainsi les chances de guérison. Des données du laboratoire ont misen avant plusieurs canaux ioniques comme étant des cibles moléculaires potentielles pour letraitement de la neuropathie aigue. Le but de ce travail de thèse a été de poursuivre les étudespermettant de comprendre le mécanisme de survenue de ces effets indésirables et de rechercherles canaux dont la modulation pharmacologique permettrait un traitement efficace et bien tolérépour ces neuropathies.Pour se faire nous avons décidé de mieux caractériser un modèle murin de neuropathiedouloureuse aigue et de mettre au point et caractériser un modèle de neuropathie chroniqueinduites respectivement, par des injections unique ou répétées d’oxaliplatine. Un travailéthologique a été entrepris chez ces modèles afin d’évaluer la survenue et le suivi longitudinaldes symptômes retrouvés en clinique : symptômes douloureux céphaliques et extracéphaliques,déficits moteurs, dépression, anxiété. Chez ces mêmes animaux, des analyses moléculaires nousont permis de sélectionner des gènes codant pour des canaux ioniques impliqués dans laphysiologie et la pharmacologie de la douleur (canaux potassiques TREK et TRAAK, canauxHCN1 & HCN2). Nous avons parallèlement entrepris de valider pharmacologiquement ces cibleschez l’animal. Les résultats de nos travaux sont présentés séquentiellement et concernent :l’étude de l’implication des canaux HCN dans l’hypersensibilité douloureuse céphalique etextracéphalique induite par l’oxaliplatine ; l’étude de l’implication des canaux TREK et TRAAKdans la neuropathie chronique induite par l’oxaliplatine et la validation pharmacologique de cescibles chez un modèle murin de cancer colorectal.Dans un premier temps, nous avons utilisé un modèle de neuropathie aigue induit parune unique injection d’oxaliplatine (6mg/kg), et avons caractérisé les symptômes décrits enclinique au niveau du territoire céphalique. Nous avons observé l’existence de symptômesdouloureux au niveau orofacial et nous avons pu les corréler à une surexpression des canauxHCN1& HCN2. Il avait précédemment été démontré une surexpression de ses mêmes canaux auniveau extracéphalique, ce qui en fait des cibles particulièrement pertinentes compte tenu desdifférences anatomiques et pharmacologiques connues entre ces deux territoires somatiques. Deplus, l’utilisation d’ivabradine (Procoralan®), antagoniste non sélectif de ces canaux, a permis deréverser l’hypersensibilité douloureuse chez ce modèle animal de neuropathie douloureuseaigue.En conclusion, l’ensemble de ces résultats expérimentaux a permis l’identification denouvelles pistes pour la compréhension et le traitement des douleurs chimio-induites àl’oxaliplatine. Dans une perspective de recherche translationnelle, cette approche préclinique esten cours de transposition dans un protocole de recherche clinique. Un essai clinique de phase II(RILUZOX) devrait débuter prochainement afin de confirmer l’intérêt du riluzole chez despatients recevant une chimiothérapie à base d’oxaliplatine. / Oxaliplatin causes neuropathies which are characterized by damage of peripheralnervous system responsible for the onset of disorders such as neuropathic pain, sensory andmotor symptoms and comorbidities. Currently, available treatments are not very effective fortreating these symptoms in acute or chronic form. However, these symptoms are theconsequence of an impaired quality of life for patients and can lead to a decrease ofchemotherapy doses used or even stopping treatment, compromising the chances of surviving.Therefore, it appears essential for further studies to understand the mechanism of occurrence ofthese pains and identify potential targets for the development of new analgesics.We decided to further characterize a mouse model of acute painful neuropathy and todevelop and characterize a chronic neuropathy model induced respectively by single or repeatedinjections of oxaliplatin. An ethological work was realized in these models to assess theoccurrence of clinical symptoms: painful symptoms at cephalic and extracephalic levels, motordeficits, depression, anxiety. In these animals, molecular analyzes allowed us to select the genesencoding ion channels involved in the physiology and pharmacology of pain (potassium channelTREK and TRAAK, HCN1 & HCN2 channels). We undertook to pharmacologically validate thesetargets in animal. The results of our work are presented sequentially and concern: the study ofthe involvement of HCN channels in the cephalic and extracephalic painful hypersensitivityinduced by oxaliplatin; the study of the involvement of TREK and TRAAK channels in chronicoxaliplatin-induced neuropathy and pharmacological validation of these targets in a mousemodel of colorectal cancer.Initially, we used a model of acute neuropathy induced by single injection of oxaliplatin(6mg / kg) to study the clinical symptoms described at the orofacial sphere. Indeed, peripheralsymptoms had already been studied and the hypothesis of the ion channels expressionremodelling was confirmed. So, we verified the existence of orofacial pain symptoms with newbehavioral tests and we have correlated these results with an over expression of HCN1 & HCN2channels. In addition, use of ivabradine (Procoralan®), a non-selective antagonist of thesechannels, allowed reversing hypersensitivity to cold at low doses and mechanicalhypersensitivity when we used higher doses.In conclusion, all of these experimental results allowed the identification of newmechanisms for understanding and treating oxaliplatin-induced neuropathy. First, involvementsof HCN1 & HCN2 channels have showed in the acute neuropathy induced by oxaliplatin for bothperipheral and oral symptoms. In particular, blocking of these channels by ivabradine has alsohelped to reverse the cold hypersensitivity. Also, the TREK-1 channel showed great interest ininvolvement in chronic neuropathy induced by oxaliplatin and riluzole showed many interestingproperties to overcome the symptoms described in this neuropathy. From the perspective oftranslational research, this preclinical approach is being transposed in a clinical researchprotocol. A phase II clinical trial (RILUZOX) should begin shortly to confirm the interest ofriluzole in patients receiving chemotherapy with oxaliplatin.
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Analyse du comportement électrochimique de matériaux d'électrodes biocompatibles, utilisables comme électrodes dans un dispositif de diagnostic médical non invasif / Electrochemical analysis of some biocompatible electrodes for non-invasive medical detectionCalmet, Amandine 27 November 2015 (has links)
Les neuropathies périphériques sont des dysfonctions causées par certaines maladies (diabète, mucoviscidose). Celles-ci peuvent être rapidement diagnostiquées par la technologie SudoscanTM. Cette technologie non invasive, est basée sur des mesures électrochimiques via l’imposition de faibles potentiels entre des électrodes appliquées sur la peau et la mesure de faibles courants. Les résultats obtenus sont liés à la composition de la sueur associée à l’innervation des glandes sudoripares. Les résultats obtenus in vivo donnent une réponse caractéristique à l’anode, qui permet de déterminer le type de maladie et son avancement. Afin de mieux comprendre les phénomènes mis en jeu aux électrodes et d’optimiser la sensibilité des mesures in vivo, des manipulations ont été réalisées in vitro dans des solutions mimant la composition de la sueur. Pour simuler la résistance du corps et réduire les densités de courant obtenues in vitro la viscosité de l’électrolyte a été augmentée. Cet électrolyte a permis d’atteindre des densités de courant in vitro proches de celles obtenues in vivo. Le second objectif de cette étude est d’analyser le comportement électrochimique in vitro de différents aciers inoxydables biocompatibles et leur résistance à la corrosion dans un milieu physiologique à pH neutre. Nous nous sommes plus précisément intéressés à l’influence de la concentration en ions chlorure sur la corrosion, aux échelles de concentrations trouvées dans la sueur, (36mM 120mM). L’objectif est de permettre une meilleure compréhension des phénomènes mis en jeu et d’analyser la sensibilité des matériaux aux ions chlorures, pour déterminer le matériau le plus prometteur pour la technologie. / A non-invasive device based on measurements of electrochemical skin conductance can detect small fiber neuropathy, a sweat gland dysfunction implicated in several diseases. In this context, the SudoscanTM technology developed by Impeto Medical provides early diagnosis, rapid and noninvasive analysis. This technology is based on measurements of skin current density via the imposition of low amplitude voltages (4 to 1.5V) between electrodes applied to the skin and measuring the low current generated. These electrodes are sensitive to the composition of the sweat produced by the eccrine glands when stimulated. The results obtained in vivo provide a characteristic response on the anode, which determines the type of disease and its progress. To better understand the phenomena involved at electrodes and to optimize the sensitivity of in vivo measurements, manipulations were performed in vitro with solutions mimicking the composition of the sweat. To simulate the resistance of the body and to reduce the current densities obtained in vitro, the viscosity of the electrolyte has been increased. This electrolyte has achieved in vitro current densities similar to those obtained in vivo. The second objective of this study is to analyze the electrochemical behavior of different biocompatible stainless steels and their corrosion resistance in a physiological medium at neutral pH. We specifically interested in the influence of the chloride ion concentration on the corrosion, in the range of sweat’s. The objective is to enable a better understanding of the phenomena involved and analyze the sensitivity of materials to chloride ions, to determine the most promising material for technology.
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Whole Exome Sequencing to Identify Disease-Causing Mutations in Lower Motor Neuron Disease and Peripheral NeuropathyWagner, Justin January 2016 (has links)
Lower motor neuron diseases and peripheral neuropathies are two groups of diseases that include multiple rare disorders where many causes are unknown and definitive treatments are unavailable. Understanding the molecular etiology of these genetic diseases provides an opportunity for rapid diagnosis, preconception genetic counseling and, in a subset, direction for the development of future treatment options. The recent introduction of whole exome sequencing (WES) marks a new era in Mendelian genetic disease research as the majority of the coding region of the genome can be sequenced in a timely and cost-effective manner. In this study, WES was used to investigate the molecular etiology of a cohort of 37 patients presenting with lower motor neuron disease or peripheral neuropathy. A molecular diagnosis was determined for seven patients informing the diagnostic utility of WES. Novel phenotypes were found for three genes originally associated with a different disorder. Finally, the foundation has been laid, through the use of functional studies and large scale data-sharing, to identify novel disease-causing genes for lower motor neuron disease and peripheral neuropathy.
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The Effects of Type 1 Diabetes Mellitus on Heat Loss During Exercise in the HeatCarter, Michael R. January 2014 (has links)
Studies show that vasomotor and sudomotor activity is compromised in individuals with Type 1 Diabetes (T1DM) which could lead to altered thermoregulatory function. However, recent work suggests that the impairments may only be evidenced beyond a certain level of heat stress. We therefore examined T1DM-related differences in heat loss responses of sweating and skin blood flow (SkBF) during exercise performed at progressive increases in the requirement for heat loss. Participants were matched for age, sex, body surface area and fitness cycled at fixed rates of metabolic heat production of 200, 250, and 300 W•m-2 of body surface area, each rate being performed sequentially for 30 min. Local sweat rate (LSR), sweat gland activation (SGA), and sweat gland output (SGO) were measured on the upper back, chest and forearm while SkBF (laser-Doppler) was measured on the forearm and upper back only.
We found that despite a similar requirement for heat loss, LSR was lower in T1DM on the chest and forearm only, relative to Control and only different at the end of the second and third exercise periods. Differences in chest LSR were due to reduced SGA whereas the decreased forearm LSR was the result of a decrease in SGO. SkBF did not differ between groups. The reduction in the sweating response in the T1DM group was paralleled by a greater increase in core temperature. We show that T1DM impairs heat dissipation as evidenced by reductions in LSR and not SkBF. A compromised thermoregulatory response during and following physical exertion is of considerable concern due to the associated increased risk of post-exertion heat-related injury.
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