My PhD research studies, described in this thesis, were designed to document the temporal development of mechanical allodynia, a hallmark symptom of painful diabetic neuropathy (PDN), as well as opioid hyposensitivity using two different rat models of diabetes mellitus (DM). Specifically, the studies were conducted using the streptozotocin (STZ)-diabetic rat model of chemically-induced Type 1 diabetes in two different rat strains, as well as the Zucker Diabetic Fatty (ZDF) rat genetic model of Type 2 diabetes. Additionally, a longitudinal investigation of the effect of basal insulin replacement therapy to restore euglycaemia from 7-days post-STZ administration, on the development of mechanical allodynia in the hindpaws of the STZ-diabetic Wistar rat model of PDN, was conducted. The studies herein also included a longitudinal study to document the temporal development of mechanical allodynia and opioid hyposensitivity in the ZDF rat, which also examined the influence of dietary composition on the time course for the development of mechanical allodynia in the hindpaws, together with opioid hyposensitivity in these animals. In the final section of this thesis, the experiments were designed to examine possible mechanisms that may contribute to the development of opioid hyposensitivity in ZDF diabetic rats. These experiments involved the quantification of opioid receptor messenger ribonucleic acid (mRNA) gene expression as well as μ-opioid receptor (MOP-r) functional responses in tissues collected from 29-wk old diabetic ZDF rats relative to 7-wk old pre-diabetic control ZDF animals. In Chapter One, diabetes mellitus and more specifically its longterm complication, PDN, the focus of this doctoral research program, has been reviewed. Specifically, possible pathogenic mechanisms underlying mechanical allodynia, the relevant diabetic rodent models of PDN, use of insulin replacement therapy in diabetic rodents and its impact on hallmark symptoms of PDN, role of opioid pharmacology, the comparative efficacy of opioids in the treatment of PDN, and possible mechanisms that may underpin the development of opioid hyposensitivity in PDN, including the impact of altered excitatory neurotransmitters, have been reviewed. In Chapter Two, a preliminary study was conducted to investigate the efficacy of 4-wks treatment with Linplants (subcutaneous (s.c.) sustained-release bovine insulin implants) alone and in combination with ActRapid® (s.c. human insulin; 0.05 U to 3.5 U/100 g/day) with respect to glycaemic control in STZ-diabetic Wistar rats, and on acute diabetes characteristics for a 5-wk post-STZ administration period. Briefly, STZ-diabetic rats were divided into three groups: (1) rats which received no insulin treatment, (2) rats which were implanted with one s.c. Linplant at Day 7 post-STZ administration, and (3) rats which received one s.c. Linplant plus a once-daily injection of ActRapid® once diabetes was confirmed at 7-days post-STZ administration. The findings were that following implantation of a single Linplant at Day 7 post-STZ administration, euglycaemia was achieved in 50% of STZ-diabetic rats, with glycaemic control maintained for up to 4-wks post-implantation. Furthermore, once-daily injection of ActRapid™ to animals whose blood glucose levels (BGLs) were not well-controlled through use of Linplants alone, failed to achieve euglycaemia. It is possible that the ActRapid™ doses administered were not sufficient to achieve euglycaemia, and that increasing the doses may provide more effective glycaemic control. However, doubling the mean ActRapid™ dose from 1.63 (+ 0.3) U administered at Day 28 to 2.56 (+ 0.6) U administered at Day 34 post-STZ administration effectively only reduced BGLs by 1.3 mM to 11.6 + 1.6 mM. This suggests that although administering additional large doses of ActRapid™ to STZ-diabetic rats may eventually achieve euglycaemia, this method would presumably not be a more efficient method in achieving euglycaemia compared with the use of dosage-adjustable s.c. Linplants. Group (1) STZ-diabetic rats which were not treated with insulin developed diabetic signs including polydipsia, hyperphagia, decreased rate of body weight gain, and mechanical allodynia. Group (2) rats in which insulin treatment from 7-days post-STZ administration restored euglycaemia and reversed polydipsia and hyperphagia, were protected against the development of mechanical allodynia and reduced weight gain for the 5-wk study duration, while rats from Group (3) with incomplete glycaemic control developed levels of polydipsia, hyperphagia, reduced weight gain and mechanical allodynia intermediate between rats in Groups (1) and (2). These findings collectively suggest a direct correlation between the level of glycaemic control and the extent to which mechanical allodynia, a defining symptom of PDN, develops. In Chapter Three, the findings from the preliminary 5-wk study in Chapter Two were used to design a 24-wk longitudinal study of the temporal development of mechanical allodynia and opioid hyposensitivity in STZ-diabetic Wistar rats for comparison with the findings of a similar study previously undertaken by our laboratory using STZ-diabetic Dark Agouti rats (Nielsen et al, 2007). Additionally, this study examined the effects of tight glycaemic control achieved through the use of insulin implants as a means of potentially preventing the development of mechanical allodynia and opioid hyposensitivity for up to 24 weeks in STZ-diabetic Wistar rats. Briefly, STZ-diabetic rats were divided into 3 groups: (1) non-insulin treated STZ-diabetic Dark Agouti rats to provide comparison data with our laboratory’s previously published data in this rat strain (Nielsen et al, 2007), (2) non-insulin treated STZ-diabetic Wistar rats to examine possible between-species differences, and (3) STZ-diabetic Wistar rats which were treated with adjustable-dose s.c. Linplants from Day 7 post-STZ administration to maintain euglycaemia for the remainder of the 24-wk study period. In this 24-wk longitudinal study in STZ-diabetic rats, body weight, 24-hr water intake, paw withdrawal thresholds (PWTs) and BGLs were monitored at fortnightly intervals in all animals in order to document possible temporal changes in the development of diabetic signs and mechanical allodynia in the hindpaws respectively. STZ-diabetic rats underwent 6-wkly opioid antinociceptive testing, using single bolus doses of each of morphine and oxycodone with a 2-3 day washout period between individual opioids in order to assess the potential influence of both diabetes and glycaemic control on opioid potency in these animals. The findings demonstrate that non-insulin treated STZ-diabetic rats of both strains exhibited a decreased rate of body weight gain and polydipsia, as well as progressive development of mechanical allodynia in the hindpaws and loss of morphine potency. Importantly, STZ-diabetic Wistar rats which were treated with insulin to maintain euglycaemia from Day 7 post-STZ administration failed to develop these diabetic symptoms for the duration of the 24-wk study period, highlighting the importance of chronic hyperglycaemia in the development of mechanical allodynia and morphine hyposensitivity in the STZ-diabetic rodent model of PDN. The research described in Chapter Four involved a 22-wk longitudinal study of the development of diabetes and its longterm sensory nerve complications, viz mechanical allodynia and opioid hyposensitivity, in the ZDF rodent model of Type 2 diabetes commencing at 7-wks of age. This study also examined the influence of four different diets fed to separate groups of ZDF rats from 7-wks age, on the time course for the development of diabetes, mechanical allodynia in the hindpaws and opioid hyposensitivity in these animals. Briefly, ZDF rats were sub-divided into four dietary groups, each of which was fed one of the four following diets for 22-wks commencing at 7-wks of age, viz: (a) Purina 5008™, (b) a domestically-produced rat chow of similar composition to Purina 5008 (termed Purina Composition diet), (c) a Diabetogenic diet, or (d) Standard Rat Chow. All rats underwent once-fortnightly measurement of BGLs, body weight, 24-hr water intake, and measurement of PWTs in the hindpaws. Additionally, ZDF rats underwent opioid antinociceptive testing, similar to that previously described for STZ-diabetic rats (Chapter Three), to investigate the influence of diabetes and dietary composition on the antinociceptive potency of single bolus doses of morphine and oxycodone administered at 6-weekly intervals over a 22-wk study period. The afore-mentioned data were compared with the respective data obtained from the pre-diabetic control group of ZDF rats that were euthanised at 7-wks of age prior to the development of hyperglycaemia. The results demonstrate that the ZDF rat develops mechanical allodynia in the hindpaws and opioid hyposensitivity in a temporal fashion, in a manner similar to that previously documented for the STZ-diabetic Wistar rat model of Type 1 diabetes (Chapter Three). For the four diets assessed, there did not appear to be significant differences between dietary groups with respect to the time course and extent of development of hyperglycaemia, mechanical allodynia or opioid hyposensitivity in the ZDF rat model of PDN. The study described in Chapter Five investigated the effect of both diabetes and dietary composition on opioid receptor mRNA expression in tissue samples collected from the five groups of ZDF rats used in the behavioural studies described in Chapter Four and outlined above. Briefly, mRNA expression for each of the - (MOP), - (DOP), and - (KOP) receptors were quantified in mid-brain and spinal cord tissues prepared from 29-wk old diabetic ZDF rats maintained on one of four diets from 7-wks age, and compared with the respective expression levels in samples prepared from pre-diabetic ZDF rats euthanised at 7-wks of age. Overall, the findings suggest that diabetes does not alter opioid receptor mRNA expression in the mid-brain or spinal cord of diabetic ZDF rats at 29-wks of age relative to the corresponding levels of mRNA expression in the mid-brain and spinal cord of pre-diabetic ZDF rats at 7-wks of age. Hence, the marked reduction in the anti-allodynic potency of morphine and oxycodone observed in diabetic ZDF rats at 29-wks of age relative to that observed in pre-diabetic ZDF rats at 7-wks of age (Chapter Four) does not appear to be associated with a decrease in opioid receptor mRNA expression. In Chapter Six, the effect of both advanced diabetes and dietary composition on opioid-agonist stimulated [35S]GTPγS binding was examined in spinal cord tissue membranes from the ZDF rat. Specifically, [35S]GTPγS binding assays were used to assess the ability of a -opioid ligand (DAMGO) to stimulate -opioid receptor coupling to inhibitory G proteins in homogenates prepared from spinal cord samples of 29-wk old ZDF rats maintained on one of four different diets from 7-wks age (Chapter Four), relative to [35S]GTPγS binding in homogenates prepared from spinal cord samples of pre-diabetic 7-wk old ZDF rats. As specific MOP agonist-stimulated [35S]GTPγS binding was significantly decreased in spinal cord homogenates from diabetic ZDF rats at 29-wks of age relative to that for pre-diabetic ZDF rats (7-wks), this may contribute, at least in part, to the morphine hyposensitivity observed in diabetic ZDF rats at 29-wks of age relative to the pre-diabetic ZDF group. However, closer examination of these data revealed that specific MOP agonist-stimulated [35S]GTPγS binding above basal did not differ significantly between the pre-diabetic group and the longterm diabetic group of ZDF rats. Instead, there was significantly lower basal [35S]GTPγS binding in the spinal cord of ZDF rats at 29-wks c.f. 7-wks of age. Together, the findings suggest that impaired basal G-protein function rather than impaired coupling of MOP-r to its inhibitory G-protein may, at least in part, underpin -opioid agonist hyposensitivity in 29-wk ZDF rats. Finally, Chapter 7 contains a brief description of the main conclusions and discussion of the relevance of this doctoral research project, including potential future research directions.

Neuropatia é a complicação mais comum e mais debilitante da Diabetes Mellitus, a longo prazo presente em mais de 50% dos pacientes que possuem a doença. A hiperglicemia induz estresse oxidativo nos neurônios de diabéticos acarretando a ativação de múltiplas vias bioquímicas, as quais são potenciais alvos terapêuticos para a neuropatia diabética. Está claro que compostos carbonil reativos são mediadores glicotóxicos do estresse oxidativo através da formação de produtos finais de glicação avançada como resultado direto da hiperglicemia. Metilglioxal e glicolaldeído são compostos carbonil reativos inevitavelmente produzidos pelo metabolismo, os quais são encontrados em maior quantidade em situações de hiperglicemia. Recentemente, tem sido dada muita atenção para o envolvimento de espécies reativas na toxicidade do metilglioxal e do glicolaldeído, e tem-se demonstrado que essas glicotoxinas têm potencial para induzir estresse oxidativo, parar o crescimento celular e promover morte por apoptose ou necrose. O metilglioxal e o glicolaldeído interagem com grupamentos sulfidril de moléculas de glutationa e de enzimas, inibindo sua atividade; entretanto, os mecanismos moleculares relacionados aos efeitos tóxicos dessas glicotoxinas e as vias pelas quais elas levam a formação de espécies reativas não estão completamente elucidados. Neste estudo nós buscamos esclarecer a relação entre o metabolismo do metilglioxal e do glicolaldeído e a produção de espécies reativas, e investigamos as possíveis rotas de morte celular envolvidas. Utilizamos a linhagem celular de neuroblastoma humano SH-SY5Y diferenciada, pois este é um modelo neuronal bem caracterizado para estudos de compostos neurotóxicos. Nós avaliamos a produção de espécies reativas induzida por metilglioxal e glicolaldeído através da técnica da diclorofluoresceína, e avaliamos, também, seus efeitos sob o conteúdo de glutationa celular. Além disso, investigamos a ativação das caspase-3, -8 e -9 e a contribuição de diferentes sistemas peroxidases (glutationa-redutase e a tioredoxina-redutase), na defesa neuronal contra essas glicotoxinas. Como resultados encontramos que o tratamento com ambas glicotoxinas rapidamente provocou um aumento na produção de espécies reativas e diminuição do conteúdo de glutationa, com concomitante ativação das caspases-8 e -9 e, posteriormente, também houve ativação da caspase-3 pelo tratamento com metilglioxal. Vimos que a tioredoxina-redutase possui um papel mais importante na defesa celular contra a toxicidade do metilglioxal do que contra o glicolaldeído, enquanto que a glutationa-redutase tem papel semelhante na defesa celular contra ambas glicotoxinas. Nossos resultados demonstraram que o estresse oxidativo é um importante mecanismo da toxicidade do metilglioxal e do glicolaldeído nas células diferenciadas SHSY5Y e, que enzimas redutoras de grupamentos sulfidril contribuem de diferentes formas na defesa celular contra cada uma dessas glicotoxinas. / Neuropathy is the most common and debilitating complication of Diabetes Mellitus present in more than 50% of the patients with long-standing disease. Hyperglycemia induces oxidative stress in neurons from diabetic patients and results in activation of multiple biochemical pathways. These activated pathways are a major source of damage and are potential therapeutic targets in diabetic neuropathy. A large body of evidence has implicated reactive carbonyl compounds as glycotoxic mediators of oxidative stress by forming advanced glycation endproducts as a direct result of hyperglycemia. Methylglyoxal and glycolaldehyde are reactive carbonil compounds inevitably produced by the metabolism, but they are found in increased rates under hyperglycemia condition. Recently, the attention has been focused on the involvement of reactive species in methylglyoxal and glycolaldehyde toxicities, resulting in oxidative stress and leading to cell growth arrest, apoptotic or necrosis death. These glycotoxins interact with sulfhydryl-groups of glutathione molecules enzymes, inhibiting their activity; however, the molecular mechanism underlying methylglyoxal and glycolaldehyde cytotoxic effects and reactive species generation are not fully understood. In this study we have pursued to establish the role of methylglyoxal and glycolaldehyde metabolisms and reactive species production, and have looked for the possible death routes involved with the toxic effects of these glycotoxins. Here we used the differentiated human neuroblastoma SH-SY5Y cells as neuronal experimental model to investigate the pathological effects of various neurotoxic compounds. We have evaluated the methylglyoxal and glycolaldehyde capacity to reactive species generation by dichlorofluorescein assay and their effects upon cellular glutathione content. Also, we have assessed the caspase-3, -8 and -9 activation and the contribution of different peroxidases systems (glutathione reductase and thioredoxin reductase) in the neuronal defense against methylglyoxal and glycolaldehyde cytotoxicities. We found that both glycotoxins promptly provoke reactive species generation and decrease the cell glutathione content, as well induce caspase-8 and -9 activation. Later caspase-3 activation was found in methylglyoxal treatment. We demonstrate that thioredoxin reductase has a most important role in cell defense against methylglyoxal toxicity than against glycolaldehyde, meanwhile there is no difference in the glutathione reductase role. Our results show that oxidative stress is an important mechanism in the methylglyoxal and glycolaldehyde toxicities and sulfhydryl reductases contributes differently in the cellular defense against these glycotoxins.

Neuropatia é a complicação mais comum e mais debilitante da Diabetes Mellitus, a longo prazo presente em mais de 50% dos pacientes que possuem a doença. A hiperglicemia induz estresse oxidativo nos neurônios de diabéticos acarretando a ativação de múltiplas vias bioquímicas, as quais são potenciais alvos terapêuticos para a neuropatia diabética. Está claro que compostos carbonil reativos são mediadores glicotóxicos do estresse oxidativo através da formação de produtos finais de glicação avançada como resultado direto da hiperglicemia. Metilglioxal e glicolaldeído são compostos carbonil reativos inevitavelmente produzidos pelo metabolismo, os quais são encontrados em maior quantidade em situações de hiperglicemia. Recentemente, tem sido dada muita atenção para o envolvimento de espécies reativas na toxicidade do metilglioxal e do glicolaldeído, e tem-se demonstrado que essas glicotoxinas têm potencial para induzir estresse oxidativo, parar o crescimento celular e promover morte por apoptose ou necrose. O metilglioxal e o glicolaldeído interagem com grupamentos sulfidril de moléculas de glutationa e de enzimas, inibindo sua atividade; entretanto, os mecanismos moleculares relacionados aos efeitos tóxicos dessas glicotoxinas e as vias pelas quais elas levam a formação de espécies reativas não estão completamente elucidados. Neste estudo nós buscamos esclarecer a relação entre o metabolismo do metilglioxal e do glicolaldeído e a produção de espécies reativas, e investigamos as possíveis rotas de morte celular envolvidas. Utilizamos a linhagem celular de neuroblastoma humano SH-SY5Y diferenciada, pois este é um modelo neuronal bem caracterizado para estudos de compostos neurotóxicos. Nós avaliamos a produção de espécies reativas induzida por metilglioxal e glicolaldeído através da técnica da diclorofluoresceína, e avaliamos, também, seus efeitos sob o conteúdo de glutationa celular. Além disso, investigamos a ativação das caspase-3, -8 e -9 e a contribuição de diferentes sistemas peroxidases (glutationa-redutase e a tioredoxina-redutase), na defesa neuronal contra essas glicotoxinas. Como resultados encontramos que o tratamento com ambas glicotoxinas rapidamente provocou um aumento na produção de espécies reativas e diminuição do conteúdo de glutationa, com concomitante ativação das caspases-8 e -9 e, posteriormente, também houve ativação da caspase-3 pelo tratamento com metilglioxal. Vimos que a tioredoxina-redutase possui um papel mais importante na defesa celular contra a toxicidade do metilglioxal do que contra o glicolaldeído, enquanto que a glutationa-redutase tem papel semelhante na defesa celular contra ambas glicotoxinas. Nossos resultados demonstraram que o estresse oxidativo é um importante mecanismo da toxicidade do metilglioxal e do glicolaldeído nas células diferenciadas SHSY5Y e, que enzimas redutoras de grupamentos sulfidril contribuem de diferentes formas na defesa celular contra cada uma dessas glicotoxinas. / Neuropathy is the most common and debilitating complication of Diabetes Mellitus present in more than 50% of the patients with long-standing disease. Hyperglycemia induces oxidative stress in neurons from diabetic patients and results in activation of multiple biochemical pathways. These activated pathways are a major source of damage and are potential therapeutic targets in diabetic neuropathy. A large body of evidence has implicated reactive carbonyl compounds as glycotoxic mediators of oxidative stress by forming advanced glycation endproducts as a direct result of hyperglycemia. Methylglyoxal and glycolaldehyde are reactive carbonil compounds inevitably produced by the metabolism, but they are found in increased rates under hyperglycemia condition. Recently, the attention has been focused on the involvement of reactive species in methylglyoxal and glycolaldehyde toxicities, resulting in oxidative stress and leading to cell growth arrest, apoptotic or necrosis death. These glycotoxins interact with sulfhydryl-groups of glutathione molecules enzymes, inhibiting their activity; however, the molecular mechanism underlying methylglyoxal and glycolaldehyde cytotoxic effects and reactive species generation are not fully understood. In this study we have pursued to establish the role of methylglyoxal and glycolaldehyde metabolisms and reactive species production, and have looked for the possible death routes involved with the toxic effects of these glycotoxins. Here we used the differentiated human neuroblastoma SH-SY5Y cells as neuronal experimental model to investigate the pathological effects of various neurotoxic compounds. We have evaluated the methylglyoxal and glycolaldehyde capacity to reactive species generation by dichlorofluorescein assay and their effects upon cellular glutathione content. Also, we have assessed the caspase-3, -8 and -9 activation and the contribution of different peroxidases systems (glutathione reductase and thioredoxin reductase) in the neuronal defense against methylglyoxal and glycolaldehyde cytotoxicities. We found that both glycotoxins promptly provoke reactive species generation and decrease the cell glutathione content, as well induce caspase-8 and -9 activation. Later caspase-3 activation was found in methylglyoxal treatment. We demonstrate that thioredoxin reductase has a most important role in cell defense against methylglyoxal toxicity than against glycolaldehyde, meanwhile there is no difference in the glutathione reductase role. Our results show that oxidative stress is an important mechanism in the methylglyoxal and glycolaldehyde toxicities and sulfhydryl reductases contributes differently in the cellular defense against these glycotoxins.

Neuropatia é a complicação mais comum e mais debilitante da Diabetes Mellitus, a longo prazo presente em mais de 50% dos pacientes que possuem a doença. A hiperglicemia induz estresse oxidativo nos neurônios de diabéticos acarretando a ativação de múltiplas vias bioquímicas, as quais são potenciais alvos terapêuticos para a neuropatia diabética. Está claro que compostos carbonil reativos são mediadores glicotóxicos do estresse oxidativo através da formação de produtos finais de glicação avançada como resultado direto da hiperglicemia. Metilglioxal e glicolaldeído são compostos carbonil reativos inevitavelmente produzidos pelo metabolismo, os quais são encontrados em maior quantidade em situações de hiperglicemia. Recentemente, tem sido dada muita atenção para o envolvimento de espécies reativas na toxicidade do metilglioxal e do glicolaldeído, e tem-se demonstrado que essas glicotoxinas têm potencial para induzir estresse oxidativo, parar o crescimento celular e promover morte por apoptose ou necrose. O metilglioxal e o glicolaldeído interagem com grupamentos sulfidril de moléculas de glutationa e de enzimas, inibindo sua atividade; entretanto, os mecanismos moleculares relacionados aos efeitos tóxicos dessas glicotoxinas e as vias pelas quais elas levam a formação de espécies reativas não estão completamente elucidados. Neste estudo nós buscamos esclarecer a relação entre o metabolismo do metilglioxal e do glicolaldeído e a produção de espécies reativas, e investigamos as possíveis rotas de morte celular envolvidas. Utilizamos a linhagem celular de neuroblastoma humano SH-SY5Y diferenciada, pois este é um modelo neuronal bem caracterizado para estudos de compostos neurotóxicos. Nós avaliamos a produção de espécies reativas induzida por metilglioxal e glicolaldeído através da técnica da diclorofluoresceína, e avaliamos, também, seus efeitos sob o conteúdo de glutationa celular. Além disso, investigamos a ativação das caspase-3, -8 e -9 e a contribuição de diferentes sistemas peroxidases (glutationa-redutase e a tioredoxina-redutase), na defesa neuronal contra essas glicotoxinas. Como resultados encontramos que o tratamento com ambas glicotoxinas rapidamente provocou um aumento na produção de espécies reativas e diminuição do conteúdo de glutationa, com concomitante ativação das caspases-8 e -9 e, posteriormente, também houve ativação da caspase-3 pelo tratamento com metilglioxal. Vimos que a tioredoxina-redutase possui um papel mais importante na defesa celular contra a toxicidade do metilglioxal do que contra o glicolaldeído, enquanto que a glutationa-redutase tem papel semelhante na defesa celular contra ambas glicotoxinas. Nossos resultados demonstraram que o estresse oxidativo é um importante mecanismo da toxicidade do metilglioxal e do glicolaldeído nas células diferenciadas SHSY5Y e, que enzimas redutoras de grupamentos sulfidril contribuem de diferentes formas na defesa celular contra cada uma dessas glicotoxinas. / Neuropathy is the most common and debilitating complication of Diabetes Mellitus present in more than 50% of the patients with long-standing disease. Hyperglycemia induces oxidative stress in neurons from diabetic patients and results in activation of multiple biochemical pathways. These activated pathways are a major source of damage and are potential therapeutic targets in diabetic neuropathy. A large body of evidence has implicated reactive carbonyl compounds as glycotoxic mediators of oxidative stress by forming advanced glycation endproducts as a direct result of hyperglycemia. Methylglyoxal and glycolaldehyde are reactive carbonil compounds inevitably produced by the metabolism, but they are found in increased rates under hyperglycemia condition. Recently, the attention has been focused on the involvement of reactive species in methylglyoxal and glycolaldehyde toxicities, resulting in oxidative stress and leading to cell growth arrest, apoptotic or necrosis death. These glycotoxins interact with sulfhydryl-groups of glutathione molecules enzymes, inhibiting their activity; however, the molecular mechanism underlying methylglyoxal and glycolaldehyde cytotoxic effects and reactive species generation are not fully understood. In this study we have pursued to establish the role of methylglyoxal and glycolaldehyde metabolisms and reactive species production, and have looked for the possible death routes involved with the toxic effects of these glycotoxins. Here we used the differentiated human neuroblastoma SH-SY5Y cells as neuronal experimental model to investigate the pathological effects of various neurotoxic compounds. We have evaluated the methylglyoxal and glycolaldehyde capacity to reactive species generation by dichlorofluorescein assay and their effects upon cellular glutathione content. Also, we have assessed the caspase-3, -8 and -9 activation and the contribution of different peroxidases systems (glutathione reductase and thioredoxin reductase) in the neuronal defense against methylglyoxal and glycolaldehyde cytotoxicities. We found that both glycotoxins promptly provoke reactive species generation and decrease the cell glutathione content, as well induce caspase-8 and -9 activation. Later caspase-3 activation was found in methylglyoxal treatment. We demonstrate that thioredoxin reductase has a most important role in cell defense against methylglyoxal toxicity than against glycolaldehyde, meanwhile there is no difference in the glutathione reductase role. Our results show that oxidative stress is an important mechanism in the methylglyoxal and glycolaldehyde toxicities and sulfhydryl reductases contributes differently in the cellular defense against these glycotoxins.

Fundação de Apoio a Pesquisa e à Inovação Tecnológica do Estado de Sergipe - FAPITEC/SE / Introduction: Sickle cell disease (SCD) is the most common genetic disease in the world. The vaso-oclusion and hemolysis are the central findings of the disease and therefore receive great attention from the scientific community, since the acute painful episodes are very frequent and responsible for a large number of hospital admissions in the country. On the sidelines of this fact so important there are chronic painful conditions that are underestimated or little studied; and perhaps this problem is the definitive diagnosis and appropriate treatment of this disease. Objectives: Identificate through a pain assessment to evaluate the occurrence of neuropathic pain (NP) and discover clinical and epidemiological characteristics of neuropathic pain (NP) in patients with SCD. Methods: A total of 554 patients, 56 were recruited from university outpatient service, which is a regional reference for the treatment of NP, and a cross-sectional study conducted between July 2015 and March 2016, after established inclusion and exclusion criteria. The instrument used in this study was to NP detection range Leeds assessment of neuropathic symptoms and signs (LANSS). Demographic and clinical data were extracted from patient records and during the interview. The scales were applied according to the rules of its creators to detect evidence of NP. Chi-square test or Fisher's exact test were applied to compare the groups with and without NP depending on variables: teens (14-18 years) versus adults (19-32 years), sex and use of hydroxyurea. Age values were compared in patients with and without NP through ANOVA and Kruskal-Wallis tests, considering significant p values <0.05. Results: The average overall age was 20.6 years and 51.8% were male. The most common site of pain is the lower back (53.6%). 14 patients (25%) had evidence of NP. Age was positively correlated with NP, with higher rates among adults compared to teens (92.9 versus 7.1%; p < 0.01). The mean age compared in the presence or absence of NP is also significantly higher than in the first group (p < 0.05). There was a positive association between patients using hydroxyurea and presence of NP (71.4 versus 28.6%; p < 0.05). An association was observed between the presence of sensory changes identified through physical LANSS the mini-exam and final score of the corresponding LANSS the NP (p < 0.01). None of the identified patients had any treatment for NP. Conclusion: In a simple way you can establish the diagnosis of NP in SCD holders and to identify the clinical and epidemiological profile of this group. / Introdução: A doença falciforme (DF) é a enfermidade genética mais prevalente em todo o mundo. A vasoclusão e hemólise são os achados centrais da doença e, por isso, recebem uma grande atenção da comunidade científica, já que os episódios dolorosos agudos são muito freqüentes e responsáveis por grande número de internações no país. À margem deste fato tão importante, há quadros dolorosos crônicos que são subestimados ou pouco estudados; e talvez, a solução deste problema esteja no diagnóstico definitivo e no tratamento adequado desta enfermidade. Objetivo: Identificar, através de um questionário de avaliação de dor, a ocorrência de dor neuropática (DN) na DF e estabelecer os perfis clínico e epidemiológico do paciente com DN e DF. Método: De um total de 554 pacientes com DF, após estabelecidos critérios de inclusão e exclusão, 56 pacientes foram recrutados em serviço ambulatorial universitário referência em DF para um estudo transversal conduzido entre julho de 2015 e março 2016. O instrumento utilizado neste estudo para detecção de DN foi a escala Avaliação de Sintomas e Sinais em Dor Neuropática (LANSS). Dados demográficos e clínicos foram extraídos do prontuário do paciente e durante a entrevista. A escala foi aplicada conforme as normas dos seu criador para detectar evidência de DN. Teste Qui-quadrado ou exato de Fisher foram aplicados para comparar os grupos com e sem DN em função das variáveis: adolescentes (14 a 18 anos) e adultos (19 a 32 anos), sexo e uso de hidroxiuréia. Valores de idade foram comparados nos grupos com e sem DN através dos testes ANOVA ou Kruskal-Wallis, considerando-se significativos os valores de p<0,05. Resultados: Dentre os pacientes, 25% tinham evidência de DN. A média de idade geral foi de 20,6 anos e 51,8% eram do sexo masculino. O local mais frequente de dor foi a região lombar (53,6%). Idade correlacionou-se positivamente com DN, com taxas maiores entre adultos em relação aos adolescentes (92,9 versus 7,1%; p<0,01). A média de idade, quando comparada em relação à presença ou ausência de DN, também é significativamente superior no grupo com DN (p<0,05). Houve associação positiva entre pacientes que usavam hidroxiuréia e presença de DN (71,4 versus 28,6%; p<0,05). Observou-se associação entre presença de alterações sensoriais identificadas através do mini-exame físico do LANSS e pontuação final do LANSS correspondente a DN (p<0.01). Nenhum dos pacientes identificados fazia qualquer tipo de tratamento para DN. Conclusão: De uma forma simples é possível estabelecer diagnóstico de DN em portadores de DF além de identificar o perfil clínico e epidemiológico deste grupo.

Introdução: A hanseníase é a principal causa infecciosa de neuropatia periférica e consequentes incapacidades em todo o mundo. Seu diagnóstico, na maioria das vezes é simples, especialmente quando as clássicas lesões cutâneas estão presentes. No entanto, alguns pacientes apresentam apenas envolvimento neural (forma neural pura - PNL) transformando o seu diagnóstico em um grande desafio. Nesses casos, mesmo quando essa possibilidade é aventada, sua confirmação pode ser extremamente difícil e muitos pacientes só serão corretamente diagnosticados tardiamente, quando uma neuropatia grave e irreversível já está estabelecida. Objetivos: Analisar as características de uma série de pacientes com diagnóstico definitivo ou provável de PNL seguidos no setor de doenças neuromusculares e dermatologia no HCRP em um período de 12 anos e reconhecer o padrão de apresentação mais frequente, suas manifestações clínicas e o padrão eletroneuromiográfico. Métodos: Estudo retrospectivo, observacional, cujos critérios de inclusão foram: evidência clínica de comprometimento de nervos periféricos na ausência de lesões de pele. O diagnóstico definitivo foi estabelecido quando o Mycobacterium leprae foi identificado na biópsia de nervo, e provável quando um quadro clínico sugestivo foi associado a pelo menos um dos seguintes: anti PGL1 positivo, padrão sugestivo na biópsia (neurite granulomatosa epitelióide, infiltrado linfomomononuclear, fibrose) e/ou padrão eletroneuromiográfico sugestivo. Para avaliar a importância da duração da doença na apresentação clínica, foram considerados dois grupos de acordo com o tempo da doença, 12 meses ou menos (grupo 1) e mais de 12 meses (grupo 2). Foram comparados os sinais, os sintomas, a gravidade da doença e o padrão da EMG para delinear o quadro de apresentação. Resultados: Dos 34 pacientes incluídos no estudo, 7 tinham diagnóstico definitivo e 24 diagnóstico provável. Os sintomas de início mais frequentes foram alterações sensitivas (91,2%), em 70,6% dos casos iniciaram nos membros superiores, sendo o nervo ulnar o local mais frequente. O padrão de distribuição intradérmico exclusivo foi observado apenas no grupo 1. A alteração da sensibilidade vibratória (p=0,07), a presença de alterações motoras (p=0,03) e hipo ou areflexia em 1 ou mais nervos (p=0,03) foram mais frequentemente observadas no grupo 2. Os nervos sensitivos mais frequentemente envolvidos foram o ulnar e fibular superficial. O nervo motor mais frequentemente afetado foi o ulnar. O padrão eletroneuromiográfico mais frequente foi de uma neuropatia sensitivo motora assimétrica com reduções focais da velocidade de condução e franco predomínio sensitivo. Conclusões: A PNL se apresenta invariavelmente de maneira assimétrica e com franco predomínio sensitivo. Na maioria das vezes o início ocorre nos membros superiores, especificamente no território do nervo ulnar. Há uma predisposição ao acometimento das fibras finas nos estágios iniciais e com a evolução da doença as fibras grossas passam a também serem afetadas. Os nervos sensitivos mais frequentemente envolvidos são o ulnar seguido pelo fibular superficial. / Backgrounds: Leprosy is the main infectious cause of peripheral neuropathy and disabilities in the world. Its diagnosis is straightforward when the classical skin lesions are present. However, some patients present only neural involvement (pure neural form-PNL) turning its diagnosis on a great challenge. Additionally, even when this possibility is suspected, confirmation may be extremely difficult and many patients are only correctly diagnosed late on the clinical course of the disease when a severe and irreversible neuropathy is already established. Objectives: To review the characteristics of a series of PNL patients followed in our institution in the last 12 years and recognize the clinical manifestations. Methods: Inclusion criteria: Clinical evidence of peripheral nerve impairment with no skin lesions. PNL diagnose were classified as definitive when the Mycobacterium leprae was identified under nerve biopsy, and probable when a suggestive clinical picture was associated to at least one of the following: positive anti PGL1, suggestive pattern biopsy represented by the presence of epithelioid granulomatous neuritis, mononuclear cell endoneuritis and fibrose and/or an EMG pattern showing a predominantly sensory mononeuritis multiplex pattern. Exclusion criteria: Two patients were excluded because of associated diabetes mellitus, one because had CMT1A and another had HNPP. To evaluate the importance of disease duration in clinical presentation, we considered two groups according to the time course, first that disease duration of 12 or fewer months (group 1) and those with disease duration over 12 months (group 2). Results: We reviewed 34 patients with PNL, including 7 with a definite and 24 with probable diagnosis. The most common onset symptoms were sensory (91.2 %), in 70.6 % of cases symptoms started in the upper limbs, the ulnar nerve being the most frequent site. Intradermal pattern was observed only in group 1. Vibration was altered more frequent in group 2 (p=0.07), the presence of motor abnormalities (p = 0.03) and deep tendon reflexes reduced or absent in 1 or more nerves (p = 0.03) were more frequently observed in group 2. Sensory nerves most frequently involved were the ulnar and superficial peroneal. The motor nerve most often affected was the ulnar. The most frequent EMG pattern was an asymmetrical sensory and motor neuropathy with focal slowing of conduction velocity. Conclusions: PNL is an asymmetrical sensory or sensory motor neuropathy. Upper limbs are most frequent affected with frequent ulnar nerve territory involvement. Small fibers seem to be affected at early stages. Larger fibers are affected with disease progression. It is unclear whether the PNL represents a stage prior to the appearance of typical skin lesions or whether it represents a different and more aggressive leprosy type. Phenotype characterization from early signs and symptoms its a powerful tool to PNL early diagnosis.

O Diabetes mellitus (DM) é o transtorno endócrino mais comum e afeta 6% da população mundial. A neuropatia diabética é uma das complicações do DM e acomete até 60% dos doentes diabéticos. Este é um estudo descritivo que teve como objetivo avaliar as condições odontológicas e a prevalência da ardência bucal nos doentes diabéticos tipo 2 com neuropatia diabética. Foram utilizados os seguintes instrumentos de avaliação: exame sensitivo padronizado da face (algiometria), questionário RDC/TMD eixos 1 e 2, (critérios de diagnóstico em pesquisa) para o diagnóstico de disfunção temporomandibular (DTM), protocolo de avaliação de dor orofacial (EDOF-HC), questionário McGill para avaliação de dor e exame periodontal (IS índice de sangramento, PCS profundidade clínica de sondagem, PCI profundidade clínica de inserção). A prevalência de dor orofacial foi 55,2% e de ardor bucal foi 17,2%. Os valores de glicemia em jejum e HbA1c apresentaram-se acima dos valores normais. Dos doentes avaliados, 44,8% eram totalmente desdentados, 41,2% dos doentes apresentaram doença periodontal avançada, prevalência de dor miofascial mastigatória foi 31%. Traços de depressão moderada e grave e de sintomas físicos inespecíficos moderados e graves foram encontrados em 51,7% dos doentes, de incapacidade baixa e moderada em 37,9%. Observou-se comprometimento da sensibilidade dolorosa em V2 lado D (p=0,007), correlacionada com os valores de HbA1c. / Diabetes mellitus (DM) is one of the most common metabolic diseases affecting 6% of the world population. Diabetic neuropathy is one of DM complications and it affects up to 60% of diabetic patients. This descriptive study aimed to evaluate the dental characteristics and burning mouth prevalence of type 2 DM patients. The following instruments were used: pain sensory test, RDC/TMD questionnaire axis 1 and 2 (research diagnostic criteria for temporomandibular disorders), EDOF-HC protocol (for orofacial pain), Mcgills pain questionnaire and periodontal evaluation (bleeding index, clinical probing depth, clinical probing insertion). Orofacial pain was found in 55,2% of the patients, burning mouth was found in 17,2%. Fasting blood glucose and HbA1c levels were found to be higher than the recommended. 44,8% of the patients were totally edentulous, severe periodontal disease prevalence was 41,2%. Masticatory myofascial pain was found in 31% of the evaluated patients. Traces of severe and moderate depression and severe and moderate nonspecific physical symptoms were found in 51,7% of the patients; moderate and low disability in 37,9%. There was significative difference in the algometry values of the V2 right side compared to the left side and to the study group (p=0,007), with positive correlation with the increased values of the HbA1c.

Introdução: A amiloidose transtiretina é uma doença autossômica dominante decorrente de uma proteína transtiretina (TTR) variante, que sofre uma mudança conformacional e origina um tetrâmero de TTR instável, passo que é decisivo para o início da formação dos depósitos amilóides em diferentes órgãos e tecidos. Na maioria dos pacientes, o sistema nervoso periférico é o alvo principal, resultando na polineuropatia amiloidótica familiar transtiretina (TTR-FAP), classicamente uma neuropatia sensitivo-motora e autonômica progressiva, evoluindo para o óbito em aproximadamente 10 anos. A mutação de ponto mais frequente no mundo, incluindo o Brasil, é a TTRVal30Met, entretanto mais de 100 mutações de ponto diferentes já foram descritas. Objetivos: descrever a epidemiologia mutacional do gene TTR na polineuropatia amiloidótica familiar e correlacionar estas mutações com seus achados clínicos e eletroneuromiográficos. Métodos: estudo de coorte, descritivo e retrospectivo de um grupo de pacientes brasileiros encaminhados para o serviço de neurogenética do HC da FMRP-USP para investigação de neuropatia periférica, cujo estudo genético identificou uma mutação no gene TTR, com posterior análise transversal dos resultados obtidos entre os subgrupos com as diferentes mutações. Resultados: um total de 128 pacientes tiveram uma mutação de ponto no gene TTR identificada, dos quais 12 (9,4%) pacientes apresentaram uma mutação não TTRVal30Met, incluindo 4 patogênicas (6 pacientes, 4,7%) e 2 não patogênicas (6 pacientes, 4,7%). A mutações não TTRVal30Met patogênicas foram TTRAsp38Tyr (2 pacientes), TTRIle107Val (2 pacientes), TTRVal71Ala (1 paciente) e TTRVal122Ile (1 paciente). Dentre as mutações não patogênicas, foram encontradas TTRGly6Ser (5 pacientes) e TTRThr119Thr (1 paciente). A mutação TTRVal30Met estava presente em 116 (90,6%) pacientes, dos quais 52 possuíam dados clínicos e eletroneuromiográficos completos: 39 (75%) tiveram início precoce e 13 (25%), início tardio. O grupo de início precoce apresentou-se como a forma clássica da PAF-TTR, sem predileção de gênero (homens: 53,8%), manifestação inicial como neuropatia de fibras finas e autonômica (82,1%) e história familiar positiva (90,3%). A ENMG estava normal em 36,7% destes pacientes. O envolvimento cardiovascular foi caracterizado mais frequentemente por alterações da condução cardíaca (84,2%), sendo menos prevalente a cardiomiopatia (11,1%). Por outro lado, o grupo de início tardio mostrou uma predominância do sexo masculino (92,3%), presença de sintomas motores na primeira consulta (38,5%), resultando numa neuropatia sensitivo-motora com acometimento de fibras grossas e história familiar negativa (69,2%). Todos apresentaram neuropatia sensitivo-motora na ENMG. Neste grupo, a cardiomiopatia estava presente em 71,4% dos pacientes. Todos os pacientes, em ambos os grupos, tiveram disautonomia em algum momento do seu seguimento clínico. Conclusões: no nosso estudo aproximadamente 5% dos pacientes com FAP-TTR tinham uma mutação não TTRVal30Met, demonstrando a importância do sequenciamento do gene TTR em pacientes com história clínica sugestiva e screening negativo para a mutação TTR Val30Met. Além disso, os pacientes brasileiros com FAP-TTRVal30Met apresentaram achados clínicos e eletroneuromiográficos similares as populações descritas com esta mutação em outros países. / Background: Transthyretin amyloidosis is an autossomal dominant disease caused by variant transthyretin, that is misfolded, originating a unstable transthyretin tetramer, a rate-limiting step in the formation of the amyloid deposits in different organs and tissues. In most patients, the peripheral nervous system is the main target, leading to transtyretin familial amyloid neuropathy (TTR-FAP), classically characterized as a progressive sensory-motor and autonomic neuropathy, that leads to death in about 10 years. TTRVal30Met is the most frequent point mutation worldwide, including Brazil, but more than 100 different point mutations has been described. Objectives: describe the mutational epidemiology of TTR gene in TTR-FAP and characterize its clinical and electrophysiological findings. Methods: a descriptive and retrospective study of a group of Brazilian patients forwarded to the Neurogenetics or Peripheral Nerve Clinics from FMRP-USP whose etiological investigation identified a mutation in the TTR gene. A cross-sectional analysis evaluating the subgroups with different mutations was also carried on. Results: we identified one hundred and twenty eight patients carrying a TTR point mutation, of whom 12 (9,4%) harbored a non-Val30Met mutation, including 4 pathogenic (6 patients, 4,7%) and 2 non-pathogenic abnormalities (6 patients, 4,7%). The non Val30Met pathogenic mutations were TTRAsp38Tyr (2 patients), TTRIle107Val (2 patients), TTRVal71Ala (1 patient) and TTRVal122Ile (1 patient). Among the non-pathogenic mutations, we found the TTRGly6Ser (5 patients) and the TTRThr119Thr (1 patient). The TTRVal30Met mutation was present in 116 (90,6%) patients, of whom 52 had a complete clinical and neurophysiological data: 39 (75%) with early-onset and 13(25%) with late-onset neuropathies. The early-onset group presented as the classic TTRFAP, with no gender predominance (male: 53,8%), the first manifestations were those of a small fiber sensory and autonomic neuropathy (82,1%) and a highly positive family history (90,3%). EMG was normal in 36,7% of these patients. The cardiovascular involvement was characterized by frequent ECG abnormalities (84,2%), less often associated with cardiomayopathy (11,1%). On the other hand, the late-onset TTRVal30Met showed a male predominance (92,3%), presence of motor complaints in the first evaluation (38,5%) resulting in a sensory-motor polyneuropathy with large fiber involvement and a negative family history (69,2%). All patients presented a sensory and motor neuropathy on EMG examination. In this group, cardiomiopathy was frequently associated with the neuropathy (71,4%). All patients, in both groups, had autonomic symptoms at some point in clinical follow up. Conclusions: In our study almost 5% of the patients with TTR-FAP have a non Val30Met pathogenic mutation, highlighting the importance of sequecing the whole TTR gene in patients with a sugestive clinical history and negative screening for TTRVal30Met mutation. In adition, the Brazilian patients we studied with early and late onset TTR-FAP, present similar findings to TTRVal30Met populations from other countries submitted to similar studies.

Le modèle murin le plus utilisé pour le diabète de type 1 est la souris NOD. L’activation des lymphocytes T autoréactifs vis à vis des cellules béta nécessite la reconnaissance par le TCR de l’auto antigène présenté par le CMH ainsi que des signaux de co stimulation. Nous apportons la preuve que la voie de costimulation ICOS/ICOSL est indispensable au développement du diabète chez la souris NOD. En effet, les souris invalidées pour le gène Icos ou IcosL sont protégées du diabète. Nous avons démontré que cette protection est liée à un défaut d’activation des LT diabétogènes. De façon inattendue, nous avons observé chez ces souris ICOS-/- et ICOSL-/- une neuromyopathie. Cette pathologie se développe parallèlement au diabète chez la souris ICOSL+/+. Sur le plan histologique, le muscle strié périphérique et le nerf périphérique est envahi par un infiltrat lymphocytaire et par des cellules présentatrices d’antigène. Nous avons démontré par des expériences de transfert adoptif que la neuromyopathie est une maladie auto-immune données, nous avons étudié les souris NOD ICOSL-/- CIITA-/-. Ces souris sont dépourvues de lymphocytes T -CD4+ et ne développent pas de neuromyopathie ni de diabète. De même, nous avons étudié les souris NOD ICOSL-/- béta2m-/-. Ces souris sont dépourvues de lymphocytes T-CD8+ et développent une neuromyopathie. Cette déviation de l’auto-immunité est liée à l’interaction entre les LT et les lymphocytes B via le signal ICOS/ICOSL. Nous avons prouvé via des expériences de transfert et de chimères que l’absence de signal ICOS/ICOSL entre les lymphocytes T et les lymphocytes B oriente l’auto-immunité vers le système nerveux périphérique et le muscle strié. Enfin, l’analyse du spectre de spécificité des anticorps présent chez la souris ICOSL-/- par western blot puis par spectrométrie de masse a précisé les cibles antigéniques de la myopathie. L’invalidation de la voie ICOS/ICOSL conduit donc à une déviation de l’auto-immunité du pancréas vers le muscle et le système nerveux périphérique. Ces données prouvent que la voie ICOS/ICOSL est indispensable à l’initiation du diabète, mais aussi au contrôle de l’auto-immunité / Costimulation pathways are described as central in T cell activation and the control of autoimmune responses. We previously reported that NOD mice that are deficient for the icosl gene are protected from diabetes, but instead develop a spontaneous autoimmune neuromyopathy. The general phenotype of the neuromyopathy observed in ICOSL-/- NOD mice is globally similar to that observed in ICOS-/- and ICOS-/-ICOSL-/- double knockout NOD mice. The neuromyopathy is observed in 100% of female mice by the age of 35 weeks. The neuropathy remains limited to the peripheral nerve tissue. The disease is characterized by an infiltration of immune cells: CD4+ T cells, CD8+ T cells, dendritic cells and B lymphocytes, but does not extend to the central nervous system. A similar infiltrate is seen in muscles. Autoimmune neuromyopathy can be transfer to naive recipients by T lymphocytes. Transfer is achieved in NOD.scid recipient mice by CD4+ T-cells, although not by CD8+ T-cells, isolated from 35 week old ICOSL-/- NOD. The predominant role of CD4+T-cells is further demonstrated in this model by the observation that CIITA-/-ICOSL-/- NOD mice do not developed the neuromyopathy. By contrast, ȕ2m-/-ICOSL-/- NOD mice develop a neuromyopathy. We obtained evidence (in chimeric mice) that the interaction between antigen-presenting cells (APC) and T lymphocytes via ICOS/ICOSL is a prerequisite to the development of diabetes, while the loss of the interaction between T lymphocytes and APC play a key role in the development of nervous and muscular autoimmunity. Finally, the spectrum analysis of antibodies specificity in mouse ICOSL-/- with Western blot and mass spectrometry indicated the antigenic targets of myopathy. Altogether, our data indicate that the deviation of autoimmunity in NOD mice from the pancreas to muscles and the peripheral nervous system in the absence of ICOS/ICOSL signal is dependent on the loss of the physiological interaction between T cells and APC

L'espèce canine (Canis lupus familiaris) rassemble plus de 350 races issues d'une sélection artificielle drastique menée par l'Homme au cours des derniers siècles. De ce fait, chaque race peut être considérée comme un isolat génétique développant chacune des affections génétiques de manière spontanée avec parfois de fortes fréquences. Ainsi, l'espèce canine constitue un modèle puissant pour identifier les gènes et allèles responsables de ces affections homologues à certaines maladies génétiques humaines. Mon premier projet a porté sur la recherche des causes génétiques de deux kératodermies plantaires chez le terrier irlandais et le dogue de Bordeaux. En combinant plusieurs analyses d'association (GWAS : « Genome Wide Association Study ») et des techniques de séquençage NGS (Next Generation Sequencing), un nouveau gène muté a été identifié pour le terrier irlandais. Concernant les dogues de Bordeaux, nous avons génotypé plus de 170 000 SNPs sur plus de 200 dogues. Une analyse de liaison génétique a permis d'identifier un locus de 20 Mb sur le chromosome 9 contenant un cluster de kératines. En combinant les données cliniques et génétiques, la kératine 16 s'avérait le meilleur gène candidat. Son séquençage complet a permis d'identifier une mutation complexe générant une protéine tronquée. Des analyses de PCR quantitatives ont révélé que ce gène muté était sous exprimé chez les individus atteints. Ce travail nous permet donc de proposer cette race de chien comme le premier modèle animal spontané de kératodermie focale non-épidermolytique (FNEPPK). En parallèle de ce projet, j'ai mené des recherches sur les bases moléculaires d'un syndrome d'automutilation acrale décrit dans plusieurs races. Cette neuropathie est homologue aux neuropathies héréditaires sensitives et autonomes (HSANs) chez l'Homme et se caractérise par une perte de sensibilité à la douleur au niveau des membres. A partir d'un GWAS, j'ai pu identifier un locus de 1,5 Mb chez le chien, dont l'orthologue humain n'est pas encore décrit chez des patients HSANs. Le séquençage complet de ce locus a mené à l'identification d'un variant situé dans une région cis-enhancer en amont d'un gène candidat. La mise en évidence d'une sous-expression du gène candidat indique que ce variant régulateur semblerait modifier l'activité de l'enhancer nous permettant de le proposer comme la mutation causale chez le chien. Cette découverte offre donc de nouvelles perspectives de recherche pour des patients humains atteints de neuropathies héréditaires sensitives. En combinant des approches génétiques complémentaires sur des modèles spontanés de maladies bien caractérisées chez le chien, j'ai pu participer à l'identification de trois nouveaux gènes, dont deux excellents candidats pour ces maladies homologues humaines. Le troisième représente un vrai modèle spontané de kératodermie K16, ouvrant ainsi des perspectives thérapeutiques qui bénéficieront à l'Homme et au chien. / The dog species (Canis lupus familiaris) contains more than 350 distinct breeds resulting from human drastic selection during the last centuries. Each breed can then be considered as a genetic isolate, developing specific spontaneous genetic diseases with high frequencies. Thus, dogs constitute a powerful model to identify new genes and alleles involved in disorders homologous to human diseases. For my thesis, I worked on two main topics. The first one focused on the search of the genetic causes of two footpad keratodermas in the Irish terrier and the dogue de Bordeaux breeds. Concerning the Irish terrier, the work was conduced by Tosso Leeb’s team in the University of Berne, in collaboration with the Antagene Company. Using a Genome Wide Association Study (GWAS) and Next Generation Sequencing (NGS), the mutation in a new gene has been identified in footpad hyperkeratosis in this breed. For the dogue de Bordeaux project, we genotyped more than 170 000 SNPs on over 200 dogs. We then performed a genetic linkage study with a subset of 68 dogs, including 14 affected dogs. We identified a significant locus of 20 Mb on the canine chromosome 9 containing one keratin cluster. Comparing clinical, histopathological and immunochemistry data, keratin 16 appeared as an excellent candidate. The sequencing of the gene revealed a complex mutation leading to a non-functional truncated K16 protein. Quantitative RT-PCR analyses showed a strong decrease of the level of KRT16 expression in affected footpads. These results led to propose the dogue de Bordeaux footpad hyperkeratosis as the first spontaneous model of focal non-epidermolytic palmoplantar keratoderma (FNEPPK). In parallel, I studied an acral mutilation syndrome described in several hunting dog breeds. This neuropathy corresponds to human hereditary sensory and autonomic neuropathies (HSANs) and is characterized by an insensitivity to pain only in the limb. With a classical GWAS strategy, using 24 affected and 30 unaffected dogs, we identified a 1.5 Mb locus in dogs, the human orthologous locus being still unknown in human patients. Targeted sequencing of this locus revealed one single variant localized in a cis-enhancer region closed to a strong candidate gene. A lower level of expression of the candidate gene in affected dogs seems to show a functional effect of the mutation in the enhancer activity. These results led to propose this variant as the causative mutation for this canine neuropathy, and so this canine model as an opportunity to identify a new human HSAN gene. Combining complementary genetic approaches on spontaneous models of well characterized canine diseases, I did participated to the identification of three new genes, two of which being novel excellent candidates for the homologous human diseases. The third represents a true homologous model of KRT16 human keratoderma, opening the field to the development of new treatments benefiting to both humans and dogs.