京都大学 / 0048 / 新制・課程博士 / 博士(人間健康科学) / 甲第21306号 / 人健博第62号 / 新制||人健||5(附属図書館) / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 黒木 裕士, 教授 恒藤 暁, 教授 坂井 義治 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

A proteína quinase dependente de RNA (PKR) é uma molécula sentinela ativada em situações de estresse celular, incluindo infecções virais. A ativação de PKR por meio de sua fosforilação aciona cascatas de sinalização intracelular envolvidas em respostas inflamatórias e inibição da síntese protéica. Dados prévios do nosso laboratório sugerem que PKR está envolvida na hiperalgesia térmica de origem inflamatória. No presente estudo, foi investigado o papel da PKR na hiperalgesia térmica induzida pelo vírus da herpes simples tipo 1 (HSV1), durante as fases herpética e pós-herpética, combinando métodos comportamentais, genéticos, farmacológicos e moleculares. Camundongos C57bl/6, PKR+/+ e PKR-/- machos foram inoculados com HSV1. Os grupos controle foram inoculados com HSV1 inativo. Alodínia mecânica e hiperalgesia térmica foram monitoradas antes da inoculação do vírus e 8, 14, 21 e 28 dias após a inoculação. A curva dose e temporesposta e o teste da capsaicina foram realizados no 8º e 21º dias após a inoculação do vírus. Também nos períodos herpético e pós-herpético, foi investigado o perfil de expressão de proteínas envolvidas nas vias de sinalização de PKR (PKR, eIF2?, PACT, IKK e PP2A?), assim como o efeito da inibição de PKR pelo monitoramento da fosforilação de PKR, IKK?/?, P38, JNK, ERK1,2 e STAT3, e expressão de CaMKII? e TRPV1 nos GRD (L3-L6) ipsilateralmente à pata inoculada. Alodínia mecânica e hiperalgesia térmica ficaram evidentes até 28 dias após a inoculação. Camundongos PKR-/- desenvolveram alodínia mecânica, mas não hiperalgesia térmica, quando comparados com animais PKR+/+. A inibição sistêmica de PKR reverteu a hiperalgesia térmica de modo tempo- e dose-dependente e preveniu o comportamento nocifensivo induzido por capsaicina, enquanto PKR-/- apresentaram resposta nocifensiva praticamente ausente em ambas as fases herpética e pósherpética. Houve aumento da expressão de PP2A? e da fosforilação de PKR, IKK?/? e eIF2?, durante os períodos herpético e pós-herpético, e de PACT na fase pósherpética. A inibição de PKR promoveu o aumento da fosforilação de P38 em ambas as fases, e redução da fosforilação de PLC?1 acompanhada do retorno da fosforilação de Akt e STAT3 ao nível do grupo controle e o aumento da expressão de Ca-MKII? na fase herpética. Já na fase pós-herpética, reduziu a fosforilação de JNK e Akt e a expressão de Ca-MKII?, retornou a fosforilação de ERK1,2, PLC?1 e STAT3 ao nível do grupo controle e aumentou a expressão de TRPV1. Nossos resultados indicam que a atividade de PKR desempenha papel essencial na hiperalgesia térmica induzida por infecção pelo HSV1 / Double stranded RNA-activated protein kinase (PKR) is a sentinel molecule activated by cellular stress conditions, including viral infections. PKR activation by phosphorylation triggers cascades involved in inflammatory response and protein synthesis suppression. Our previous data suggest that PKR is involved in the inflammatory thermal hyperalgesia. Here we investigated the role played by PKR on thermal hyperalgesia induced by herpes simplex virus type-1 (HSV-1), during herpetic and post-herpetic phases, by combining behavioral, genetic, pharmacological, and molecular methods. Adult male C57bl/6, PKR+/+ and PKR-/- mice were inoculated with HSV-1. Control groups were inoculated with inactive (mock) HSV1. Mechanical allodynia and thermal hyperalgesia were monitored before virus inoculation and 8, 14, 21, and 28 days post-inoculation. The dose- and timeresponse curve and the capsaicin test were performed at 8th and 21st days post virus inoculation. Also in the herpetic and post-herpetic periods, was investigated the expression profile of proteins involved in the PKR signaling pathways (PKR, eIF2?, PACT, IKK and PP2A?), and the effect of PKR inhibition by monitoring PKR, IKK?/?, P38, JNK, ERK1,2, and STAT3 phosphorylation, and Ca-MKII? and TRPV1 expression in the dorsal root ganglia (L3-L6) ipsilaterally to the inoculated paw. Mechanical allodynia and thermal hyperalgesia became evident until 28 days postinnoculation. PKR-/- mice developed mechanical allodynia but not thermal hyperalgesia, when compared with PKR+/+ mice. Systemic PKR inhibition reversed thermal hyperalgesia in a dose and time-dependent manner, and prevented the capsaicin-induced nocifensive behavior, whereas PKR-/- showed no nocifensive behavior almost absent in both herpetic and post-herpetic phases. There was increased expression of PP2A? and the phosphorylation of PKR, IKK?/?, and eIF2?, during herpetic and post-herpetic periods, and PACT in the post-herpetic phase. PKR inhibition increased P38 phosphorylation in both phases, and reduction of PLC?1 phosphorylation together with the return of the Akt and STAT3 phosphorylation to the control group level, and enhanced Ca-MKII? expression in the herpetic phase. At the post-herpetic phase, suppressed JNK and Akt, and Ca-MKII? expression returned ERK1,2, PLC?1 and STAT3 phosphorylation to control group level and increased TRPV1 expression. The data indicate that PKR activity plays an essential role in the HSV-1 infection-induced thermal hyperalgesia

La chimiothérapie néoadjuvante (CNA) est une avancée majeure dans les traitements des cancers du sein. La CNA réduit nettement la tumeur primaire et permet une chirurgie conservatrice à tous les stades du cancer. Malheureusement, elle s’accompagne de dysfonctionnements neurologiques qui limitent le succès du traitement anti-tumoral. La prise en charge médicale de ces troubles neurologiques est problématique compte tenu de l’inefficacité des neuroprotecteurs disponibles. De plus, la recherche de nouvelles thérapeutiques est handicapée par l’absence de modèles expérimentaux reproduisant fidèlement les symptômes évoqués par la CNA séquentielle « épirubicine (EPI)/docétaxel (DO) » ou CNA-[EPI-DO] fréquemment utilisée. Ce travail de thèse a permis de caractériser pour la première fois un modèle animal pertinent de troubles neurologiques périphériques et centraux évoqués par la CNA-[EPI-DO]. Ce modèle préclinique a été utilisé pour révéler l’efficacité d’un traitement concomitant de duloxétine et d’alloprégnanolone contre la neuropathie périphérique douloureuse induite par la CNA-[EPI-DO]. Nos travaux montrent aussi que la duloxétine exerce une action bénéfique contre les déficits cognitifs évoqués par la CNA-[EPI-DO]. La thèse ouvre des perspectives prometteuses à explorer pour le développement de thérapies efficaces contre les altérations neurologiques CNA-induites. / Neoadjuvant chemotherapy (NAC) represents a major progress in breast cancer therapy. By shrinking significantly, the tumor volume, NAC allows conservative surgery at all stages of breast cancer. Unfortunately, NAC also induces neurological dysfunctions that jeopardize the chances of success of anti-tumor treatments. Therapeutic management of these neurological disorders remains a major concern because neuroprotective drugs currently available are not effective. Furthermore, investigations to characterize novel effective therapeutics are hampered by the lack of reliable experimental models mimicking the neurological symptoms evoked by the sequential epirubicin (EPI)/docetaxel (DO)-NAC or [EPI-DO]-NAC frequently used in humans. The present thesis work allowed the first characterization of a relevant animal model of [EPI-DO]-NAC-induced peripheral and central neurological disorders. This preclinical model has successfully been used to demonstrate the efficacy of duloxetine and allopregnanolone concomitant treatment against [EPI-DO]-NAC-evoked painful peripheral neuropathy. Our results also reveal a beneficial action of duloxetine against [EPI-DO]-NAC-induced cognitive deficits. The thesis opens promising perspectives to be explored for the development of effective therapies against [EPI-DO]-NAC-induced neurological alterations.

A proteína quinase dependente de RNA (PKR) é uma molécula sentinela ativada em situações de estresse celular, incluindo infecções virais. A ativação de PKR por meio de sua fosforilação aciona cascatas de sinalização intracelular envolvidas em respostas inflamatórias e inibição da síntese protéica. Dados prévios do nosso laboratório sugerem que PKR está envolvida na hiperalgesia térmica de origem inflamatória. No presente estudo, foi investigado o papel da PKR na hiperalgesia térmica induzida pelo vírus da herpes simples tipo 1 (HSV1), durante as fases herpética e pós-herpética, combinando métodos comportamentais, genéticos, farmacológicos e moleculares. Camundongos C57bl/6, PKR+/+ e PKR-/- machos foram inoculados com HSV1. Os grupos controle foram inoculados com HSV1 inativo. Alodínia mecânica e hiperalgesia térmica foram monitoradas antes da inoculação do vírus e 8, 14, 21 e 28 dias após a inoculação. A curva dose e temporesposta e o teste da capsaicina foram realizados no 8º e 21º dias após a inoculação do vírus. Também nos períodos herpético e pós-herpético, foi investigado o perfil de expressão de proteínas envolvidas nas vias de sinalização de PKR (PKR, eIF2?, PACT, IKK e PP2A?), assim como o efeito da inibição de PKR pelo monitoramento da fosforilação de PKR, IKK?/?, P38, JNK, ERK1,2 e STAT3, e expressão de CaMKII? e TRPV1 nos GRD (L3-L6) ipsilateralmente à pata inoculada. Alodínia mecânica e hiperalgesia térmica ficaram evidentes até 28 dias após a inoculação. Camundongos PKR-/- desenvolveram alodínia mecânica, mas não hiperalgesia térmica, quando comparados com animais PKR+/+. A inibição sistêmica de PKR reverteu a hiperalgesia térmica de modo tempo- e dose-dependente e preveniu o comportamento nocifensivo induzido por capsaicina, enquanto PKR-/- apresentaram resposta nocifensiva praticamente ausente em ambas as fases herpética e pósherpética. Houve aumento da expressão de PP2A? e da fosforilação de PKR, IKK?/? e eIF2?, durante os períodos herpético e pós-herpético, e de PACT na fase pósherpética. A inibição de PKR promoveu o aumento da fosforilação de P38 em ambas as fases, e redução da fosforilação de PLC?1 acompanhada do retorno da fosforilação de Akt e STAT3 ao nível do grupo controle e o aumento da expressão de Ca-MKII? na fase herpética. Já na fase pós-herpética, reduziu a fosforilação de JNK e Akt e a expressão de Ca-MKII?, retornou a fosforilação de ERK1,2, PLC?1 e STAT3 ao nível do grupo controle e aumentou a expressão de TRPV1. Nossos resultados indicam que a atividade de PKR desempenha papel essencial na hiperalgesia térmica induzida por infecção pelo HSV1 / Double stranded RNA-activated protein kinase (PKR) is a sentinel molecule activated by cellular stress conditions, including viral infections. PKR activation by phosphorylation triggers cascades involved in inflammatory response and protein synthesis suppression. Our previous data suggest that PKR is involved in the inflammatory thermal hyperalgesia. Here we investigated the role played by PKR on thermal hyperalgesia induced by herpes simplex virus type-1 (HSV-1), during herpetic and post-herpetic phases, by combining behavioral, genetic, pharmacological, and molecular methods. Adult male C57bl/6, PKR+/+ and PKR-/- mice were inoculated with HSV-1. Control groups were inoculated with inactive (mock) HSV1. Mechanical allodynia and thermal hyperalgesia were monitored before virus inoculation and 8, 14, 21, and 28 days post-inoculation. The dose- and timeresponse curve and the capsaicin test were performed at 8th and 21st days post virus inoculation. Also in the herpetic and post-herpetic periods, was investigated the expression profile of proteins involved in the PKR signaling pathways (PKR, eIF2?, PACT, IKK and PP2A?), and the effect of PKR inhibition by monitoring PKR, IKK?/?, P38, JNK, ERK1,2, and STAT3 phosphorylation, and Ca-MKII? and TRPV1 expression in the dorsal root ganglia (L3-L6) ipsilaterally to the inoculated paw. Mechanical allodynia and thermal hyperalgesia became evident until 28 days postinnoculation. PKR-/- mice developed mechanical allodynia but not thermal hyperalgesia, when compared with PKR+/+ mice. Systemic PKR inhibition reversed thermal hyperalgesia in a dose and time-dependent manner, and prevented the capsaicin-induced nocifensive behavior, whereas PKR-/- showed no nocifensive behavior almost absent in both herpetic and post-herpetic phases. There was increased expression of PP2A? and the phosphorylation of PKR, IKK?/?, and eIF2?, during herpetic and post-herpetic periods, and PACT in the post-herpetic phase. PKR inhibition increased P38 phosphorylation in both phases, and reduction of PLC?1 phosphorylation together with the return of the Akt and STAT3 phosphorylation to the control group level, and enhanced Ca-MKII? expression in the herpetic phase. At the post-herpetic phase, suppressed JNK and Akt, and Ca-MKII? expression returned ERK1,2, PLC?1 and STAT3 phosphorylation to control group level and increased TRPV1 expression. The data indicate that PKR activity plays an essential role in the HSV-1 infection-induced thermal hyperalgesia

Title: Electromyographic and clinical evaluation of vincristine-induced peripheral neuropathy in pediatric patients after treatment of acute lymphoblastic leukemia and correlation with the Bruinkins-Oseretsky Test of Motor Proficiency Second Edition Objectives: The aim of this work was to characterize the neurological consequences of vincristine-induced peripheral neuropathy (VIPN) clinically and electromyographically and to evaluate motor skills of pediatric patients after the end of treatment of acute lymphoblastic leukemia. We also determined the relationship between the results of the clinical and electromyographic evaluation of VIPN and the correlation with the results of motor skills tests. Methods: The study involved 35 probands (19 girls and 16 boys) with a mean age of 10.7 years (SD ± 4.3) and a mean time since the last dose of vincristine of 2.3 years (SD ± 1.2). VIPN was assessed using a clinical pediatric-modified Total Neuropathy Score (ped-mTNS) and nerve conduction studies (NCS). Motor skills were assessed using the Bruinkins-Oseretsky Test of Motor Proficiency, Second Edition (BOT-2). Results: The clinical presence of VIPN, according to ped-mTNS, was found in 20 % of probands. Abnormalities in nerve conduction studies were reported by 60.9 % of probands. Of these, 92.9 % had motor...

Microtubule targeting agents (MTAs) are chemotherapeutics commonly used in the treatment of breast, ovarian, lung, and lymphoma cancers. There are two main classes of MTAs based upon their effects on microtubule stability. The two classes are the destabilizing agents, which include the drug vincristine, and the stabilizing agents, which include paclitaxel and epothilone B. These drugs are highly effective antineoplastics, but their use is often accompanied by several side effects, one of which is peripheral neuropathy. Peripheral neuropathy can be characterized by burning pain, tingling, loss of proprioception, or numbness in the hands and feet. In some patients, the MTA-induced peripheral neuropathy is debilitating and dose-limiting; however, there are no effective prevention strategies or treatment options for peripheral neuropathy as the mechanisms mediating this side effect are unknown. The goal of this work was to investigate MTA-induced effects on neuronal activity and morphology in order to elucidate the underlying mechanisms involved in the development of MTA-induced peripheral neuropathy. As an indicator of sensory neuronal activity, the basal and stimulated release of the putative nociceptive peptide, calcitonin gene-related peptide (CGRP), was measured from sensory neurons in culture after exposure to the MTAs paclitaxel, epothilone B, and vincristine. Neurite length and branching were also measured in sensory neuronal cultures after treatment with these MTAs. The results described in this thesis demonstrate that MTAs alter the stimulated release of CGRP from sensory neurons in differential ways depending on the MTA agent employed, the CGRP evoking-stimulus used, the concentration of the MTA agent, the duration of exposure to the MTA agent, and the presence of NGF. It was also observed that MTA agents decrease neurite length and branching, independent of the concentration of NGF in the culture media. Thus, this thesis describes MTA-induced alterations of sensory neuronal sensitivity and neurite morphology and begins to elucidate the underlying mechanisms involved in MTA-induced alterations of sensory neurons. These findings will undoubtedly be used to help elucidate the mechanisms underlying MTA-induced peripheral neuropathy.

Diabetic Peripheral Neuropathy (DPN) is one of the most common types of diabetes that can affect the cornea. An accurate analysis of the corneal epithelium nerve structures and the corneal endothelial cell can assist early diagnosis of this disease and other corneal diseases, which can lead to visual impairment and then to blindness. In this thesis, fully-automated segmentation and quantification algorithms for processing and analysing sub-basal epithelium nerves and endothelial cells are proposed for early diagnosis of diabetic neuropathy in Corneal Confocal Microscopy (CCM) images. Firstly, a fully automatic nerve segmentation system for corneal confocal microscope images is proposed. The performance of the proposed system is evaluated against manually traced images with an execution time of the prototype is 13 seconds. Secondly, an automatic corneal nerve registration system is proposed. The main aim of this system is to produce a new informative corneal image that contains structural and functional information. Thirdly, an automated real-time system, termed the Corneal Endothelium Analysis System (CEAS) is developed and applied for the segmentation of endothelial cells in images of human cornea obtained by In Vivo CCM. The performance of the proposed CEAS system was tested against manually traced images with an execution time of only 6 seconds per image. Finally, the results obtained from all the proposed approaches have been evaluated and validated by an expert advisory board from two institutes, they are the Division of Medicine, Weill Cornell Medicine-Qatar, Doha, Qatar and the Manchester Royal Eye Hospital, Centre for Endocrinology and Diabetes, UK.

Peripheral neuropathies are a set of common diseases that affect the peripheral nervous system, causing damage to vital connections between various parts of the body and the brain and spinal cord. Different clinical conditions are known to selectively impact various size nerve fibers, which often makes it difficult to diagnose which peripheral neuropathy a patient might have. The nerve conduction velocity diagnostic test provides clinically useful information in the diagnosis of some peripheral neuropathies. This method is advantageous because it tends to be minimally invasive yet it provides valuable diagnostic information. However, this test does not determine characteristics of peripheral nerve fiber size distributions, and therefore does not show any detailed information regarding the nerve fibers within the nerve trunk. Being able to determine which nerve fibers are contributing to the evoked potential within a nerve trunk could provide additional information to clinicians for the diagnosis of specific pathologies of the peripheral nervous system, such as chronic inflammatory demyelinating polyneuropathy or early diabetic peripheral neuropathy. In this study, three rat sciatic nerves are sectioned and stained with hematoxylin and eosin in order to measure the nerve fiber diameters within the nerve trunk. Stained samples are viewed using brightfield microscopy and images are analyzed using ImageJ. Histograms were created to show the frequency of various nerve fiber diameters. The nerve fiber diameters measured during this research are consistent with the range of previously published diameter values and will be used to support continuing research for a novel method to characterize peripheral nerve fiber size distributions using group delay.