京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19593号 / 医博第4100号 / 新制||医||1014(附属図書館) / 32629 / 京都大学大学院医学研究科医学専攻 / (主査)教授 松岡 雅雄, 教授 朝長 啓造, 教授 西渕 光昭 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Cystoisospora canis is an Apicomplexan parasite of the small intestine of dogs. C. canis produces monozoic tissue cysts (MZT) that are similar to the polyzoic tissue cysts (PZT) of Toxoplasma gondii, a parasite of medical and veterinary importance, which can reactivate and cause toxoplasmic encephalitis. We hypothesized that C. canis is similar biologically and genetically enough to T. gondii to be a novel model for studying tissue cyst biology. We examined the pathogenesis of C. canis in beagles and quantified the oocysts shed. We found this isolate had similar infection patterns to other C. canis isolates studied. We were able to superinfect beagles that came with natural infections of Cystoisospora ohioensis-like oocysts indicating that little protection against C. canis infection occurred in these beagles. The C. canis oocysts collected were purified and used for future studies. We demonstrated in vitro that C. canis could infect 8 mammalian cell lines and produce MZT. The MZT were able to persist in cell culture for at least 60 days. We were able to induce reactivation of MZT treated with bile-trypsin solution. In molecular studies, we characterized C. canis genetically using ITS1 and CO1 to build phylogenetic trees and found C. canis was most similar to C. ohioensis-like with ITS1 and more similar to T. gondii than any other coccidia using ITS1 and CO1. We identified genes and proteins involved with virulence, cyst wall structure, and immune evasion of T. gondii and examined the DNA of C. canis for orthologs. C. canis had orthologs with 8 of 20 T. gondii genes examined. Monoclonal and polyclonal antibody and lectin studies demonstrated similar tissue cyst wall proteins on C. canis MZT and T. gondii PZT. Our findings in vitro and using genetic characterization of C. canis indicated the presence of similar genes and proteins, and its close phylogenetic location with T. gondii demonstrate that C. canis may serve as a model to examine tissue cyst biology. The system we described provides a simple model to produce tissue cysts and to study host factors that cause reactivation of tissue cysts. / Ph. D.

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) are major human pathogens. HSV establishes latency in the nervous system and reactivates to cause recurrent disease, resulting in transmission of progeny virions to naïve individuals. Though HSV-1 and HSV-2 share similar structure and genes, they have distinctive recurrence profiles. Generally, HSV-1 reactivation is associated with disease 'above the waist' and HSV-2 reactivation is associated with disease 'below the waist'. This phenomenon was described decades ago but still remains unexplained. The mechanism of HSV latent infection in the peripheral nervous system (PNS) has been extensively investigated, especially with in sensory neurons. Another component of the peripheral nervous system (PNS), autonomic neurons, were also known to be infected with HSV productively and latently, but largely ignored because of the assumption that there is no difference in the pathogenesis of HSV in the neurons and that both HSV-1 and HSV-2 behave in the same way in different types of neurons. However, autonomic neurons differ in physiological function compared to sensory neurons. Activation factors of autonomic neurons, such as emotional stress, trauma and hormonal fluctuation, are also known HSV reactivation triggering factors. Therefore, I hypothesized that autonomic neurons innervating the site of HSV infection are responsible the different reactivation frequencies of HSV-1 and HSV-2 after peripheral invasion. In this report, the role of autonomic neurons in HSV pathogenesis were examined using the female guinea pig reactivation model. Major findings of this report are that 1) parasympathetic ganglia innervating the ocular region support latent infection of HSV-1 selectively, thus contributing the more frequent HSV-1 reactivation, 2) mixed autonomic ganglia in the genital area support HSV-2 latent infection selectively, and 3) sympathetic neurons in the genital region supported productive and latent infection of HSV-1 and HSV-2 differently. All of the results in this report indicate that autonomic neurons play a distinctive role in HSV pathogenesis compared to the sensory neurons and are responsible for the different reactivation frequencies of HSV-1 and HSV-2. This report raises the importance of autonomic neurons in HSV pathogenesis and challenges the paradigm of HSV pathogenesis. / Ph. D.

The contrasting infection strategy of herpes simplex virus (HSV) consists of an initial primary lytic infection in epithelial cells, followed by establishment of lifelong latency in sensory and autonomic neurons of the peripheral nervous system that innervate the site of infection. Any cellular stress trigger, ranging from external stimuli such as UV radiation or nerve injury to psychological and physiological stress, can reactivate HSV from latency in the neurons, resulting in recurrent disease episodes. Stress hormones and deprivation of neurotrophic factor (NTF) both have a strong correlation with HSV reactivation from neurons. However, neuronal signaling pathways cardinal to HSV latency and reactivation are still not clear. This dissertation provides new understanding of HSV latency and reactivation in response to two orthogonal stress stimuli, viz. stress hormones epinephrine (EPI) and corticosterone (CORT), as well as NTF deprivation that simulates a nerve injury in primary neuronal cultures. In this dissertation, we demonstrate that physiological stress hormones EPI and CORT differentially regulate HSV-1 and HSV-2 reactivation in adult neurons. Both EPI and CORT treatment reactivated only HSV-1 in sympathetic superior cervical ganglia (SCG) neurons, while HSV-2 was reactivated only by CORT in both sensory trigeminal ganglia (TG) neurons and sympathetic superior cervical (SCG) neurons. EPI utilized the combination of α and β adrenergic receptor complex, while CORT signaled through glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) to reactivate HSV in the neurons. NTFs are tissue-target derived growth factors required for neuronal protection and survival. Neurotrophins are also required for maintaining HSV latency, as NTF deprivation reactivates both HSV-1 and HSV-2 in adult sensory TG and sympathetic SCG neurons. In addition, assessing the temporal kinetics of HSV gene expression showed differential expression profiles of viral immediate-early (IE) genes ICP0, ICP4, ICP27 and trans-activator VP16 following treatment with stress hormones and NTF deprivation in HSV-1 and HSV-2 infected neurons. We also show that different molecular mechanisms are involved in HSV latency and reactivation, which are dependent on the stimuli and the type of neurons. Tyrosine kinase receptor-mediated PI3K-Akt-mTORC signaling cascades have been studied for their role in maintaining HSV latency. Activation of β-catenin signalosome expression has also been implicated during HSV latency and following reactivation. GSK3β is a key effector molecule that inter-connects Akt and β-catenin mediated pathways, forming an Akt-GSK3β-β-catenin signaling axis. Analyzing the Akt-GSK3β-β-catenin signaling in response to stress hormone and NTF deprivation revealed significant differences in protein expression levels between HSV-1 and HSV-2 infected sensory and sympathetic neurons. In HSV-1 infected neurons, the Akt-GSK3β-β-catenin maintains the signal transmission in order to keep the neurons alive, but HSV-2 infections obliterated the entire axis in the adult neurons, particularly in sympathetic neurons. In summary, we demonstrate that HSV-1 and HSV-2 do not have a 'one for all' infection mechanism. Establishment of latency and reactivation by HSV is virus specific, stimulus specific and neuron specific. / Doctor of Philosophy / Herpes simplex viruses (HSVs) are common global viral pathogens that are responsible for causing lifelong painful infections and debilitating disease. The two serotypes of HSV include HSV-1, which is associated with oral or ocular disease but can also cause genital disease, and HSV-2, which is predominantly associated with genital herpes. Once infected, both HSV-1 and HSV-2 are present as lifelong reservoirs in our peripheral neurons. Stress stimuli mediated by our stress hormones or external triggers, such as nerve trauma or an axonal injury, can periodically reactivate the latent virus to cause recurrent disease. Clinical manifestation of HSV recurrences range from asymptomatic viral shedding to painful blisters, cold sores, or herpetic keratitis. In some cases, the virus can spread to the central nervous system, causing encephalitis or recurrent meningitis. No vaccines have been approved yet, and the current treatment utilizes nucleoside analogs, such as acyclovir and its prodrug valacyclovir, to ameliorate the symptoms of HSV infection by halting viral replication and if taken as a daily prophylaxis, reduces the chances of clinical recurrence. Given the route and transmission efficiency of HSV, it is practically impossible to prevent herpes infection. To develop strategic therapeutic interventions to lock the virus in its latent phase in the neurons and prevent it from reactivation, a better understanding of neuronal signaling pathways cardinal to HSV latency and reactivation is necessary. However, neuronal signaling pathways cardinal to HSV latency and reactivation are still not clear. In this dissertation, we make contributions to better understand HSV latency and reactivation in response to stress stimuli. We show that different stress stimuli exert preferential reactivation between HSV-1 and HSV-2, and are further dependent upon the neurons where they establish latency. Our study specifically focuses on three neuronal stressors that have been associated with HSV recurrences: two stress hormones, epinephrine (EPI) and corticosterone (CORT), as well as deprivation of neurotrophic factors (NTF) that simulates nerve injury. We also focused on a neuronal signaling cascade involved in the response to all of these stimuli, Akt-GSK3β-β-catenin, and viral gene transcripts that respond to these stimuli during reactivation. Comprehensive understanding of the neuronal processes and viral gene transcripts involved during HSV-1 and HSV-2 reactivation in neurons will help the herpes virology field towards development of targeted therapies and vaccines to prevent reactivation and recurrent disease.

Le sélénium est reconnu comme un micronutriment important pour l'homme et les animaux. Plusieurs études ont montré qu'une supplementation en sélénium dans le régime alimentaire pourrait être bénéfique contre les cancers du foie, du colon, du pancréas et de la prostate. Le mécanisme anti-carcinogène du sélénium se produit au niveau systémique, cellulaire et nucléaire. Ces processus peuvent également impliquer le système immunitaire et ne doivent pas être interprétés par un seul mécanisme. Jusqu'à présent son mécanisme d'action est encore inconnu. L'objectif de cette étude était d'étudier l'effet des composés du sélénium, à faibles concentrations, sur la capacité de réparation de l'ADN dans les cellules du cancer de la prostate LNCaP (p53 compétentes). Ce travail est divisé en trois parties. La première partie du travail a été consacrée à étudier l'effet des deux composés du sélénium (SS et SM) sur les propriétés cytotoxiques et génotoxiques de différents stress oxydatifs et non oxydatifs. Les résultats ont montré qu'un prétraitement avec une faible dose en Se stimulait la synthèse des sélénoprotéines, et protègait contre la toxicité et les dommages oxydatifs à l'ADN induites par les UVA ou H2O2, mais pas par MMS ou UVC. La deuxième partie a été consacrée à l'influence de la supplementation en sélénium sur la capacité de réparation de l'ADN. Notre travail a clairement montré l'augmentation de l'efficacité d'excision de certaines glycosylases que n'est pas nécessairement corrélée à une augmentation de l'expression génique et /ou protéiques. Enfin, la troisième partie de notre travail a été dédiée à l'optimisation de la technique Host Cell Reactivation (HCR) qui nous a permis d'étudier la capacité de réparation de l'ADN in cellulo, afin de cibler les partenaires impliqués dans la voie de signalisation affectées par la supplémentation en sélénium. En conclusion, nous pourront penser que le mécanisme d'action du sélénium est représenté par un délicat équilibre entre l'activation et la répression de l'activité de certaines protéines qui induit des changements conformationnels plus ou moins directement impliqués dans la réparation de l'ADN et la progression de la croissance cellulaire. / Selenium was recognized as an important micronutrient for both humans and animals. Several studies showed that increased selenium in the diet might be beneficial against liver, colon, pancreas and prostate cancer. The anticarcinogenic actions of Se occur at the systemic, cellular and nuclear level. These actions may also involve the immune system and thus cannot be interpreted by a single mechanism. Until now its mechanisms of action are not well understood. The objective of this study was to investigate the effect of selenocompounds at low doses on DNA repair capacity in the p53-proficient LNCaP prostate cancer cells. This work is divided into three parts. The first part of the work was devoted to study the effect of two selenocompounds (SS and SM) on the cytotoxic and genotoxic properties of various oxidative and non oxidative stresses. The results showed that low doses of Se pre-treatment stimulates selenoprotein synthesis, protects against toxicity and oxidative DNA damage induced by UVA or H2O2 but not by MMS or UVC. The second part of our investigation was devoted to the influence of selenium supplementation on DNA repair capacity. Our work clearly showed an increase in excision efficiency of the glycosylases activity that was not necessarily correlated with an increase of gene expression and/or protein levels. Finally, the third part of our work was devoted to the optimization of Host Cell Reactivation assay (HCR) to study the DNA repair capacity in cellulo, in order to target the partners involved in the signalling pathway affected by selenium supplementation. In conclusion, we could image that the mechanism of action of selenium is represented by a delicate balance between activation and repression of protein activity that induces conformational changes of several proteins more or less directly involved in DNA repair and progression of cell growth.

Les travaux présentés dans ce manuscrit concernent le développement d‘une approche originale permettant la détermination de plusieurs composés (principalement aflatoxines et pesticides organophosphorés), à l‘aide de biocapteurs électrochimiques basés sur l‘inhibition de l‘acétylcholinestérase. Dans un premier temps, un nouveau biocapteur potentiométrique utilisant des transistors à effet de champ sensibles au pH (pH-FETs) comme transducteurs a été développé pour la détermination de l‘aflatoxine B1 (AFB1) et différent paramètres d‘élaboration et de fonctionnement du biocapteur ont été optimisés. Le biocapteur proposé est caractérisé par une stabilité opérationnelle élevée and bonne reproductibilité du signal en cours d‘utilisation et de stockage. Le biocapteur a ensuite été évalué pour l‘analyse d‘échantillons réels (blé, sésame, noix et pois) et une simulation mathématique de la réponse du biocapteur potentiométrique à l‘AFB1 a été proposée pour la première fois et validée. Dans un deuxième temps, un biocapteur conductimétrique utilisant des microélectrodes interdigitées en or a été développé. La sensibilité de ce biocapteur aux aflatoxines ainsi qu‘à d‘autres classes de substances toxiques, tels que les pesticides organophosphorés, les métaux lourds, les glycoalkaloïdes, et les surfactants, a été déterminée. Une nouvelle procédure originale, permettant la détermination sélective de toxines multiclasses par application successive de solutions de réactivation visant spécifiquement des inhibiteurs irréversibles ou réversibles, a été finalement proposée. En utilisant cette méthode, il a été montré que les biocapteurs enzymatiques pouvaient être appliqués à l‘analyse des aflatoxines et des pesticides organophosphorés, ainsi qu‘à la détermination de la toxicité globale des échantillons / Investigations reported in this manuscript are focused on the development of an original approach for the detection of several toxic compounds, mainly aflatoxins and organophosphorus pesticides, using acetylcholinesterase (AChE)-based inhibitory electrochemical biosensors. In a first step, a new potentiometric biosensor using pH Sensitive Field-Effect Transistors (pH-FETs) as transducers was investigated for aflatoxin B1 (AFB1) determination and different elaboration and working parameters were optimized. The proposed biosensor was characterized by high operational stability and reproducibility of the signal during the work as well as during the storage. The biosensor was further evaluated for real samples analysis (wheat, sesame, walnuts and peas) and a mathematical simulation of the potentiometric biosensor response to aflatoxin B1 was proposed for the first time and validated. In a second step, a conductometric biosensor using interdigitated gold microelectrodes was developed. The sensitivity of the biosensor to aflatoxins and other classes of toxic substances, such as organophosphorus pesticides, heavy metals ions, glycoalkaloids, and surfactants, was determined. A new and original procedure, enabling the selective determination of multiclass toxins by applying successive reactivation solutions targeting either irreversible or reversible inhibitors, was finally proposed. Using this method, the electrochemical enzyme inhibitory biosensors could be applied to the analysis of aflatoxins and organophosphorus pesticides, as well as for the determination of total toxicity of the samples

Lyme disease is a tick-transmitted illness caused by Borrelia burgdorferi sensu lato (s.l.), a group of spirochetes with at least three human pathogenic species, B. burgdorferi sensu stricto, B. afzelii and B. garinii. These spirochetes cycle between vertebrate reservoirs, mainly rodents, and ixodid ticks. Both terrestrial birds and seabirds can be infected with B. burgdorferi s.l. but the function of birds as reservoirs is largely unknown, even though they are potentially important epidemiologically due to their ability to carry ectoparasites and microorganisms over long distances. This thesis describes the role of birds in Lyme disease Borrelia biology in general and Borrelia ecology and epidemiology in particular. B. burgdorferi s.l. has previously been detected in the seabird tick Ixodes uriae and an enzootic Borrelia cycle distinct from terrestrial Borrelia cycles has been described. In this study B. garinii was isolated from the proposed seabird reservoirs and the tick I. uriae infesting them. The strains isolated did not show evident differences from human pathogenic B. garinii strains, indeed 7/8 strains had an ospC allele associated with Borrelia causing disseminated Lyme disease. Antibodies against B. burgdorferi s.l. were detected in people frequently bitten by I. uriae. Thus the marine enzootic Borrelia cycle may be a risk for humans, either by direct transfer of the spirochete from /. uriae or via introduction of Borrelia into a terrestrial enzootic Borrelia cycle. In order to investigate the role of passerine (Passeriformes) birds as amplification hosts in the terrestrial Borrelia cycle, experimental infections of canary finches (Serinus canaria) and redwing thrushes (Turdus iliacus) were carried out. The result showed that B. burgdorferi s.l. can persist for several months in passerine birds and the infection in redwing thrushes can be reactivated in response to migration. Thus, birds may be more infectious to ticks during their migration and therefore important long-range disseminators of B. burgdorferi s.l. Migration in birds is associated with elevated stress hormones that in turn can cause reactivation of latent infections. Lyme disease in humans could perhaps be activated when the immune response is modulated by stress. Herein I describe a patient with a stress activated latent Borrelia infection, which supports this hypothesis. The seabird tick I. uriae has a circumpolar distribution in both the northern and southern hemispheres and in this study identical B. garinii flagellin gene (flaB) sequences were detected in I. uriae from these hemispheres, indicating a transequatorial transport of B. garinii. Parsimony analysis of I. uriae ITS2 and 16S rDNA sequences suggested that northern and southern I. uriae might be reproductively separated. Therefore passive transport of infected ticks between the polar regions is unlikely and instead seabirds probably carry an active Borrelia infection during their migration. In conclusion, this work shows that migrating seabirds and passerine birds probably are important for the long-range dispersal of B. burgdorferi s.l., and that this mechanism of dispersal could be important for the distribution of human Lyme disease. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 2001, härtill 6 uppsatser</p> / digitalisering@umu