Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Campus access only. Vita. Bibliography: 176-188.

Background: Gliomas are the most frequent primary brain tumours in adults with two main histological subtypes: astrocytoma and oligodendroglioma. Translocator protein (TSPO) is a pro-inflammatory molecule over-expressed predominantly in activated microglia under pathological conditions. In astrocytoma samples, TSPO has also been found to be up-regulated and correlated with the malignancy of the tumours. [11C]-(R)PK11195 is a selective radioligand for the TSPO widely applied in clinical PET studies. We used [11C]-(R)PK11195 PET to investigate in vivo cerebral TSPO expression and microglial activation in patients with gliomas of different histological subtypes and grades. Methods: 24 glioma patients and 10 healthy volunteers underwent volumetric MRI and dynamic [11C]-(R)PK11195 PET scans. Tissue time-activity curves (TACs) were extracted from tumour regions and normal grey and white matter of the brains. Parametric maps of binding potential (BPND) were generated with the simplified reference tissue model. Co-registered MRI/PET was used to guide tumour biopsies. Tumour tissue was quantitatively assessed for TSPO expression and microglial infiltration by immunohistochemistry. Results: Three types of tumour TAC were observed in gliomas (grey matter-like kinetics, white matter-like kinetics and mixed kinetics), which differed between low-grade astrocytomas and low-grade oligodendrogliomas but were independent of the tumour grade. [11C]-(R)PK11195 BPND also differed between the two subtypes of low-grade gliomas, and low-grade gliomas demonstrated lower BPND than high-grade gliomas. 4 cases of high-grade glioma with minor or no contrast enhancement on MRI showed pronounced [11C]-(R)PK11195 binding. Immunohistochemistry confirmed that expression of TSPO correlated with [11C]-(R)PK11195 BPND of the tumour. It was related mainly to expression by neoplastic cells while the contribution from tumour-infiltrating microglia was minimal. When compared with control subjects, increased [11C]-(R)PK11195 BPND was found in patients’ normal appearing cerebral structures, being more prominent in the tumour-bearing than the tumour-free hemisphere. This extra-tumoral [11C]-(R)PK11195 binding was correlated with the duration of epileptic seizures, the symptom shared by the majority of our patients. Conclusions: Gliomas show differences in [11C]-(R)PK11195 kinetics and binding that are related to histological subtype and grade. Neoplastic cells rather than activated microglia are the main cellular sources expressing TSPO and determine the [11C]-(R)PK11195 binding within the tumours. [11C]-(R)PK11195 PET has the potential to detect malignant transformation of non-enhancing gliomas and facilitate the targeting of more aggressive areas in tumour biopsy. The high extra-tumoral [11C]-(R)PK11195 binding indicates widespread microglial activation in otherwise normal appearing cerebral structures of glioma patients. It is associated with epilepsy and could open up novel therapeutic perspectives for seizure control in this patient population.

La sclérose en plaques (SEP) est une maladie auto-immune inflammatoire chronique qui détériore la myéline centrale des patients. Des progrès ont été réalisés dans le traitement de la SEP mais il n'existe pas encore de neuroprotecteurs efficaces contre les dommages axonaux entraînant un lourd handicap. Ce travail de thèse a évalué le potentiel du XBD173, un ligand du translocator protein contrôlant la neurostéroïdogenèse et la neuroinflammation, à améliorer les symptômes dans un modèle de SEP induit par la protéolipide-protéine (souris EAE-PLP) qui mime la forme rémittente-récurrente de la SEP (85% des patients). Nos résultats montrent qu'à faible dose, le XBD173, qui diminue les taux sériques d’interleukines pro-inflammatoires et empêche la répression des marqueurs myélinique et axonal, améliore aussi la symptomatologie des souris EAE-PLP. L'effet bénéfique du XBD173 contre les signes cliniques est reproduit chez les souris EAE-MOG mimant la forme progressive de la SEP. La thèse ouvre des perspectives pour l'élaboration de stratégies efficaces contre la SEP sans induire d'effets secondaires majeurs. / Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease affecting the central myelin. Progress has been made for MS treatment, but effective neuroprotective drugs against MS-evoked axonal damages and severe disability are still missing. This thesis assessed the potential of XBD173, a ligand of the translocator protein controlling neurosteroidogenesis and neuroinflammation, to improve symptoms in the proteolipid protein-induced MS model (EAE-PLP mice) that mimics the relapsing-remitting form affecting 85% of MS patients. Our results show that XBD173 at low dose decreases serum levels of pro-inflammatory interleukins, prevents the repression of myelin and axonal markers, and also ameliorates clinical deficits in EAE-PLP mice. The beneficial effect of XBD173 against clinical symptoms was also observed in the EAE-MOG mice mimicking the progressive MS. Promising perspectives are open for the development of effective strategies against MS with little or no serious side effects.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on Sept. 4, 2009). Includes bibliographical references.

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京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13378号 / 論医博第2212号 / 新制||医||1047(附属図書館) / (主査)教授 井上 治久, 教授 髙橋 良輔, 教授 渡邉 大 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

The Type III Secretion (T3S) system is a system utilized by many pathogenic bacteria to inject proteins into host cells during an infection. Effector proteins enter the host cell by passing through the proteinaceous T3S translocon, which forms a pore on the host cell membrane. Pseudomonas aeruginosa is an opportunistic pathogen that utilizes the T3S system, and very little is known about how the P. aeruginosa translocon forms. The proteins PopB and PopD are believed to assemble into the P. aeruginosa translocon. A pore-forming heterocomplex of PopB and PopD has been reconstituted in model membranes, however this heterocomplex has not been assessed in its relation to the translocon formed on the host cell. The interaction of this heterocomplex with other T3S system components was measured to determine if this complex acts similarly to the translocon. Initial assays that can be used to compare the molecular weight of the translocon isolated from eukaryotic cells after P. aeruginosa contact to the calculated molecular weight of the heterocomplex were developed as well. This study provides insight into how the PopB:PopD heterocomplex formed in model membranes relates to the translocon formed during a P. aeruginosa infection.

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O aumento da expectativa de vida da população mundial tem se associado com uma maior prevalência de doenças neurodegenerativas. A Doença de Alzheimer (DA) é a doença neurodegenerativa mais comum e a principal causa de demência em indivíduos com mais de 60 anos, sendo caracterizada por um declínio progressivo na memória e função mental dos pacientes. Esses sintomas são acompanhados por alterações histopatológicas no cérebro desses indivíduos, incluindo a presença de uma grande quantidade de placas senis, formadas pela deposição do peptídeo beta-amiloide (Aβ), e de emaranhados neurofibrilares formados pela hiperfosforilação da proteína Tau. Estudos indicam que a deposição do Aβ é uma das principais responsáveis pelo desenvolvimento da DA, causando dano neuronal através da ativação de várias vias pró-apoptóticas e dando origem aos sintomas de demência típicos dessa doença. Até o momento, não existem tratamentos eficazes para o combate à DA, de forma que a maior parte das intervenções farmacológicas é destinada apenas ao tratamento de alguns de seus sintomas. A proteína translocadora (TSPO) se localiza em pontos de contato entre as membranas mitocondriais interna e externa e está relacionada com o transporte de colesterol para o interior da mitocôndria e com a regulação da esteroidogênese e da apoptose. Estudos mostram que ligantes da TSPO apresentam efeitos neuroprotetores em diferentes modelos experimentais de lesão cerebral e doenças neurodegenerativas. Especificamente em relação à DA, um estudo indicou que o 4’-clorodiazepam (4’-CD), um ligante da TSPO, apresenta efeitos neuroprotetores em um modelo animal dessa doença, sendo um possível candidato para o seu tratamento. Dessa forma, o objetivo desse estudo foi verificar o efeito neuroprotetor do 4’-CD em diferentes modelos in vitro de toxicidade induzida pelo Aβ, além de seus efeitos sobre o desenvolvimento de neurônios hipocampais. Inicialmente, demonstramos que o 4’-CD reduziu a morte celular de células SH-SY5Y expostas a um modelo de toxicidade induzida pela administração de Aβ. Esses efeitos estiveram associados com a redução da expressão da proteína pró-apoptótica Bax e com um aumento da expressão da survivina, uma proteína anti-apoptótica. A expressão das proteínas Bcl-xl e procaspase-3, por outro lado, não foi alterada pelos tratamentos. Posteriormente, estudamos os efeitos neuroprotetores do 4’-CD contra a toxicidade induzida pela administração do Aβ em culturas organotípicas de hipocampo. Nesses experimentos, foi demonstrado que o 4’-CD reduz a morte celular de culturas organotípicas de hipocampo expostas ao Aβ através de um aumento na expressão da enzima SOD, sem alterar, no entanto, a expressão das proteínas Akt e procaspase-3. Por fim, foi avaliado o efeito do 4’-CD sobre o desenvolvimento de culturas primárias de neurônios hipocampais de camundongos machos e fêmeas. Foi observado que as culturas de neurônios hipocampais das fêmeas apresentaram um desenvolvimento mais rápido do que as dos machos. O 4’-CD acelerou a maturação e aumentou a ramificação neurítica dos neurônios hipocampais dos machos, mas não exerceu qualquer efeito sobre os neurônios das fêmeas. Em suma, foi observado que o 4’-CD apresenta efeitos neuroprotetores contra o Aβ em células SH-SY5Y e em culturas organotípicas do hipocampo, apresentando-se como um fármaco em potencial para o tratamento da DA. Além disso, foi observado que o 4’-CD exerceu um efeito dependente do sexo sobre o desenvolvimento de culturas primárias de neurônios hipocampais, estimulando o desenvolvimento e a ramificação neurítica de neurônios hipocampais de machos, mas não de fêmeas. / The increase in life expectancy of the world population has been associated with a higher prevalence of neurodegenerative diseases. The Alzheimer’s Disease (AD) is the most common neurodegenerative disorder and the main cause of dementia among people over 60 years, being characterized by a progressive decline in the memory and mental function of the patients. These symptoms are associated with histopathological changes in the brain of these patients, including the presence of senile plaques, formed by the deposition of amyloid-beta (Aβ), and neurofibrillary tangles, which are related to the hyperphosphorylation of Tau protein. Studies indicate that Aβ deposition is a major contributor to AD progression, promoting neuronal damage through the activation of different pro-apoptotic pathways and giving rise to the typical dementia symptoms of this disease. To date, there are no effective treatments for AD, so that most of the pharmacological intervention is intended for the treatment of some of its symptoms. The translocator protein (TSPO) is located in contact sites between the outer and the inner mitochondrial membranes and is involved in the cholesterol transport into the mitochondria and in the regulation of steroidogenesis and apoptosis. Studies show that TSPO ligands present neuroprotective effects in different experimental models of brain injury and neurodegenerative diseases. Specifically regarding AD, a study indicated that 4’-chlorodiazepam (4’-CD), a TSPO ligand, is neuroprotective in an animal model of this disease, being a possible candidate for its treatment. Therefore, the aim of this study was to evaluate the neuroprotective effect of 4’-CD in different experimental models of Aβ- induced neurotoxicity in vitro, as well as its effects on the development of hipocampal neurons. First, it was demonstrated that 4’-CD decreased the cell death of SH-SY5Y cells exposed to the Aβ. This effect was associated with the inhibition of the Aβ-induced upregulation of Bax, a pro-apoptotic protein, and downregulation of survivin, a prosurvival protein. On the other hand, the expression of Bcl-xl and procaspase-3 was not change by the treatments. After, it was studied the neuroprotective effects of 4’-CD against Aβ in organotypic hipocampal cultures. In these experiments, it was shown that 4’-CD decreases the cell death of organotypic hippocampal slices exposed to the Aβ by increasing the protein expression of SOD, but without changing the expression of Akt and procaspase-3. Finally, due to the importance of the processes of neuronal development and maturation in the regeneration of CNS after injury, it was evaluated the effect of 4’-CD on the development of primary hippocampal neurons of male and female mice. It was observed that female primary hippocampal neurons presented an increased rate of development than male neurons. 4’-CD stimulated the development and increased the neuritic branching of male but not from female neurons. In summary, it was observed that 4’-CD presented a neuroprotective effect against Aβ in SH-SY5Y cells and in rat organotypical hippocampal slices, presenting itself as a promising agent for the treatment of AD. Also, it was observed that 4’-CD modulates the development of hippocampal neurons in a sex-dependent manner, stimulating the development of male but not from female cells.