京都大学 / 新制・論文博士 / 博士(医学) / 乙第13501号 / 論医博第2260号 / 新制||医||1061(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小林 恭, 教授 中本 裕士, 教授 黒田 知宏 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

PET/CT with prostate-specific membrane antigen (PSMA)-targeted tracers has been used in the diagnosis and staging of patients with clear cell renal cell carcinoma (ccRCC). For ccRCC primary tumors, PET parameters were shown to predict histologic grade and features. The aim of this study was to correlate PSMA PET/CT with histopathological findings in patients with metastatic recurrence of ccRCC. Patients with ccRCC who underwent PSMA-targeted PET/CT and subsequent histopathological evaluation of suspicious lesions were included. Specimens underwent immunohistochemical marking. Lesion diameter, volume and tracer uptake were correlated with the extent and intensity of molecular PSMA expression and with clinical findings. Twelve PET-positive lesions of nine patients were evaluated. Eleven ccRCC metastases and one prostate carcinoma were detected histopathologically. Molecular PSMA expression was detected in all lesions, which intensity and distribution did not correlate with PET parameters. PSMA-targeted PET/CT is a feasible tool for the evaluation of patients with ccRCC but cannot reliably predict histologic features of metastases. PSMA may also be expressed in malignant lesions other than ccRCC, leading to incidental detection of these tumors.

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La réaction inflammatoire post-ischémique est dominée par les cellules mononuclées phagocytaires : la microglie, cellules immunocompétentes du parenchyme cérébral, et les macrophages dérivant du sang et infiltrant le tissu cérébral lésé. L’imagerie cellulaire par IRM avec injection de nanoparticules superparamagnétiques d’oxydes de fer (USPIO) a contribué à la description dynamique de cette infiltration macrophagique tardive. Cependant, la séquence spatio-temporelle de l’activation microgliale et du recrutement macrophagique, intriqués avec des altérations de la barrière hématoencéphalique au cours des premières heures post-ischémie, reste mal élucidée. Nous avons tenté de mieux comprendre la relation entre les signaux IRM après injection d’USPIO et la réaction phagocytaire aux temps précoces post-ischémie dans un modèle d’accident vasculaire cérébral chez la souris. Nos résultats suggèrent que les modifications précoces de signal après injection d’USPIO reflètent principalement des mécanismes non spécifiques d’entrée des particules dans le tissu lésé. Pour étudier les interactions entre les cellules neurales et les macrophages périphériques au cours de la réaction inflammatoire post-ischémique précoce, nous avons développé un modèle in vivo d’ischémie globale chez la souris et un modèle in vitro d’hypoxie sur cultures organotypiques d’hippocampe. Ce dernier modèle nous a permis d’analyser les effets de co-cultures macrophagiques sur la survie des cellules neurales au sein du parenchyme cérébral ischémié et d’analyser les profils d’expressions cytokiniques impliqués dans ces interactions à médiation humorale. Nous avons observé un effet neuroprotecteur sur la perte neurale post-hypoxique des co-cultures macrophagiques. Cet effet à médiation humorale est associé à un profil d’activation macrophagique de type alternatif (M2). / Clinical outcome in cerebral ischemia may be influenced by innate immune cells of myeloid lineage : central nervous system (CNS)-infiltrating peripheral macrophages and CNS-resident microglia. Noninvasive monitoring of these cells may improve the understanding of postischemic inflammation. Accumulation of Ultrasmall Superparamagnetic Particles of Iron Oxide (USPIO) has been observed in infarcted areas at the subacute stage of experimental stroke. However, the exact route of USPIO uptake and early brain distribution remain elusive, hampering the interpretation of USPIO-relatedsignals. Therefore, we compared MRI signal changes after intravenous USPIO injection with the histological distribution of iron particles and macrophagic cells 6 to 24 hours after permanent middle cerebral artery occlusion in mice. Our results suggest that in this model, early USPIO-related MR signal changes are mainly related to passive diffusion of free USPIO through a damaged blood-brain barrier and to intravascular trapping rather than peripheral phagocyte infiltration. To understand the complex interactions between microglia, hypoxic neurons and CNS-infiltrating macrophages, we setup an in vitro model where primary macrophages were co-cultured with hippocampal slices submitted to hypoxia and glucose deprivation (OGD). Our results indicate that under these experimental conditions, cultured macrophages engage in a M2 activation pattern and afford partial protection from OGD-induced neuronal loss through paracrine mechanisms. We also conclude that microglia is susceptible to hypoxia-induced cell death in vitro and in vivo

Toxocara canis is endoparasitic geohelminth of canids. In its life cycle it uses paratenic host (even humans) and can cause severe problems called cerebral toxocariasis when attacks central nervous system. The exact mechanism of pathogenicity in nervous system is unknown and experimental studies examines rather the acute phase of toxocariasis, therefore we characterized the course of cerebral toxocarosis in mice from acute to chronic phase in this master thesis. We found larvae of the parasite in the brains of mice. The larvae in the tissues occurred both, individually and in clusters. The presence of larvae was observed in regions that affect both movement and memory. We did not find any visible injury nor inflammation surrounding the larvae in the tissue. However, histological examination showed brain tissue pathologies in all mice, namely local necrosis, hemorrhages, thickened vessel walls, cell infiltrates in tissue and around vessels and abnormal angiogenesis. The mice showed neurological symptoms with increasing frequency from the 9th week post infection. Production of specific antibodies was also monitored. The level of antibodies in reinfected mice was higher compared to antibody levels of mice with a single infection. No correlation with the presence of neurological symptoms was shown. Key...

Orientador: Maria Cecília Rui Luvizotto / Coorientador: Heitor Flávio Ferrari / Banca: Daniela Bernadete Rozza / Banca: Livia Castanhas Breganó / Resumo: O tumor mamário canino (TMC) é a neoplasia que mais comumente acomete cadelas idosas não castradas. Na análise histopatológica, 41 a 53% dos TMCs são classificados como malignos. Os receptores de estrógeno α (REα) são receptores nucleares importantes na transcrição de fatores e transdução de sinais hormonais, considerados um dos fatores prognósticos para o tumor mamário humano, participam de forma ativa na carcinogênese mamária. Durante o desenvolvimento do TMC, macrófagos teciduais compõe o microambiente tumoral, com a função de estimular a angiogênese, aumentando a possibilidade de metástases, sendo fundamental para o prognóstico de neoplasia mamária na cadelae na mulher. O objetivo deste trabalho foi pesquisara expressão e a provável correlação de REα com a distribuição de macrófagos tumorais (TAMs) em TMCs por meio de imunohistoquímica. Foram analisados 40 tumores mamários malignos, classificados histologicamente em quatro grupos distintos e um grupo controle composto por 08 glândulas mamárias caninas sem alteração anatomopatológica e seus respectivos linfonodos regionais. Os resultados mostraram que a imunomarcação positiva ao REα variou de um a três casos dentre os grupos histológicos estudados, havendo nestes, menor número de metástase para linfonodo. A positividade para os TAMs mostrou associação com o tipo histológico, variando de moderada a acentuada nos carcinomas mamários de alto grau histológico que apresentaram necrose, invasão linfática e formação tubular. Não ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The canine mammary tumor (CMT) is the neoplasia that most commonly affects uncastrated elderly female dogs. On histopathological analysis, 41 to 53% of CMTs are classified as malignant. Estrogen receptors α (REα) are important nuclear receptors for transcription factors and signal transduction of hormones, considered prognosis factor for breast cancer in human, and recognized as actively involved in breast carcinogenesis. During the development of mammary cancer in canine species, macrophages composes the tumor microenvironment, stimulating angiogenesis, facilitating the local dissemination of neoplastic cells and increasing the possibility of metastasis, being fundamental for mammary neoplasia prognosis in dogs and women. This study aimed to evaluate the expression and the probable correlation of REα with the distribution of tumor associated macrophages (TAMs) in CMTs by immunohistochemistry. Forty CMTs were analyzed histologically in four distinct groups and a control group composed of eight canine mammary glands without alteration anatomopathological and their respective regional lymph nodes. The results showed that the REα positive immunostaining varied from one to three cases among the histological groups studied, and there was less lymph node metastasis. The positivity for TAMs showed a positive correlation with the histological type, ranging from moderate to severe in mammary carcinomas of high histological grade that presented necrosis, lymphatic invasion and tubular ... (Complete abstract click electronic access below) / Mestre

När ett vävnadspreparat har genomgått en otillräcklig dehydrering måste det åtgärdas innan provet kan gå vidare i den histotekniska processen. I dagsläget finns det två åtgärdande metoder på Patologilaboratoriet, Region Jönköpings län. Den ena metoden går ut på att provet placeras i smält paraffin för mer stadga. Den andra metoden går ut på att provet rehydreras för att sedan dehydreras på nytt. Syftet med denna studie var att utvärdera en tredje metod, ”dumpingmetoden” som går ut på att vävnadspreparaten dehydreras på nytt direkt utan att först bli rehydrerade.  Denna metod utförs i nuläget redan på vissa hud- och obduktionspreparat på Patologilaboratoriet. ”Dumpingmetoden” skulle kunna förenkla arbetet på laboratoriet då den är snabbare, kräver färre kemikalier och är automatiserad till skillnad från befintliga metoder. Resultaten från studien visade att ett otillräckligt dehydrerat vävnadspreparat aldrig kan bli lika bra som om det hade hanterats korrekt från början. Det är dock trots allt möjligt att åtgärda ett otillräckligt dehydrerat preparat i den graden att det blir bedömbart. ”Dumpingmetoden” förenklade snittningen av alla preparat som testades. Hos kolon- och tonsillpreparat verkade ”dumpingmetoden” ge bättre kvalitet av hematoxylin- och eosinfärgningen än de befintliga metoderna. För hudpreparat gav dock metoden där vävnadspreparat rehydrerats för att sedan dehydreras på nytt bäst kvalité av infärgningen. / A tissue that has been insufficiently dehydrated has to be fixed before it can continue through the histotechnological process. Currently, there are two established measures at the Pathology Laboratory, Department of Laboratory Medicine, Region Jönköping County. One of the methods is to put the sample in melted paraffin to give the tissue more support. The other method is to rehydrate the tissue followed by a new dehydration. The purpose of this study was to evaluate a third method called the “dumping method”. The “dumping method” involves a directly dehydration of the tissue, excluding rehydration. This method is currently being used on some of the skin and autopsy samples at the Pathology Laboratory. The “dumping method” would ease the work at the Laboratory since it is faster, requires fewer chemicals and can be automated, unlike the current available methods. The results from the study showed that an insufficiently dehydrated tissue can’t be restore to its original state. Never less it is possible to restore the tissue sufficiently for diagnosis. The “dumping method” eased the sectioning of all the tissue samples included in this study. For colon and tonsil tissues, the “dumping method” ensured higher quality of the haematoxylin and eosin staining than the current available methods. For skin tissues, however, the method of rehydration followed by a new dehydration resulted in the highest staining quality

A doença da bananeira \'mal-do-Panamá\', causada pelo fungo Fusarium oxysporum f. sp. cubense (Foc) é uma das doenças mais destrutivas da bananeira e é considerada uma das seis doenças economicamente mais importante da história da humanidade. Algumas cultivares resistentes, como a \'BRS Platina\', foram lançadas pela Embrapa, porém para a sustentabilidade da resistência é necessário entender os mecanismos moleculares envolvidos na resposta de resistência e defesa. O objetivo deste estudo foi caracterizar o processo de infecção pelo Foc raça 1 em três cultivares contrastantes para a resistência e analisar o padrão transcricional no início da interação. A análise histopatológica indicou que o Foc raça 1 penetra pela raízes laterais e principal, colonizando os espaços inter e intracelular do córtex nas três cultivares. Foram visualizadas, hifas \'globosas\' na cultivar suscetível \'Maçã\' com a formação de estruturas de resistência, como clamidósporos. Na cultivar resistente \'BRS Platina\', foi observado por microscopia óptica no período inicial da interação (24 horas após inoculação) a indução de respostas de defesa da planta, como formação de zona de cicatrização, e aos 15 dias após inoculação, formação de tilose, presença de cristais de oxalato de cálcio e deposição de calose. Foi utilizada a tecnologia Illumina para sequenciamento massal de RNA e abordagens de bioinformática para identificar genes diferencialmente expressos (DE) relacionados com a resposta de defesa de bananeira em interações compatíveis e incompatíveis. O sequenciamento paired-end gerou um total de 113.632.486 fragmentos (reads) com alta qualidade. Do total de reads alinhados no genoma referência (\'DH-Pahang\'), 55.555.480 alinharam-se com genes conhecidos e anotados no genoma referência, sendo utilizados para a análise DE inoculado x não inoculado, permitindo detectar 2.307 genes para as três cultivares. Os genes anotados de cada cultivar foram comparados, sendo identificados quatro genes comuns para as três cultivares, dez compartilhados entre \'Maçã\' e \'Prata-anã\', 21 compartilhados entre \'BRS Platina\' e \'Maçã\', 114 compartilhados entre \'BRS Platina\' e \'Prata-anã\', além de 75 serem exclusivos de \'Maçã\', 599 de \'BRS Platina\' e 1484 de \'Prata-anã\'. O mecanismo de resistência/defesa ao Foc em \'BRS Platina\', ocorre em nível de percepção precoce na presença do patógeno desencadeando resposta de defesa inexistente em \'Maçã\', e com cinética distinta da cultivar com resposta intermediária (\'Prata-anã\'). Dessa forma, os resultados permitiram propor um modelo da resposta de defesa/resistência ao Foc raça 1 em bananeira, baseando-se no nível de indução de genes que codificam para proteínas de reconhecimento do patógeno (receptor like kinase), fatores de transcrição (WRKY e MYB); reforço e síntese de parede celular, degradação da parede celular do fungo (quitinase e glucanases), heat shocks, enzimas antioxidantes e na resposta visualizada pela histologia na cultivar \'BRS Platina\'. Sendo assim, este trabalho fornece novas perspectivas para estudos de análise funcional, identificação e anotação de novos genes relacionados a resposta de defesa e resistência ao Foc raça 1. / The banana Panama disease, caused by fungus Fusarium oxysporum f. sp. cubense (Foc), is one of the most destructive disease of the industry, and it is considered one of the six most economically important of all times. A few cultivars, such as \'BRS Platina\', were released, but it is still necessary to understand molecular mechanisms involved in defense response and resistance. The objective of this study was to characterize the infection process by Foc in three banana cultivars contrasting for resistance to Foc and to analyze the transcriptional profile at the beginning of interaction. In this way, Foc race 1 penetrated the main and lateral roots, colonizing inter- and intracellular spaces of the root cortex in the three cultivars. Hyphae were globose in the susceptible cultivar \'Maçã\' with the formation of resilience structure, such as chlamydospores. In the resistant cultivar \'BRS Platina\', during the initial period of interaction (24 hours after inoculation), induced of plant defense responses, such as a healing zone, tylosis formation, presence of calcium oxalate and callose deposition. The Illumina technology were applied to sequence RNA, followed by bioinformatic tools to identify genes differentially expressed (DE) related to resistance and defense response in the compatible and incompatible interactions. Pair-end sequencing generated a total of 113,632,486 reads with high quality. From the total of aligned reads to the banana reference genome (\'DH-Pahang\'), 55,555,480 aligned with gene models annotated in the reference genome. The aligned contigs were analysed for DE, comparing inoculated x non-inoculated, enabling the detection of 2307 genes for the three cultivars. Each annotated gene from each cultivar was compared: four common genes to the three cultiars; 10 genes were shared between \'Maçã\' and \'Prata-anã\'; 21 shared between \'BRS Platina\' and \'Maçã\'; 114 shared between \'BRS Platina\' and \'Prata-anã\', plus 75 exclusive to \'Maçã\'; 599 exclusive to \'BRS Platina\' and 1,484 to \'Prata-anã\'. The mechanism of resistance/defense in \'BRS Platina\', level of perception occurs early in the presence of the pathogen defense response triggering nonexistent in \'Maçã\' and with kinetics distinct cultivar with intermediate response (\'Prata-anã\'). Thus, the results have provided a model of defense response/resistance to Foc race 1 in banana, based on the level of gene induction that encode recognition proteins (Receptor-like Kinase, RLK), transcription factors (WRKY and MYB), cell wall synthesis and reinforcement, degradation of fungal cell wall (chitinases and glucanases), heat shocks , proteins;anto-oxidative enzymes and visualized by histologcal in response cultivar \'BRS Platina\'. The present work offer new perspectives to functional analyses, identification and annotation of new genes related to resistance and defense response to Foc race 1.

Imiquimod é uma imidazoquinolina, agonista do TLR-7, ativador de células dendríticas (DC) e indutor da transcrição de diversas citocinas. O objetivo deste estudo é avaliar o crescimento tumoral, a formação de metástases em órgãos internos, a atividade do sistema imune e o tipo de morte celular induzido devido ao tratamento tópico. Camundongos foram inoculados com células de melanoma murino B16F10. O fármaco, na dose de 5mg, foi aplicado no local da inoculação uma vez por dia até o fim do experimento. Foram estudados quatro grupos experimentais, dois inoculado com células tumorais, sendo um tratado e outro não, e outros dois grupos, um recebeu somente o tratamento com o fármaco e o outro não recebeu tratamento. Os animais foram pesados e seus tumores medidos periodicamente. Exemplares dos grupos foram sacrificados em quatro tempos diferentes, imediatamente submetidos à necropsia para a análise de massas tumorais por citometria de fluxo com caspase-3 e anexina-v para investigar a indução de apoptose e/ ou necrose tumoral, e remoção do tumor, pele, Baço, linfonodo e pulmão, que foram fixados, inclusos em parafina, cortados e corados com HE, para análise histológica. / Imiquimod is a TLR-7 agonist and activator of dendritic cells (DC), and induces transcription of several cytokines. This study`s objective is to evaluate tumor growth, development of metastasis within internal organs and the immune system`s function, due to the topical treatment. Two groups of C57BL/6J mice were inoculated with B16F10 melanoma cells subcutaneously, and only one of the groups was treated, applied topically at the injection site once a day, followed by a second round of two groups, one treated and the other as a healthy group. Every two tumors and weight were measured. In order to observe the tumor progression and treatment effects, groups of animals were sacrificed on four days apart and immediately submitted to necropsy for a tumor analysis by flow cytometry, to evaluate the induction of apoptosis and necrosis, and to macroscopically observe the tumor development within internal organs, vascularization and posterior inclusion of the organs in paraffin for histological analysis. Tumor, skin, spleen, limph node and lung were colored with HE for histological analysis of migrating immune system cells and tumoral cells in the organs and describing its integrity.