Orientador: Helio Amante Miot / Resumo: FUNDAMENTOS: Melasma é hipermelanose crônica, focal, adquirida decorrente de patogênese não totalmente compreendida, resultado da alteração funcional e arquitetural dos melanócitos. A predisposição genética, aspectos hormonais e exposição à radiação solar são os elementos mais associados ao desenvolvimento da doença e essenciais para entendimento da fisiopatologia. É bem estabelecida a relação entre a exposição à radiação solar e a piora do quadro, entretanto, o efeito independente do UVB, UVA, assim como a atuação da luz visível (LV) são pouco estudados. OBJETIVOS: Padronização de um modelo de cultura organoide cutâneo primário para estudo da melanogênese da pele com melasma e pele normal adjacente, à irradiação com diferentes comprimentos de onda (UVB, UVA, LV). MÉTODOS: Etapa 1: Amostras de pele da região retroauricular (punch 3mm), de 10 voluntários foram seccionados longitudinalmente, e cultivados em meio DMEM segundo protocolo estabelecido por Ayres, para padronização de viabilidade e dosimetria das radiações induzindo melanogênese. Um dos fragmentos foi irradiado e o outro mantido ao abrigo da luz por 72h. Foram avaliados aspectos morfológicos e arquiteturais da histologia (H&E e Fontana-Masson) e por rt-PCR para comparação da expressão quantitativa de gene constitucional (GAPDH) entre as peles recém-coletadas e as cultivadas. Foram padronizadas as doses de radiação e tempo de cultura que promovessem viabilidade da amostra e aumento de 10% na intensidade de pigmentação... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: FUNDAMENTALS: Melasma is chronic hypermelanosis, focal, acquired due to pathogenesis not fully understood, resulting from the functional and architectural alteration of melanocytes. Genetic predisposition, hormonal aspects and exposure to solar radiation are the elements most associated with the development of the disease and essential for understanding the pathophysiology. The relationship between exposure to solar radiation and worsening of the condition is well established, however, the independent effect of UVB, UVA, as well as the action of visible light (VL) are poorly studied. OBJECTIVES: Standardization of a primary cutaneous organoid culture model to study melanogenesis of skin with melasma and adjacent normal skin to irradiation with different wavelengths (UVB, UVA, VL). METHODS: Step 1: Skin samples from the retroauricular region (punch 3mm) from 10 volunteers were longitudinally sectioned and cultured in DMEM medium according to the protocol established by Ayres, for viability standardization and radiation dosimetry inducing melanogenesis. One of the fragments was irradiated and the other kept in the dark for 72h. Morphological and architectural aspects of histology (H&E and Fontana-Masson) and rt-PCR were evaluated for comparison of quantitative constitutional gene expression (GAPDH) between newly collected and cultured skins. Radiation doses and culture time that promoted sample viability and a 10% increase in basal layer pigmentation intensity were standardized... (Complete abstract click electronic access below) / Mestre

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22886号 / 医博第4680号 / 新制||医||1048(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 髙橋 良輔, 教授 井上 治久, 教授 伊佐 正 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Nowadays, the organoids structures has become more popular as suitable model systems for clinical research, particularly for development of new medication and drug screening. The standard study approaches include invazive biochemical or molecular-biology analysis as well as non-invasive optical approaches. Among optical methods, various microscopy techniques can give a very detailed information about the structure of organoids. However, the microscopy is time consuming as well as it puts a great demand on instrumentation. Therefore, the microscopy is not suitable for high content analysis of multiple samples. This work is focused on the development of the device and experimental technique for high-throughput screenings of organoids structures for biomedical applications based on microtitrate plates. Literatre search for non-invasive optical methods, suitable for analysis of organoid structures. The necessary adjustements of existing system for algae phenotypization are discussed. An experiment was made to test functionality of designed system. Practical use for clinical use is tested by the experiment of spheroids reaction to selected cytostatics. The results and findings are discussed in the conclusion.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21341号 / 医博第4399号 / 新制||医||1031(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 小川 誠司, 教授 坂井 義治, 教授 武藤 学 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Matrin3 (MATR3) is an RNA binding protein involved in many roles in the nucleus, such as chromatin architecture and gene expression regulation, modulating transcriptional and post-transcriptional processes as RNA splicing and mRNA stabilization. Nevertheless, some functions of MATR3 within the cells are not entirely clear. MATR3 has been associated with Amyotrophic Lateral Sclerosis (ALS), a neurodegenerative disease that damages motor neuron (MN) cells and leads to progressive muscle paralysis and respiratory failure. A better understanding of MATR3 activity within cell physiology could represent an essential breakthrough for studying MATR3-associated pathologies. Using MATR3-silenced human pluripotent stem cell (hiPSC) line model, we collected data on the MATR3 role in the pluripotency and in the neural induction and differentiation. We found that the downregulation of MATR3 alters the expression level of crucial self-renewal factors such as OCT4, NANOG, KLF4, and LIN28A. We observed MATR3 acts at multiple levels of the gene expression, i.e. regulating YTHDF1 expression, and in RNA metabolism, having a role in mRNA stabilization and translation. The reduction of stemness potential caused by MATR3 downregulation creates a defect during the neurodifferentiation process, which does not arrest motor neurons formation but induces selective alterations that may affect motor neurons functionality. Indeed, several morphological and molecular abnormalities were observed during the neuronal differentiation, such as the alterations of the formation of neuroepithelial rosettes that arise in a reduction of neurite lengths and arborization in neuronal cells. On this basis, we investigated neuronal differentiation in the brain organoids grown from iPSCs derived from ALS patients fibroblasts. We show, for the first time, that MATR3 is a critical factor in orchestrating the stemness network through transcriptional, post-transcriptional, and translational regulation, therefore affecting the differentiation of mature neurons.

Retinoids are a class of compounds derived from the metabolism of vitamin A and β-carotene. Retinoid signaling has vital functions in both vertebrate and invertebrate embryogenesis such as the formation of body axes and the control of organogenesis. Genetic evidence suggests a role of retinoids in cell fate decision, maturation and homeostasis of the prostate epithelium. Knockout (KO) of the retinoic acid receptor gamma (RARG) gene in mice leads to growth deficits and male sterility due to squamous metaplasia and keratinization of the seminal vesicles and prostate. Noteworthy, synergistic antitumor effects of retinoids and vitamin D have been described in prostate cancer cell lines, although a mechanistic link between retinoic acid (RA) signaling and prostate epithelium differentiation and tumorigenesis has not yet been elucidated. Here, taking advantage of mouse prostate organoids (mPrOs), we report an essential role for RA in the differentiation and integrity of the periurethral and proximal luminal compartments of the prostate epithelium. Mechanistically, RA, through the activation of RARγ, promotes the expression of Foxa1, a pioneer transcription factor that cooperates with androgen receptor (AR) in directing progenitor cells towards the luminal lineage. Reduced RA signaling in organoids leads to downregulation of key structural and polarity proteins along with a loss of luminal identity, a phenotype that is fully rescued by constitutive expression of exogenous Foxa1. Overall, our study demonstrates the importance of RA signaling in prostate epithelium differentiation and homeostasis. In addition to the tumorigenic role of Foxa1 mutations recently described in several human cancers, alteration in RA pathway due to altered uptake/absorption/metabolism of vitamin A and β-carotene, or depending on specific molecular dysfunctions (e.g., epigenetic RARB silencing), could represent a critical rheostat for prostate tumorigenesis.

To optimize neoadjuvant radiochemotherapy of pancreatic ductal adenocarcinoma (PDAC), the value of new irradiation modalities such as proton therapy needs to be investigated in relevant preclinical models. We studied individual treatment responses to RCT using patient-derived PDAC organoids (PDO). Four PDO lines were treated with gemcitabine, 5-fluorouracile (5FU), photon and proton irradiation and combined RCT. Therapy response was subsequently measured via viability assays. In addition, treatment-naive PDOs were characterized via whole exome sequencing and tumorigenicity was investigated in NMRI Foxn1nu/nu mice. We found a mutational pattern containing common mutations associated with PDAC within the PDOs. Although we could unravel potential complications of the viability assay for PDOs in radiobiology, distinct synergistic effects of gemcitabine and 5FU with proton irradiation were observed in two PDO lines that may lead to further mechanistical studies. We could demonstrate that PDOs are a powerful tool for translational proton radiation research.