As shown by recent reports, number of obese people in recent years has been on the increase, there are about 4 million people in Taiwan who are considered to be overweight. World Health Organization (WHO) and United States Center for Disease Control and Prevention (CDC) publicly announced that: Obesity will be the greatest health killer of this century, its damage to personal health is comparable to that of cigarettes. Obesity can cause heart problems, diabetes, artery diseases, high blood pressure, increased chances of cancer occurrence, condition increase and deteriora- tion of Alzheimer¡¦s disease, gall bladder diseases, and shortening of life span. The cause of obesity is due to a fault in adipocytes metabolism functions, and because of this, research into adipocytes molecular regulation is becoming more popular and valued. The process of adipogenesis, the formation of adipose tissue, has become better understood by the studies of several cell types that can be induced to undergo differentiation into adipocytes. The first, and the best characterized, model of adipogenesis in vitro is the 3T3-L1 cell line, a substrain of Swiss 3T3 mouse cell line. 3T3-L1 cells propagated under normal conditions have a fibroblastic phenotype. However, when treated with a combination of dexamethasone, isobutylmethylxanthine (IBMX or MIX) and insulin, 3T3-L1 cells adopt a rounded phenotype and within 5 days begin to accumulate lipids intracellularly in the form of lipid droplets. Treatment of cells with dexamethasone activates the transcription factor CCAAT/enhancer -binding protein £] (C/EBP£]). IBMX inhibits soluble cyclic nucleotide phosphodiesterases and results in increased intracellular cAMP levels. At the nuclear level, treatment with IBMX results in activation of the related transcription factor C/EBP£_. Immediately after exposure to exogenous inducers, the gene expression of C/EBP£] and C/EBP£_ significantly and transiently increases, C/EBP£] and C/EBP£_ may also regulate the expression of C/EBP£\ and PPAR£^. C/EBP£\ and PPAR£^ are considered to play a prominent role in regulating the gene expression of proteins necessary for the development fo the functional mature adipocyte. Within 3 days of exposure to inducers, the cells undergo two rounds of mitosis, termed mitotic clonal expansion, which are required for differentiation. Insulin or insulin-like growth factor-1 promote adipocyte differentiation by activating PI3-kinase and Akt activity. Modulation of the activity of the forkhead transcription factor Foxo1 appears to be necessary for insulin to promote adipocyte differentiation. C/EBP£\ and PPAR£^ direct the final phase of adipogenesis by activating expression of adipocyte-specific genes, such as fatty acid synthetase, fatty acid binding protein, leptin and adiponectin. The identification of regulators of adipogenesis raises the prospect of preventing or reversing obesity through pharmacological means. My research is aimed at investigating the adipocytes differentiation and regeneration adaptive mechanisms of mice 3T3L-1 preadipocytes and human processed lipoaspirate cells (PLA). By using adipocytes culture techniques in conjunction with adipocytes growth induction and gene delivery techniques to further study obesity related genes, POMC and PTEN, and downstream regulators , PPAR£^ and Adiponectin, in regards to their roles in the process of adipocytes differentiation.

Macrophage-conditioned medium (MacCM) inhibits the differentiation of rodent and human preadipocytes. Previous studies report that murine J774A.1-MacCM inhibits clonal expansion (early required phase of adipogenesis), including Rb phosphorylation. I hypothesized that MacCM induced alterations in cyclins and/or cyclin-dependent kinases (CDKs) were responsible for impairing Rb phosphorylation. My first objective was to assess the effect of J774A.1-MacCM on CDK4, CDK2, and their regulatory cyclins. Murine 3T3-L1 preadipocytes were differentiated with control medium or J774A.1-MacCM. Expression of cyclin D and A was inhibited by J774A.1-MacCM. Inhibition of cyclin A expression was associated with reduced differentiation-induced CDK2 activity. My second objective was to assess the expression patterns of cell cycle proteins in differentiating human abdominal subcutaneous preadipocytes, which do not undergo clonal expansion in culture. Cyclin E expression increased with differentiation. THP-1-MacCM (a human macrophage cell line) further enhanced this increase. My studies suggest MacCM leads to alterations in cyclin/CDK regulation during adipogenesis in murine and human preadipocyte models.

Macrophage-conditioned medium (MacCM) inhibits the differentiation of rodent and human preadipocytes. Previous studies report that murine J774A.1-MacCM inhibits clonal expansion (early required phase of adipogenesis), including Rb phosphorylation. I hypothesized that MacCM induced alterations in cyclins and/or cyclin-dependent kinases (CDKs) were responsible for impairing Rb phosphorylation. My first objective was to assess the effect of J774A.1-MacCM on CDK4, CDK2, and their regulatory cyclins. Murine 3T3-L1 preadipocytes were differentiated with control medium or J774A.1-MacCM. Expression of cyclin D and A was inhibited by J774A.1-MacCM. Inhibition of cyclin A expression was associated with reduced differentiation-induced CDK2 activity. My second objective was to assess the expression patterns of cell cycle proteins in differentiating human abdominal subcutaneous preadipocytes, which do not undergo clonal expansion in culture. Cyclin E expression increased with differentiation. THP-1-MacCM (a human macrophage cell line) further enhanced this increase. My studies suggest MacCM leads to alterations in cyclin/CDK regulation during adipogenesis in murine and human preadipocyte models.

Macrophage-conditioned medium (MacCM) inhibits the differentiation of rodent and human preadipocytes. Previous studies report that murine J774A.1-MacCM inhibits clonal expansion (early required phase of adipogenesis), including Rb phosphorylation. I hypothesized that MacCM induced alterations in cyclins and/or cyclin-dependent kinases (CDKs) were responsible for impairing Rb phosphorylation. My first objective was to assess the effect of J774A.1-MacCM on CDK4, CDK2, and their regulatory cyclins. Murine 3T3-L1 preadipocytes were differentiated with control medium or J774A.1-MacCM. Expression of cyclin D and A was inhibited by J774A.1-MacCM. Inhibition of cyclin A expression was associated with reduced differentiation-induced CDK2 activity. My second objective was to assess the expression patterns of cell cycle proteins in differentiating human abdominal subcutaneous preadipocytes, which do not undergo clonal expansion in culture. Cyclin E expression increased with differentiation. THP-1-MacCM (a human macrophage cell line) further enhanced this increase. My studies suggest MacCM leads to alterations in cyclin/CDK regulation during adipogenesis in murine and human preadipocyte models.

Macrophage-conditioned medium (MacCM) inhibits the differentiation of rodent and human preadipocytes. Previous studies report that murine J774A.1-MacCM inhibits clonal expansion (early required phase of adipogenesis), including Rb phosphorylation. I hypothesized that MacCM induced alterations in cyclins and/or cyclin-dependent kinases (CDKs) were responsible for impairing Rb phosphorylation. My first objective was to assess the effect of J774A.1-MacCM on CDK4, CDK2, and their regulatory cyclins. Murine 3T3-L1 preadipocytes were differentiated with control medium or J774A.1-MacCM. Expression of cyclin D and A was inhibited by J774A.1-MacCM. Inhibition of cyclin A expression was associated with reduced differentiation-induced CDK2 activity. My second objective was to assess the expression patterns of cell cycle proteins in differentiating human abdominal subcutaneous preadipocytes, which do not undergo clonal expansion in culture. Cyclin E expression increased with differentiation. THP-1-MacCM (a human macrophage cell line) further enhanced this increase. My studies suggest MacCM leads to alterations in cyclin/CDK regulation during adipogenesis in murine and human preadipocyte models.

Fungal infections of animals have become an increasingly important global issue. White-Nose Syndrome is an ongoing fungal epizootic of North American hibernating bats, caused by epidermal infections of the fungus, Pseudogymnoascus destructans. Infections emerged early in 2006 in New York State and have since spread to 35 US States and seven Canadian Provinces, with rates of mortality exceeding 90% in some bat colonies. As an emerging outbreak in North America, the transmission of P. destructans is assumed to occur in a radial fashion outwards from the point of origin. In addition, the factors that may influence P. destructans transmission have been postulated, but not tested before. Lastly, as reproduction is assumed to be strictly clonal in North America, invasive populations should have low genetic diversity, and may even accumulate deleterious mutations over time. The aim of my PhD research is to test these assumptions regarding the spread, evolution, and adaptation of P. destructans using combination of genotyping methods. My results showed how P. destructans isolates have shifted in terms of phenotypes and physiological capabilities since being introduced. In addition, I describe patterns of connectivity across the landscape, which are more consist with the level of anthropogenic activity than variation in climate. The mutations common to all invasive strains of P. destructans are associated with adaptations that have occurred since being introduced from Europe, some with relevant metabolic functions that fit their pathogenic lifestyle. Together, my results revealed significant phenotypic and genotypic changes during the spread of P. destructans in North America. The factors identified here that influence the phenotypic and genotypic changes should help developing better management strategies against the White-Nose Syndrome pathogen. / Thesis / Doctor of Philosophy (PhD)

O câncer de mama é uma doença heterogênea que é caracterizada por células epiteliais de mama malignas. As células-tronco cancerígenas (CST) no câncer de mama podem aumentar o potencial de agressividade através do tumor. O objetivo deste estudo é avaliar a expansão clonal do microambiente tumoral e a diferenciação celular após o estímulo com células estaminais mesenquimais. As células MSC derivadas da geléia de Wharton foram co-cultivadas com MCF- 7 em proporções de 1%, 10%, 30%. A co-cultura de MCF-7 com MSC mostrou alteração na localização da e-caderina para o citoplasma e, de preferência, o núcleo. Para a n-caderina, a co-localização foi predominantemente na membrana após a exposição do MSC. As células MCF-7 apresentaram colocalização do citoplasma e do núcleo no biomarcador de ?-catenina. Este fenômeno é confirmado à WB com o aumento dos níveis de proteínas de ecaderina no citoplasma no MCF-7 após o estímulo do MSC. A morfologia das transições amênico-mesenquimatosas foi mostrada no ensaio 3D em algumas colônias, no entanto esta morfologia não é predominante. A co-cultura de MCF- 7 com MSCs aumenta o número de mammosferes e influencia o aumento de CD44 + / CD24-, esse fenômeno foi possível sob estimulação de 30% das células MSC. A linhagem celular de câncer de mama MCF-7 em associação com MSC pode aumentar o potencial de agressividade através do tumor. Essa interação no microambiente do tumor é determinante para a expansão clonal e diferenciação celular, que são mecanismos relevantes no processo de disseminação metastática. / Breast cancer is a heterogeneous disease that is characterized by malignant breast epithelial cells. Cancer stem cells (CST) in breast cancer can boost a potential for aggressiveness trough the tumor. The goal for this study is to evaluated the tumor microenvironment clonal expansion and cellular differentiation after stimulus with mesenchymal stem cells. MSC cells derived from Wharton\'s jelly were co-cultured with MCF-7 in proportions 1%,10%,30%. The co-culture of MCF-7 with MSC showed alteration on localization of ecadherin to the cytoplasm and preferably nucleus. For n-cadherin, the colocalization were predominantly in membrane after MSC exposition. MCF-7 cells showed cytoplasm and nucleus co-localization on ?-catenin biomarker. This phenomenon is confirmed to WB with increasing the proteins levels of ecadherin on cytoplasm in MCF-7 after MSC stimulus. Amoeboid to mesenchymal transitions morphology were showed on 3D assay in some colonies, however this morphology is not predominant. The co-culture of MCF-7 with MSCs increase in the number of mammospheres and influence the increase of CD44+/CD24-, this phenomenon was possible under stimulation of 30% of MSC cells. The breast cancer cell line MCF-7 in association with MSC can boost a potential for aggressiveness trough the tumor. This interaction on tumor microenvironment is determinant for the clonal expansion and cellular differentiation, which are relevant mechanisms in the process of metastatic dissemination.

For the first time in human history, more people worldwide suffer from obesity than are undernourished. Numerous health complications are associated with obesity including cardiovascular disease, Type 2 Diabetes, cancers of reproductive tissues, stroke, depression, anxiety disorders, and Alzheimer’s disease. A deeper understanding of the anti-adipogenic effects and mechanism of action of sesquiterpene lactones may have pharmacological import in the continuing search for therapeutic modalities to ameliorate the effects of this global obesity epidemic. Dehydroleucodine (DhL), 11,13-dihydro-dehydroleucodine (DH-DhL), and dehydroparashin-B (DhP), sesquiterpene lactones extracted from or derived from compounds extracted from Artemisia douglasiana, were investigated for their anti-adipogenic effects on 3T1-L1 preadipocytes. Dehydroleucodine inhibited the expression of C/EBPa and PPARg, and also strongly blocked the expression of C/EBPβ, an early stage biomarker of early adipogenesis, in a concentration-dependent manner. Dehydroleucodine arrested the cell cycle at the G0/G1 phase, increased p27 and decreased both cyclins A and D and their partners (e.g., CDK2 and CDK4). Furthermore, DhL downregulated expression of histone demethylase JMJD2 as well as repressed the expression of histone methyltransferase MLL4, which in turn diminished the expression of C/EBPb and PPARg, respectively. 11,13-dihydro-dehydroleucodine blocked the accumulation of lipid droplets and inhibited the expression of PPARγ and C/EBPβ. Collectively, the results indicate that the inhibition of early stage preadipocyte differentiation by DH-DhL may be associated with cell cycle arrest at the G0/G1 phase. Dehydroparashin-B significantly decreased the accumulation of lipid content and downregulated the expression of CEBPβ, PPARγ and CEBPα as well as FAS. Interestingly, the addition of DhP inhibited the number as well as the size of the lipid droplets during the differentiation of 3T3-L1 preadipocytes. Taken together, this data suggests that DhP has an important inhibitory effect on cellular pathways regulating adipocyte differentiation.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22036号 / 医博第4521号 / 新制||医||1038(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 滝田 順子, 教授 松田 道行, 教授 山田 亮 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The human gamma-delta (gd) TCR+ T-cell subset may undergo specific antigen-driven activation and clonal expansion, in the context of systemic sclerosis (SSc) pathogenesis. The purpose of this study was; 1) To determine whether gd TCR+ T-cells are clonally expanded in skin biopsies and peripheral blood from patients with SSc; and 2) To develop approaches for identification of the antigens recognized by these clonally-expanded gd TCR+ T-cells. Total RNA was isolated from the skin biopsies and peripheral blood of patients with SSc (n=8). After cDNA synthesis, the g- and d-chain TCR transcripts were amplified by PCR, cloned and sequenced for analysis. Full length copies of the TCR transcripts were constructed, expressed in a TCR-negative Jurkat T-cell line using retroviral gene transduction, and verified by RT-PCR and flow cytometry for gd TCR expression. Putative antigen recognition, by the transduced gd TCR+ Jurkat T-cell lines, was assessed via; 1) Measuring intracellular calcium flux in the transduced cells after stimulation with putative SSc antigens, including DNA topoisomerase I, centromere proteins A and B, hsp 27, hsp 90 and the viral lysate of human cytomegalovirus; and 2) Cytotoxicity against human endothelial cell lines (HUVEC and HLMVEC) via measurement of lactate dehydrogenase release from the targets. We report the presence of substantial, statistically-significant, proportions of identical g- and d-chain transcripts in skin biopsies and PBMC of patients with SSc, demonstrating the presence of antigen-driven clonal expansions. Jurkat T-cells, transduced with the clonally-expanded gd TCR transcripts from a patient, showed no evidence of cytotoxicity against the human endothelial cell lines, or calcium flux in response to stimulation with the putative SSc antigens assessed. In conclusion, extensive clonal expansions of g- and d-chain TCR transcripts were identified in skin biopsies and peripheral blood of patients with SSc, demonstrating the presence of oligoclonal populations of gd TCR+ T-cells in these patients. These gd TCR+ T-cells have undergone proliferation and clonal expansion in vivo in response to as yet unidentified antigens. Furthermore, an approach has been developed for the identification of the antigens recognized by the clonally-expanded gd TCR transcripts, which can be expanded to additional patients with SSc. / Microbiology and Immunology