京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第22283号 / 理博第4597号 / 新制||理||1659(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)准教授 佐藤 ゆたか, 教授 高橋 淑子, 准教授 秋山 秋梅 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Yes / Ca2+ release from the Golgi apparatus regulates key functions of the organelle, including vesicle trafficking. However, the signaling pathways that control this form of Ca2+ release are poorly understood and evidence of discrete Golgi Ca2+ release events is lacking. Here, we identified the Golgi apparatus as the source of prolonged Ca2+ release events that originate from the nuclear ‘poles’ of primary cardiac cells. Once initiated, Golgi Ca2+ release was unaffected by global depletion of sarcoplasmic reticulum Ca2+, and disruption of the Golgi apparatus abolished Golgi Ca2+ release without affecting sarcoplasmic reticulum function, suggesting functional and anatomical independence of Golgi and sarcoplasmic reticulum Ca2+ stores. Maximal activation of β1-adrenoceptors had only a small stimulating effect on Golgi Ca2+ release. However, inhibition of phosphodiesterase (PDE) 3 or 4, or downregulation of PDE 3 and 4 in heart failure markedly potentiated β1-adrenergic stimulation of Golgi Ca2+ release, consistent with compartmentalization of cAMP signaling within the Golgi apparatus microenvironment. β1-adrenergic stimulation of Golgi Ca2+ release involved activation of both Epac and PKA signaling pathways and CaMKII. Interventions that stimulated Golgi Ca2+ release induced trafficking of vascular growth factor receptor-1 (VEGFR-1) from the Golgi apparatus to the surface membrane. These data establish the Golgi apparatus as a juxtanuclear focal point for Ca2+ and β1-adrenergic signaling, which functions independently from the sarcoplasmic reticulum and the global Ca2+ transients that underlie the primary contractile function of the cell.

Medulloblastomas are tumours of cerebellar origin and are thought to arise from the malignant transformation of progenitor cells in the developing cerebellum. A number of developmental signalling pathways are required for the precise cell specification, proliferation, migration and differentiation involved in forming the mature cerebellum and it is the dysregulation of these processes that can lead to the eventual formation of a tumour. Genes encoding components of the canonical Wnt/β-catenin signaling pathway are mutated in around 15% of medulloblastomas and germline mutations that activate this pathway are known to predispose to medulloblastoma. Despite this, the contribution of Wnt/β-catenin signaling to normal cerebellum development is not yet well understood and the developmental origins of medulloblastoma arising from activation of this pathway are only beginning to be revealed. Therefore, the aims of this thesis were to characterise the spatio-temporal nature of Wnt/β-catenin signalling during cerebellum development and to investigate its function, with the broad goal of informing our understanding of how medulloblastoma arises from oncogenic activation of Wnt/β-catenin signalling. To address the first aim I utilised a LacZ expressing Wnt/β-catenin signalling reporter mouse to characterize the spatio-temporal pattern of Wnt/β-catenin pathway activation during cerebellum development. Analysis of LacZ reporter expression revealed a pattern of transient Wnt/β-catenin activity in discrete cell populations throughout cerebellum development. I found that Wnt/β-catenin activity is present during the early specification of granule cells at the cerebellar rhombic lip but not during the expansion of this cell population at later stages. During perinatal development Wnt/β-catenin activity shifts to the cerebellar ventricular zone, a known germinal centre for GABAergic interneurons and glia, and was observed in cells radiating out from this region. By early postnatal development the expression of the Wnt/β-catenin reporter became progressively restricted to the developing Bergmann glia population. To investigate the function of Wnt/β-catenin in these cell lineages and how its dysregulation could contribute to medulloblastoma, I used a combination of ex vivo organotypic culture, in utero electroporation and tissue-specific gene targeting to manipulate components of the pathway. Culturing slices of E18.5 cerebellum in the presence of small molecule activators of the Wnt/β-catenin pathway revealed a reduction in the expression of glial markers Sox9 and GFAP. In addition, interneuron lineage marker Pax2 was also reduced, supporting the conclusion that dysregulation of Wnt/β-catenin signalling affects the generation of cell lineages from the ventricular zone. To investigate this hypothesis further, I constitutively activated the Wnt/β-catenin signalling pathway in the developing cerebellum using Cre-Lox gene targeting to knock out Apc, a negative regulator of the pathway, in ventricular zone derived lineages. Cre-induced recombination of Apc resulted in nuclear accumulation of β-catenin, a sign that the pathway had become ectopically activated. Furthermore, a reduction in the expression of Sox9 and Pax2 was also observed in these mutant cells. From these data, I conclude a potential role for Wnt/β-catenin signaling in the regulation of glial/interneuron progenitors. Combined, these data support a model where Wnt/β-catenin signalling could perform multiple functions in specification of the granule lineage, regulation of glial/interneuron progenitors and in glial differentiation/maturation. Importantly, dysregulation of progenitor self-renewal and differentiation is widely acknowledged to promote tumourigenesis. Thus, the data in this thesis support a potential mechanism for the development of medulloblastoma from the dysregulation of ventricular zone progenitors.

No description available.

Master of Science in Biomedical Sciences / Department of Anatomy and Physiology / Yongming Sang / Animal metabolic and immune systems integrate and inter-regulate to exert effective immune responses to distinct pathogens. The signaling pathway mediated by mechanistic target of rapamycin (mTOR) is critical in cellular metabolism and implicated in host antiviral responses. Recent studies highlight the significance of the mTOR signaling pathway in the interferon (IFN) response. Type I IFNs mediate host defense, particularly, against viral infections, and have myriad roles in antiviral innate and adaptive immunity. In addition to their well-known antiviral properties, type I IFNs also affect host metabolism. However, little is known about how animal type I IFN signaling coordinates immunometabolic reactions during antiviral defense. Therefore, understanding the interaction of mTOR signaling and the type I IFN system becomes increasingly important in potentiating antiviral immunity. Tissue macrophages (MФs) are a primary IFN producer during viral infection, and their polarization to different activation statuses is critical for regulation of immune and metabolic homeostasis. Using porcine reproductive and respiratory syndrome virus (PRRSV) as a model, we found that genes in the mTOR signaling pathway were regulated differently in PRRSV-infected porcine alveolar MФs at different activation statuses. Therefore we hypothesize that: 1) the mTOR signaling pathway involves host anti-PRRSV regulation; 2) mTOR signaling interacts with IFN signaling to modulate the antiviral response; and 3) different type I IFN subtypes (such as IFN-α1 and IFN-β) regulate mTOR signaling differently. We show that modulation of mTOR signaling regulated PRRSV infection in MARC-145 cells and porcine primary cells, in part, through regulating production and signaling of type I IFNs. In addition, expression and phosphorylation of two key components in the mTOR signaling pathway, AKT and p70 S6 kinase, were regulated by type I IFNs and PRRSV infection. Taken together, we determined that the mTOR signaling pathway, a key pathway in regulation of cell metabolism, also mediates the type I IFN response, a key immune response in PRRSV infection. Our findings reveal that the mTOR signaling pathway potentially has a bi-directional loop with the type I IFN system and implies that some components in the mTOR signaling pathway can serve as targets for augmentation of antiviral immunity and therapeutic designs.

As the most frequently diagnosed cancer in North American men, prostate cancer can progress to the androgen independent stage after initial response to androgen ablation therapy. The molecular mechanisms involved in the hormonal progression of prostate cancer are not completely understood. Here, we analyze changes in the transcriptome of prostate cancer cells at different stages of progression to reveal potential mechanisms. Applying Affymetrix GeneChip technology, we identified the transcriptomes in response to stimulation of androgen and PKA pathways in human prostate cancer cells. In addition to PSA, other common target genes were identified. Genes differentially expressed in response to androgen and stimulation of the PKA pathway in vitro were also differentially expressed during hormonal progression in vivo. Upon androgen stimulation, androgen receptor binds to a functional androgen response element within the promoter region of SESN1, a p53 targeted gene, and represses its expression. The expression of SESN1 was induced by castration in LNCaP xenografts, but the expression was eventually suppressed again in the androgen independent stage of prostate cancer. Knockdown of SESN1 promoted the proliferation of prostate cancer cells. Expression patterns of androgen-regulated genes in androgen independent tumours were revealed to be more similar to that from before castration than to the tumors under androgen ablation. The β-catenin, a potent coactivator of the androgen receptor, and Wnt pathway was deregulated in androgen-independent tumours. There was increased nuclear colocalization and interaction of androgen receptor and β-catenin with hormonal progression of prostate cancer. This study provides insight into hormonal effects on prostate cancer and possible pathways involved in the development of androgen independent disease, as well as potential therapeutic targets.

As the most frequently diagnosed cancer in North American men, prostate cancer can progress to the androgen independent stage after initial response to androgen ablation therapy. The molecular mechanisms involved in the hormonal progression of prostate cancer are not completely understood. Here, we analyze changes in the transcriptome of prostate cancer cells at different stages of progression to reveal potential mechanisms. Applying Affymetrix GeneChip technology, we identified the transcriptomes in response to stimulation of androgen and PKA pathways in human prostate cancer cells. In addition to PSA, other common target genes were identified. Genes differentially expressed in response to androgen and stimulation of the PKA pathway in vitro were also differentially expressed during hormonal progression in vivo. Upon androgen stimulation, androgen receptor binds to a functional androgen response element within the promoter region of SESN1, a p53 targeted gene, and represses its expression. The expression of SESN1 was induced by castration in LNCaP xenografts, but the expression was eventually suppressed again in the androgen independent stage of prostate cancer. Knockdown of SESN1 promoted the proliferation of prostate cancer cells. Expression patterns of androgen-regulated genes in androgen independent tumours were revealed to be more similar to that from before castration than to the tumors under androgen ablation. The β-catenin, a potent coactivator of the androgen receptor, and Wnt pathway was deregulated in androgen-independent tumours. There was increased nuclear colocalization and interaction of androgen receptor and β-catenin with hormonal progression of prostate cancer. This study provides insight into hormonal effects on prostate cancer and possible pathways involved in the development of androgen independent disease, as well as potential therapeutic targets.

No description available.

Understanding biochemical reactions inside cells of individual organisms is a key factor for improving our biological knowledge. Signaling pathways provide a road map for a wide range of these chemical reactions that convert one signal or stimulus into another. In general, each signaling pathway in a cell involves many different proteins, each with one or more specific roles that help to amplify a relatively small stimulus into an effective response. Since proteins are essential components of a cells activities, it is important to understand how they work and in particular, to determine which of species proteins participate in each role. Experimentally determining this mapping of proteins to roles is difficult and time consuming. Fortunately, many individual pathways have been annotated for some species, and the pathways of other species can often be inferred using protein homology and the protein properties.

abstract: Given the process of tumorigenesis, biological signaling pathways have become of interest in the field of oncology. Many of the regulatory mechanisms that are altered in cancer are directly related to signal transduction and cellular communication. Thus, identifying signaling pathways that have become deregulated may provide useful information to better understanding altered regulatory mechanisms within cancer. Many methods that have been created to measure the distinct activity of signaling pathways have relied strictly upon transcription profiles. With advancements in comparative genomic hybridization techniques, copy number data has become extremely useful in providing valuable information pertaining to the genomic landscape of cancer. The purpose of this thesis is to develop a methodology that incorporates both gene expression and copy number data to identify signaling pathways that have become deregulated in cancer. The central idea is that copy number data may significantly assist in identifying signaling pathway deregulation by justifying the aberrant activity being measured in gene expression profiles. This method was then applied to four different subtypes of breast cancer resulting in the identification of signaling pathways associated with distinct functionalities for each of the breast cancer subtypes. / Dissertation/Thesis / M.S. Computer Science 2011