Myelin is critical for the operation of a functional vertebrate nervous system, allowing for rapid saltatory conduction and providing trophic support to axons. In multiple sclerosis (MS), the immune system attacks myelin sheaths, leading to de-myelination of axons. De-myelinated axons not only lose their ability to conduct rapid nerve impulses, but are themselves susceptible to damage and loss. Long term demyelination leads to neuronal loss and the devastating symptoms of secondary stages of MS. One therapeutic approach which has been suggested is to improve the ability of oligodendrocyte precursor cells (OPCs) to differentiate into mature re-myelinating oligodendrocytes. This process is known to occur in vivo, however, the myelin produced appears reduced and the efficiency with which OPCs differentiate into myelinating oligodendrocytes (OLs) varies greatly. For example, the ability of OPCs from older mice to differentiate is reduced compared to those from young mice. This fact taken alongside the presence of many OPCs in some MS lesions which have failed to re-myelinate makes identifying compounds which can increase OPC differentiation into OLs a key goal for MS drug development. In this work, I use OPC to OL differentiation during zebrafish development as a model for differentiation of OLs more generally. Zebrafish are widely used for chemical screening, with recent developments in genetic manipulation, such as CRISPR/Cas9 gene editing and Tol2 transgenesis, allowing for production of targeted mutations and fluorescent reporter lines respectively. Retinoid X receptor-γ (RXRγ) has previously been identified as being transcriptionally upregulated during re-myelination. Moreover, treatment with 9-cis-retinoic acid, an agonist for the receptor, has been shown to improve remyelination in vivo in rats with toxin induced focal demyelination. I first present a manual chemical screen of a library of compounds designed to target RXRγ, from which I identify several compounds which reproducibly increase OLs in zebrafish. In order to assess whether any of the hit compounds could be acting as agonists for RXRγ, I have created a double knockout zebrafish lacking both genes coding for the zebrafish RXRγ homologues (rxrga and rxrgb). This line has been used to test the activity of hit compounds in a RXRγ loss of function background. Following this first chemical screen, it was clear that great improvements could be made to both throughput and robustness if the screen was automated. Using a commercially available fish handling robot which automates the imaging of plates of zebrafish embryos, known as the VAST BioImager, the throughput of our assay was increased from ~40 fish per day to up to around 300 to 400. We combined the VAST BioImager with a state of the art spinning disk confocal microscope, giving us (to the best of our knowledge) the world's fastest in vivo vertebrate screening system capable of orienting fish and imaging at sub-cellular resolution. This significant increase in rate of fish processing led to the need for an increase in the rate of image analysis. Much of the gains in throughput would be lost to time counting cells, and so I developed software to automate the image processing and analysis. The software developed is shown to closely match the abilities of a human to identify compounds which give significant increases in OLs, with very little human intervention required. In the final section of this work I present an example screen performed using the VAST BioImager in combination with the automated cell counting software, which I developed. The hits from this screen highlight our ability to automatically identify compounds that increase the number of OLs in the developing zebrafish. This method is broadly applicable to other central nervous system cell types and other methods of analysis can be integrated into the presented screening software.

Master of Science / Biochemistry and Molecular Biophysics / Zhilong Yang / Poxvirus infections continue to threaten human health despite the eradication of smallpox, which was one of the most lethal infectious diseases in human history. Our objectives were to identify the host cell components/functions that are important for poxvirus infection and to gain insights into the molecular mechanism of poxvirus replication, ultimately guiding novel anti-viral development. Using vaccinia virus, the prototype poxvirus, we screened inhibitors of viral replication from over 3,000 chemical compounds, most of which have known cellular targets. This screening revealed numerous JAK/STAT3 inhibitors that could inhibit the replication of vaccinia virus. We further used multiple inhibitors of the JAK/STAT3 pathway and tested their effects on the replication of vaccinia virus in multiple primary and transformed cells through reporter assay and viral infectious particles measurement. The JAK/STAT3 inhibitors being tested were: SC144, an inhibitor of the interleukin 6(IL-6), a receptor of the JAK/STAT3 signaling pathway, AZ960 (a JAK2 inhibitor), Stattic and niclosamide (inhibitors of STAT3). Overall, our data indicate the JAK/STAT3 inhibitors could repressed vaccinia virus replication in multiple cell types, suggesting that the JAK/STAT3 signaling pathway is required for the efficient replication of vaccinia virus. Moreover, we observed that STAT3 was enriched in the cell nucleus, although the phosphorylation level of STAT3 was downregulated in vaccinia virus-infected cells during the early stages of infection. This study demonstrates an important role of the JAK/STAT3 signaling pathway in the replication of vaccinia virus, providing a possible novel direction by which to intervene in poxvirus infection and related diseases.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24090号 / 医博第4866号 / 新制||医||1059(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 岩田 想, 教授 林 康紀, 教授 松田 道行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Pigment patterns of zebrafish are a beautiful example in which to study key processes of vertebrate development such as neural crest cell migration and patterning of neural crest-derived cell types. This can for example be achieved by characterizing mutants like parade in which pigment cell development is abnormal. Here, we present a chemico-genetic study of the pigment pattern mutant parade; uniquely, this mutant displays ectopic pigment cells in the ventral medial pathway of the trunk but the characteristic stripe pattern of zebrafish embryos is unaffected. Using a positional cloning approach, we have identified the parade gene as the cell surface receptor ednra2. This was further confirmed in transient knock-down assays. Combined sequencing data from three different parade alleles strongly indicates that the mutation disrupts ednra2 receptor function by deleting C-terminal regulatory and structural residues. To expand the available molecular tools in pigment cell research, notably to chemically dissect the parade phenotype, we have participated in small molecule screening for inhibitors of pigment cell development. From this, we have isolated 57 compounds which robustly alter the development of melanophores and iridophores in wild-type embryos; 26 of these compounds additionally affect the parade phenotype, primarily by rescuing the ectopic pigment cells. Notably, chemical rescue has shown that the MEK pathway is important for the development of the parade phenotype. Our study therefore adds to our understanding of pigment pattern formation in zebrafish embryos and reveals novel functions for ednra2 in dorso-ventral patterning and cell type specification of neural crest derivatives.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23565号 / 医博第4779号 / 新制||医||1054(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 武藤 学, 教授 万代 昌紀, 教授 上杉 志成 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21006号 / 医博第4352号 / 新制||医||1028(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 高橋 淳, 教授 濵﨑 洋子, 教授 渡邊 直樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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