This research programme investigated the expression of prolyl hydroxylase (PHD) proteins in rodent tissues. The importance of PHD enzymes lies in their ability to render oxygen sensitivity to Hypoxia inducible factor (HIF), the principal mediator of intracellular oxygen homeostasis. The first part of this study focused on developing and validating anti-sera capable of detecting PHD proteins in rodent tissues. With these reagents, it was possible to assess the relative expression of each PHD protein in a number of different rat tissues. PHD2 was the most abundant isoform in all tissues studied. In contrast, an abundance of PHD1 was observed only in testis and skeletal muscle. A number of different tissue species of PHD3 were identified and their abundance was found to vary between different tissues. These observations provide further evidence of the principal role of PHD2 in regulating HIF in vivo, but also point towards additional roles for PHD1 and PHD3 in selected tissues. They highlight the potential for there being a complex interplay between different PHD enzymes which could, in the future, prove potential targets for therapeutic manipulation. This study also provides additional insights into the mechanisms underlying the phenotypes observed in PHD deletional mouse models which appear, in many cases, to be directly related to the abundance of a given PHD isoform. The emerging role of PHD3 as a promoter of sympathetic lineage apoptosis prompted further study of PHD3 expression in rat neuronal tissues. An abundance of PHD3 was demonstrated throughout the rat sympathetic nervous system, a finding which appeared at odds with its known role as a promoter of neuronal apoptosis and resulted in a series of collaborative studies which demonstrated a sympatho-adrenal phenotype in wild type compared to PHD3-/- mice. Further collaborative studies utilising wild type mice and those deleted of specific PHD isoforms, were carried out to assess the significance of the abundance of PHD3 and PHD1 noted here in rat hippocampus and testis respectively. While neither study demonstrated statistically significant phenotypes, these observations remain of interest and areas for future research.

RNA Binding Motif (RBM) domain proteins RBM5 and RBM10 have been shown to influence apoptosis, cell cycle arrest and splicing in transformed cells. In this study, RBM5 and RBM10 were examined in non-transformed cells in order to gain a wider range of knowledge regarding their function. Expression of Rbm5 and Rbm10, as well as select splice variants, was examined at the mRNA and protein level throughout H9c2 skeletal and cardiac myoblast differentiation. Results suggest that Rbm5 and Rbm10 may (a) be involved in regulating cell cycle arrest and apoptosis during skeletal myoblast differentiation and (b) undergo post-transcriptional or translational regulation throughout myoblast differentiation. All in all, the expression profiles obtained in the course of this study will help to suggest a role for Rbm5 and Rbm10 in differentiation, as well as possible differentiation-specific target genes with which they may interact.

Analyses of protein sequences from diverse genomes have revealed the ubiquitous nature of multi-domain proteins. They form up to 70% of proteomes of most eukaryotic organisms. Yet, our understanding of protein structure, folding and evolution has been dominated by extensive studies on single-domain proteins. We provide quantitative treatment and proof for prevailing intuitive ideas on the strategies employed by nature to stabilize otherwise unstable domains. We find that domains incapable of independent stability are stabilized by favourable interactions with tethered domains in the multi-domain context. Natural variations (nsSNPs) at these sites alter communication between domains and affect stability leading to disease manifestation. We emphasize this by using explicit all-atom molecular dynamics simulations to study the interface nsSNPs of human Glutathione S-transferase omega 1. We show that domain-domain interface interactions constrain inter-domain geometry (IDG) which is evolutionarily well conserved. The inter-domain linkers modulate the interactions by varying their lengths, conformations and local structure, thereby affecting the overall IDG. These findings led to the development of a method to predict interfacial residues in multi-domain proteins based on difference evolutionary information extracted from at least two diverse domain architectures (single and multi-domain). Our predictions are highly accurate (∼85%) and specific (∼95%). Using predicted residues to constrain domain–domain interaction, rigid-body docking was able to provide us with accurate full-length protein structures with correct orientation of domains. Further, we developed and employed an alignment-free approach based on local amino-acid fragment matching to compare sequences of multi-domain proteins. This is especially effective in the absence of proper alignments, which is usually the case for multi-domain proteins. Using this, we were able to recreate the existing Hanks and Hunter classification scheme for protein kinases. We also showed functional relationships among Immunoglobulin sequences. The clusters obtained were functionally distinct and also showed unique domain-architectures. Our analysis provides guidelines toward rational protein and interaction design which have attractive applications in obtaining stable fragments and domain constructs essential for structural studies by crystallography and NMR. These studies enable a deeper understanding of rapport of protein domains in the multi-domain context.

By cross-linking B7-DC on dendritic cells (DC) the human IgM antibody (B7-DC XAb) shifts polarized immune responses from Th2 to Th1 in an antigen-specific manner. The molecular determinants governing the ability of DC to reprogram the polarity of T cell recall responses are not yet known. In addition to the expected role of T-bet expressed by T cells in regulating Th1 responses, we find using in vitro assays and an established in vivo model of allergic airway inflammation that T-bet expression by DC is also required for the polarity shift promoted by B7-DC XAb. T-bet expression by both T cells and DC is critically important for B7-DC XAb-induced down-regulation of IL-4, up-regulation of IFN-γ and suppression of allergic airway inflammation. Moreover, retroviral reconstitution of T-bet expression in T-bet-deficient DC rescued their ability to modulate both naive and memory T-cell responses from Th2 to Th1. Our observations further our understanding of the critical mediators controlling the ability of DC to modify the responses of previously activated T cells and reveal the interesting use of the same transcription factor to regulate the inductive phenotype of DC and the inducible phenotype of T cells.

京都大学 / 0048 / 新制・論文博士 / 博士(理学) / 乙第12911号 / 論理博第1547号 / 新制||理||1590(附属図書館) / 32121 / 京都大学大学院理学研究科・物理学・宇宙物理学専攻 / (主査)講師 市川 正敏, 教授 山本 潤, 教授 田中 耕一郎 / 学位規則第4条第2項該当 / Doctor of Science / Kyoto University / DGAM

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Submitted by Maria Cristina (library@lncc.br) on 2015-09-23T19:19:26Z No. of bitstreams: 1 dissertacao-vitor.pdf: 1691206 bytes, checksum: fef620f128c4346f7ff506439cdffdfd (MD5) / Approved for entry into archive by Maria Cristina (library@lncc.br) on 2015-09-23T19:20:00Z (GMT) No. of bitstreams: 1 dissertacao-vitor.pdf: 1691206 bytes, checksum: fef620f128c4346f7ff506439cdffdfd (MD5) / Made available in DSpace on 2015-09-23T19:20:12Z (GMT). No. of bitstreams: 1 dissertacao-vitor.pdf: 1691206 bytes, checksum: fef620f128c4346f7ff506439cdffdfd (MD5) Previous issue date: 2015-03-31 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Splicing Alternativo (SA) é um mecanismo pós-transcricional que produz mais de um produto de gene, através da combinação de diferente éxons do lócus genômico, gerando grande parcela da variedade do proteoma dos eucariotos. Ao longo da última década, como seu papel regulatório tornou-se mais e mais evidente, SA tornou-se um fator chave para criação de complexidade de diferentes organismos dado um repertório constante de genes através das gerações. Através da inserção, exclusão ou substituição de partes da sequência do transcrito, SA pode obviamente também ter um impacto nos domínios funcionais de proteínas. Apesar de algumas tentativas que tem sido principalmente prejudicadas por questões técnicas causada pela redundância nas sequências transcritos alternativos, os efeitos em larga escala do SA em nível funcional tem sido pouco estudados até os dias de hoje. Este projeto descreve o desenvolvimento de uma ferramenta computacional chamada ASTAFUNK - Alternative Splicing Trancriptional Analyses with FUNctional Knowledge - um programa stand-alone automatizado e eficiente para estudar como a diversidade de determinado transcriptoma é traduzido em variação funcional, baseado em um padrão de anotação de transcriptomas (em GTF, Gene Transfer Format) e perfis de domínios (no formato do Pfam). Resumidamente, ASTAFUNK traduz as regiões que sofreram splicing alternativo de open read frames em sequências de aminoácidos, que subsequentemente são alinhadas com o profile Hidden Markov Model da base de dados do Pfam, empregando programação dinâmica padrão (algoritmo de Viterbi) com alguns refinamentos técnicos (isto é, abordagem branch-and-bound). Em contraste com ferramentas convencionais de predição de domínios (por exemplo, HMMER), o algoritmo de ASTAFUNK foi projetado para evitar escaneamentos redundantes de sequências em transcriptomas com alto grau de SA. Neste trabalho, aspectos teóricos e práticos da abordagem do ASTAFUNK são avaliados, e a eficiente implementação em JAVA é disponível livremente na internet sob BSD 3-clause open source license. / Alternative splicing (AS) is a mechanism that produces more than one gene product at the transcriptional level, by combining different exons of a gene, generating a major part of the proteome diversity in eukaryotes. Over the last decade, as the regulatory role of splicing has become more and more evident, AS turned a key factor in creating different organism complexity given a rather constant repertoire of genes across generations. By inserting, deleting or substituting part of the transcript sequence, AS can obviously also have an impact on functional protein domains. Despite some attempts that have mainly been hampered by technical issues caused by the redundancy in alternative transcript sequences, the large-scale effects of AS on the functional level has been poorly studied so far. This project describes the development of a computational tool called ASTAFUNK - Alternative Splicing Trancriptional Analyses with FUNctional Knowledge - an automated and efficient stand-alone program to study how diversity of a custom transcriptome translates into functional variation, based on standard transcriptome annotations (in GTF, Gene Transfer Format) and domain profiles (in Pfam format). In a nutshell, \afunk{} translates the alternatively spliced parts of open reading frames on the fly into amino acid sequences, which subsequently are aligned with the profile Hidden Markov Models from Pfam employing standard dynamic programming (Viterbi's algorithm) with some technical refinements (i.e., a branch-and-bound approach). In contrast to conventional domain prediction tools (e.g., the HMMER aligner), the ASTAFUNK algorithm has been designed to avoid redundant sequence scans in AS-enriched transcriptomes. In this work, theoretical and practical aspects of the ASTAFUNK approach are evaluated, and the efficient JAVA implementation is made freely available over the internet under the BSD 3-clause open source license.

Ebola virus (EBOV) is a member of the Filoviridae family of highly pathogenic viruses that cause severe hemorrhagic fever and is the causative agent of the 2014 West Africa outbreak. Currently, there are no approved filovirus vaccines or treatments to combat these sporadic and deadly epidemics. One target for EBOV antiviral therapy is to block viral entry into host cells. Recently, phosphatidylserine (PtdSer) receptors, primarily known for their involvement in the clearance of dying cells, were shown to mediate entry of enveloped viruses including filoviruses. The PtdSer receptors, T-cell immunoglobulin mucin domain-1 (TIM-1) and family member TIM-4, serve as filovirus receptors, significantly enhancing EBOV entry. TIM-dependent virus uptake occurs via apoptotic mimicry by binding to PtdSer on the surface of virions through a conserved PtdSer binding pocket within the amino terminal IgV domain. TIM-4 is expressed on antigen presenting cells (APCs), including macrophages and dendritic cells (DCs), which are critical in early EBOV infection. My studies are the first to define the molecular details of virion/TIM-4 interactions and establish the importance of TIM-4 for EBOV infection of murine resident peritoneal macrophages. In addition, previous work has utilized only in vitro models to establish the importance of the TIM proteins in EBOV entry. My studies are the first to demonstrate the importance of TIM-1 and TIM-4 for in vivo EBOV pathogenesis and to confirm them as relevant targets of future filovirus therapeutics. Macrophage phenotypes can vary greatly depending upon chemokine and cytokine signals from their microenvironment. Historically, macrophages have been classified into two major subgroups: classically activated macrophages (M1) and alternatively activated macrophages (M2). Macrophages are a critical early target of EBOV infection and my work primarily focused on interferon gamma-stimulated (M1) macrophages since this treatment profoundly inhibited EBOV infection of human and murine macrophages. Interferon gamma treatment blocked EBOV replication in macrophages, reducing viral RNA levels in a manner similar to that observed when cultures were treated with the protein synthesis inhibitor, cycloheximide. Microarray studies with interferon gamma-treated human macrophages identified more than 160 interferon-stimulated genes. Ectopic expression of a select group of these genes inhibited EBOV infection. These studies provide new potential avenues for antiviral targeting as these genes that have not previously appreciated to inhibit infection of negative strand RNA viruses including EBOV. In addition and most exciting, using MA-EBOV, we found that murine interferon gamma, when administered either 24 hours before or after infection, protects lethally challenged mice and significantly reduces morbidity. Our findings suggest that interferon gamma, an FDA-approved drug, may serve as a novel and effective prophylactic or treatment option.

Axonal and synaptic degeneration is a hallmark of peripheral neuropathy, brain injury, and neurodegenerative disease. Axonal degeneration has been proposed to be mediated by an active autodestruction program, akin to apoptotic cell death; however, loss-of-function mutations capable of potently blocking axon self-destruction have not been described. Using a forward genetic screen in Drosophila, we identified that loss of the Toll receptor adaptor dSarm (sterile a/Armadillo/Toll-Interleukin receptor homology domain protein) cell-autonomously suppresses Wallerian degeneration for weeks after axotomy. Severed mouse Sarm1 null axons exhibit remarkable long-term survival both in vivo and in vitro, indicating that Sarm1 prodegenerative signaling is conserved in mammals. Our results provide direct evidence that axons actively promote their own destruction after injury and identify dSarm/Sarm1 as a member of an ancient axon death signaling pathway. This death signaling pathway can be activated without injury by loss of the N-terminal self-inhibitory domain, resulting in spontaneous neurodegeneration. To investigate the role of axon self-destruction in disease, we assessed the effects of Sarm1 loss on neurodegeneration in the SOD1-G93A model of amyotrophic lateral sclerosis (ALS), a lethal condition resulting in progressive motor neuron death and paralysis. Loss of Sarm1 potently protects motor axons and synapses from degeneration, but only extends animal survival by 10%. Thus, there appears to be at least two driving forces in place during ALS disease progression: (1) Sarm1 mediated axon death, and (2) cell body destruction via some unknown mechanism.

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