Neuroplasticity research yields mixed results for the differential contribution of perilesional and contralesional brain areas to language recovery in aphasia. This paper will outline variables that mediate the presence and degree of right hemisphere activity and may account for some of the inconsistent research findings. Factors include the site and size of left hemisphere lesions, the phase of recovery, and the language task type and complexity. The performance accuracy of tasks also will be explored to further qualify the nature of homologous activity. Results found right hemisphere activation to be modulated by the damage and preservation of specific brain areas as well as by the presence of large left hemisphere lesions. Right hemisphere activity also was more consistently evident in the acute phase of recovery and returned to the left hemisphere in the chronic stage. Additionally, homologous areas tended to be more active during comprehension-based language tasks and during tasks of greater difficulty. In qualifying the nature of contralesional mechanisms, the activity appears to be more linguistic-oriented in less-recovered individuals with aphasia and more related to cognitive effort in well-recovered individuals. The nature of homologous activation depends on the brain’s ability to reactivate left hemisphere language networks. / text

Behaviors are a product of underlying neural circuits, yet there is a paucity of mechanistic information about how nervous systems contribute to the repeated evolution of similar behaviors. Theoretical studies have predicted that the same behavioral output can be generated by neural circuits with different properties. Here, we test the theory in biological circuits by comparing the central pattern generator (CPG) circuits underlying swimming behaviors in nudibranchs (Mollusca, Gastropoda, Euthyneura, Nudipleura). In comparative studies of neural circuits, neurotransmitter content can serve as landmarks or molecular markers for neuron types. Here, we created a comprehensive map of GABA-immunoreactive neurons in six Nudipleura species. None of the known swim CPG neurons were GABA-ir, but they were located next to identifiable GABA-ir neurons/clusters. Despite strong conservation of the GABA-ergic system, there were differences, particularly in the buccal ganglia, which may represent adaptive changes. We applied our knowledge of neurotransmitter distribution along with morphological traits to identify the neuron type Si1 in Flabellina, a species that swims via whole body left-right (LR) flexions and in Tritonia, a dorsal-ventral (DV) swimming species. Si1 is a CPG member of the LR species Melibe, whereas its homologue in the LR species Dendronotus is not. In Flabellina, Si1 was part of the LR CPG and despite having similar synaptic connections as Flabellina and Melibe, Si1 in Tritonia was not part of its DV swim CPG. Side by side circuit comparison of Flabellina, Melibe and Dendronotus revealed different combinations of circuit architecture and modulation resulting in different circuit configurations for LR swimming. This includes differences in the role and activity pattern of Si1, sensitivity to curare and the effect of homologues of C2, a DV CPG neuron, on the LR motor pattern. These results collectively reveal three different circuit variations for generating the same behavior. It suggests that the neural substrate from which behaviors arise is phylogenetically constrained. While this neural substrate can be configured in multiple different ways to generate the same outcome, the possibilities are finite and, as seen here, similar structural and functional neural motifs are used in the evolution of these circuits.

Holliday junctions are formed as an intermediate during DNA recombination as the two strands come together. Recombination occurs during meiosis, and also during DNA double strand repair. Trapping this branched intermediate could prevent DNA repair from occurring in cells which would prove beneficial during cancer treatment. There are many enzymes that cleave Holliday junctions. One such enzyme, T7 Endonuclease I, was specifically chosen to detect ligand binding at the core of the junction since its binding and cleavage of cruciforms is well documented. Specialized bifunctional ligands were studied in this project that were designed to bind DNA structures that are held in close proximity to one another. These compounds have two identical binding modules that are connected by a linker of various length and rigidity, with each module binding very weakly; however, when both modules bind the binding affinity is greatly enhanced. The interactions of these compounds with cruciforms are currently being studied.

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京都大学 / 新制・課程博士 / 博士(医学) / 甲第24280号 / 医博第4896号 / 新制||医||1061(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 森田 智視, 教授 松田 道行, 教授 波多野 悦朗 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Successful replication of the genome during each cell cycle requires that every replication fork merge with its opposing fork. However, lesions in the template DNA or protein-DNA barriers often impede replication forks and threaten the timely completion of genome duplication. If a fork encounters a replication fork barrier (RFB), it can be subject to a variety of fates. In some cases the replisome is maintained in a manner such that it can resume DNA synthesis when the barrier is removed. Alternatively the stalled fork is simply held in a competent state to merge with the opposing fork when it arrives. However, fork stalling can also precipitate dissociation of the replisome (fork collapse) or even fork breakage. If this happens the recombination machinery can intervene to restore DNA integrity and restart replication, albeit with a risk of causing deleterious genetic change if ectopic homologous sequences are recombined. I have exploited a site-specific RFB in fission yeast termed RTS1 to investigate the consequences of perturbing a single replication fork. RTS1 is a polar RFB (i.e. it blocks fork progression in a unidirectional fashion), enabling replication to be completed by the opposing fork. Despite this, fork blockage at RTS1 triggers a strong recombinational response that is able to restart DNA synthesis, which at least initially is highly error prone. Here, I present my work in establishing a live cell imaging approach to visualizing the recombinational response at the RTS1 RFB, demonstrating that the majority of cells initiate recombination-dependent replication (RDR). RDR begins within a few minutes of fork blockage and is only curtailed by the arrival of the opposing fork. It depends on the Rad52 protein, which remains associated with the restarted fork and whose presence correlates with its infidelity. I also illustrate the significance of various genetic factors, including Rad51, the Rad51 mediators, Fml1 helicase, Rad54 translocase, Pfh1 sweepase, and Cds1 checkpoint kinase, in modulating Rad52 localization and block-induced recombination at the RTS1 RFB.

Coronaviruses have caused high pathogenic epidemics within the human population on two occasions; in 2003 a coronavirus (HCoV-SARS) caused severe acute respiratory syndrome and in 2012 a novel coronavirus emerged named Middle East respiratory syndrome (HCoV-MERS). Four other species of coronavirus circulate endemically in the human population (HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1), which cause more benign respiratory disease than either HCoV-SARS or HCoV-MERS. The emergence of HCoV-MERS provides an additional opportunity to study the characteristics of coronaviruses. Reverse genetics can be used to study an organism’s phenotype by logical mutation of its genotype. Construction of an infectious clone construct provides a means to investigate the nature of HCoV-MERS by reverse genetics. An HCoV-MERS infectious cDNA clone system was constructed to use for reverse genetics by homologous recombination of bacterial artificial chromosomes (BACs). This system should aid in answering remaining questions of coronavirus genetics and evolution as well as expedite the development of vaccines and prophylactic treatments for HCoV-MERS. / October 2015

Mutations in the breast cancer susceptibility 1 (BRCA1) gene are linked to breast as well as ovarian cancers. However, most cancer-causing mutations within the BRCA1 gene have been found in the N’ and C’ terminal regions of the BRCA1 protein, both believed to be important for DNA double-strand break (DSB) repair. The BRCA1 C’ terminal (BRCT) repeats have been implicated in phospho-serine protein binding whereas the N’ terminal RING domain interacts with the BARD1 protein to form a hetero-dimeric complex with E3 ubiquitin ligase activity. The BRCA1 BRCT domain binds CtIP, BACH1, and RAP80, all of which have been directly implicated in homologous recombination repair (HRR). Lysine 1702 (K1702) of BRCA1 resides within the phospho-serine binding pocket of the first BRCT repeat of BRCA1. To determine the effect of manipulating the ability of BRCA1 to bind CtIP and other phospho-proteins binding to the BRCA1 BRCT domain on DSB repair, and specifically HRR, we introduced a K1702M mutation into BRCA1 known to impair BRCT binding to a pSer-X-X-Phe peptide representing BACH1. Surprisingly, instead of impairing HRR, we found that BRCA1 K1702M resulted in hyper-recombination with > 3-fold higher levels of HRR compared to wild-type BRCA1 using an HRR assay based on GFP expression in BRCA1-defective HCC1937 cells. This hyper-recombinogenic phenotype coincided with cell-cycle arrest in S/G2 suggesting that the potential lack of binding of critical proteins to the BRCA1 BRCT domain results in abnormal HRR by priming cells to undergo more HRR which is enhanced during the S and G2 phases of the cell-cycle. In line with the increased HRR seen with the HRR/GFP assay, HCC1937 cells expressing BRCA1 K1702M showed increased levels of RAD51 foci and nuclear staining suggesting that HRR was highly elevated. Interestingly, the hyper-recombinogenic phenotype of BRCA1 K1702M could be reduced to normal levels with a second mutation (I26A) in BRCA1 that affects BRCA1 and CtIP ubiquitination. These results reveal a hierarchal regulation of HRR with ubiquitination having a dominate role in DSB repair by BRCA1 and suggests that targeted disruption of BRCT-CtIP binding increases HRR that is in turn controlled by ubiquitination. In addition, we provide evidence that BRCA1 serine 1387 phosphorylation within the SQ cluster region of BRCA1 is involved in the cell survival and DNA damage response to IR. The BRCA1 S1387A mutant only partially increased the radiosurvival of HCC1937 cells compared to cells expressing wild-type BRCA1 and immunocytochemical analysis revealed wild-type BRCA1 was located in the nucleus whereas the S1387A mutant was cytoplasmic in response to IR. We also show that BRCA1 SQ cluster serine phosphorylation in addition to serine 1387 is involved in HRR. Altogether, these findings reveal the importance of various regions of BRCA1 in DSB repair and may lead to multiple strategies of modulating BRCA1 function in response to DNA damage.

Orientador: Gilberto Aparecido Coclete / Coorientador: Alvimar Lima de Castro / Banca: Leda Maria Pescinini Salzedas / Banca: Ana Lúcia Álvares Capelozza / Resumo: Enxerto ósseo tem sido realizado objetivando prevenir ou corrigir defeitos ósseos oriundos de traumas ou intervenções cirúrgicas. Existem várias maneiras de se obter o osso doador, sendo o autógeno e o homógeno os que parecem ter uma melhor resposta tecidual. O estudo do tecido ósseo por métodos não invasivos se tornou possível a partir da descoberta dos raios X em 1895 e, com o advento da tomografia em meados de 1970, ocorreu uma evolução na radiologia convencional. A partir daí, disponibilizando-se a obtenção de cortes sem a sobreposição das estruturas, permitindo assim uma melhor resolução da imagem radiográfica. O presente experimento teve como propósito analisar o processo de reparo ósseo em imagens obtidas por tomografia espiral em suínos submetidos a enxerto de osso homógeno congelado, comparando-o com enxerto de osso autógeno. Os 12 animais foram sacrificados aos 7, 30, 60, e 90 dias pós-operatórios, quando as mandíbulas foram removidas para a realização das tomografias espirais. Observou-se que em todos os tempos pós-operatórios o enxerto autógeno foi o que obteve melhor resultado. Mostrava-se mais reabsorvido e unido ao tecido ósseo e com radiopacidade semelhante à do tecido ósseo adjacente, além de ausência de reabsorção óssea ao redor dos mesmos. Em contrapartida, o enxerto homógeno se apresentava menos reabsorvido e na maioria das vezes, com sinal de reabsorção do tecido ósseo adjacente. Além dos melhores resultados com o enxerto autógeno, o estudo permitiu concluir que, a tomografia espiral foi um exame competente para avaliação dos enxertos, bem como o modelo experimental (suíno), foi perfeitamente viável para o estudo do tecido ósseo. / Abstract: Bone graft has been accomplished aiming to prevent or correct bone defects originating of traumas or surgical interventions. Several techniques exist of obtaining the bone donor, being the autogenous and the homogenous then that seem have a better tissue reaction. The study of the bone tissue for methods non invasive became possible as from the discovery of the rays X in 1895 and, with the coming of the tomography in the middle of 1970, happened an evolution in the conventional radiology. Since then, making possible the obtainment of cuts without the overexposure of the structures, allowing a better resolution of the image radiographic. The present experiment had as purpose to analyze by the spiral tomography the process of bone repair in swine submitted to graft of bone frozen homogenous, being compared with graft of autogenous bone. The 12 animals were sacrificed to the 7, 30, 60, and 90 postoperative days, when the mandibulae were removed for the accomplishment of the spiral tomography. It was observed that in every postoperative time the autogenous graft was what obtained better result. It showed more reabsorbed and united to the bone tissue and with radiopacity similar to the one of the adjacent bone tissue, besides absence of bone reabsorption about of the same ones. In compensation, the graft homogenous came less reabsorbed and most of the time, with sign of reabsorption of the adjacent bone tissue. Besides best results with the autogenous graft, the study allowed to conclude that the spiral tomography went a competent exam to evaluation of the grafts, as well as the experimental model (swine), was practicable perfect for study bone tissue. / Mestre