Estudos preliminares através de microarray nos mostraram que a E3 ligase Mdm2 foi regulado positivamente no músculo de animais hipertireoideos. Dessa forma, nós inferimos uma possível relação de Mdm2 com a atrofia causada por T3. Para testar nossa hipótese, ratos foram induzidos ao hipertireoidismo para análises subsequentes. Concomitante com a perda de massa muscular foi confirmado um aumento da expressão de Mdm2 tanto no nível gênico (p<0.05) quanto protéico. Interessantemente, Mdm2 foi preferencialmente expresso em fibras tipo I, mostrando maior sensibilidade dessas fibras ao T3. Além disso, foi observado uma diminuição severa na expressão de Pax7/MyoD associado à superexpressão de Mdm2, sugerindo inatividade das células satélites. Surpreendentemente, a inibição de Mdm2 em miotubos cultivados provocou uma diminuição severa no diâmetro destes (~35%, p<0.05), ou seja, tal inibição foi incapaz de minimizar a proteólise muscular causada por T3. Portanto, nós concluímos que a responsividade de Mdm2 ao T3 agiria como um mecanismo compensatório numa tentativa de minimizar a proteólise muscular causada pelo hipertireoidismo. Esta conclusão é reforçada pela atrofia observada em miotubos durante a inibição de Mdm2 sem a presença de T3. / Previous studies in our lab through microarray assay observed Mdm2, an E3 ligase, up regulated in soleus muscle from hyperthyroid rats. In this sense, we inferred that Mdm2 could be related to muscle atrophy caused by T3. To test our hypothesis, rats were induced to experimental hyperthyroidism for subsequent analysis. Along the muscle mass loss, the increase on Mdm2 gene expression was confirmed (p<0.05) as well as protein expression by RT-PCR and Western Blot, respectively. Interestingly, Mdm2 was expressed predominantly in fiber I type during T3 treatment, demonstrating a higher sensibility when compared to type II fiber. Moreover, it was observed a severe decrease in Pax7/MyoD labeling, associated to an increase on Mdm2 labeling, suggesting that T3 could be associated with inactivation of satellite cells. Surprisingly, Mdm2 inhibition in myotubes have induced severe decrease on myotubes diameter (~35%, p<0.05), in other words, Mdm2 inhibition was not able to decrease muscle proteolysis during high levels of T3. Thus, the increase on Mdm2 levels could be a compensatory effect to reduce the muscle mass loss during T3 treatment. This conclusion is highlighted by the myotubes atrophy observed during the Mdm2 inhibition without T3 treatment.

Neste estudo, testamos dois novos moduladores seletivos do receptor de glicocorticóide, nomeados L5 e L7, em comparação com o dexametasona, sobre aspectos estruturais, funcionais e moleculares no músculo sóleo. Ratos Wistar foram tratados com doses progressivas de dexametasona, L5 e L7 em 1 ou 7 dias. A massa corporal e a ingestão alimentar apresentaram queda após o tratamento com dexametasona em todas as doses; os tratamentos com L5/L7 mostraram resposta semelhante aos controles. O peso do músculo foi diminuído pelo dexametasona, efeito não observado nos tratamentos com L5/L7. Apenas o tratamento com dexametasona causou uma diminuição na área de secção transversa dos tipos de fibra muscular analisada. A força tetânica do sóleo foi diminuída pela dexametasona, nos tratamentos com L5/L7 este parâmetro também não foi afetado. A expressão gênica de MAFbx/Atrogin-1 e MuRF-1 foi elevada pela dexametasona; por outro lado, L5/L7 não elevaram a expressão destes genes. Concluímos que o L5/L7, em contraste com o dexametasona, preveniu o músculo esquelético da atrofia. / In this study, we have tested two new selective modulators named L5 and L7 along with dexamethasone in skeletal muscle structural, functional and molecular aspects. Male Wistar rats were treated with progressive doses of dexamethasone, L5 and L7 for 1 and 7 days. While body weight and food intake were decreased by the dexamethasone treatment in all doses, L5/L7 treatments induced gain in body weight similarly to controls. Muscle weight was decreased by dexamethasone, while L5/L7 were ineffective. Only the dexamethasone treatment caused a decrease in the analyzed cross sectional area of the skeletal muscle fiber types. Soleus tetanic force was decreased by the dexamethasone treatment, while L5/L7 treatments did not alter this parameter. MAFbx/Atrogin-1 and MuRF-1 gene expressions were elevated by dexamethasone; on the other hand, L5/L7 did not modulate any expression of those genes. We conclude that L5/L7, in contrast to dexamethasone, spare skeletal muscle from structural and functional loss, and molecular changes, reinforcing their role as a therapeutic device.

PDFには「高橋 由佳子」と記載 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21955号 / 医博第4497号 / 新制||医||1037(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 川上 浩司, 教授 古川 壽亮, 教授 富樫 かおり / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Interest in hippocampus increased sharply after his significance in the process of learning and retention of information was published. In particular, considerable interest was in its volume changes and their effect on Alzheimer’s disease. Understanding the structure and function hippocampus would contribute to a more accurate diagnosis of this disease. In this work was created a method of hippocampal segmentation using active contours. With its help, the data composed of both healthy and a diseased patients was segmented and the results were then statistically analyzed using statistical methods such as Kruskal-Walis test, Mann-Whitney test. The level of significance given by results of analysis supports alternative hypothesis that attaches significance of the difference in volume of the hippocampus between studied groups.

U5 small ribonucleoprotein particle (U5 snRNP) is a crucial component of the spliceosome, the complex responsible for pre-mRNA splicing. Despite the importance of U5 snRNP, not much is known about its biogenesis. When we depleted one of the core U5 components, protein PRPF8, the other U5-specific proteins do not associate with U5 snRNA and the incomplete U5 was accumulated in nuclear structures known as Cajal bodies. To further clarify the role of PRPF8 in U5 snRNP assembly, we studied PRPF8 mutations that cause an autosomal dominant retinal disorder, retinitis pigmentosa (RP). We prepared eight different PRPF8 variants carrying RP-associated mutations and expressed them stably in human cell culture. We showed that most mutations interfere with the assembly of snRNPs which consequently leads to reduced efficiency of splicing. The mutant PRPF8 together with EFTUD2 are stalled in the cytoplasm in a form of U5 snRNP assembly intermediate. Strikingly, we identified several chaperons including the HSP90/R2TP complex and ZNHIT2 as new PRPF8's interactors and potential U5 snRNP assembly factors. Our results further imply that these chaperons preferentially bind the unassembled U5 complexes and that HSP90 is required for stability of...

Skeletal muscle is a highly adaptable tissue that comprises approximately 40% of total body weight while accounting for up to 90% of whole-body oxygen consumption and energy expenditure during exercise. The loss of skeletal muscle protein and subsequent decrease in muscle mass (atrophy) that accompanies disuse results primarily from a decrease in intracellular protein synthesis combined with an increase in proteolytic activity. Interestingly, these processes of skeletal muscle atrophy are amplified by changes in mitochondrial capacity, with evidence suggesting that the maintenance of mitochondria during periods of disuse protects skeletal muscle against atrophy. Remarkably, rodents with denervated muscle are protected against muscle atrophy following whole-body heat stress. The mechanism of protection appears to be tied to the observed increases in heat shock protein (HSP) and PGC-1α, which accompany the heat stress. Without any published observations as to whether such heat-induced protection against muscle atrophy would translate to human muscle, the aim of this project was to determine the extent to which deep tissue heating (via pulsed shortwave diathermy) might provide protection against skeletal muscle atrophy.

Indiana University-Purdue University Indianapolis (IUPUI) / Glucocorticoid excess is a leading cause of osteoporosis. The loss of bone mass and strength corresponds to the increase in fractures exhibited after three months of glucocorticoid therapy. Glucocorticoids induce the bone cellular responses of deceased bone formation, increased osteoblast/osteocyte apoptosis, and transient increased bone resorption, which result in rapid bone loss and degradation of bone microarchitecture. The current standard of care for osteoporosis is bisphosphonate treatment; however, these agents further suppress bone formation and increase osteonecrosis and low energy atypical fracture risks. Thus, there is an unmet need for interventions that protect from glucocorticoid therapy. The purpose of these studies was to investigate novel mechanisms that potentially interfere with glucocorticoid-induced bone loss. We chose to explore pathways that regulate endoplasmic reticulum stress, the canonical Wnt pathway, and Pyk2 activity. Pharmacologic reduction of endoplasmic reticulum stress through salubrinal administration protected against glucocorticoid-induced bone loss by preservation of bone formation and osteoblast/osteocyte viability. In contrast, inhibition of Wnt antagonist Sost/sclerostin and inhibition of Pyk2 signaling did not prevent glucocorticoid-induced reductions in bone formation; however, both Sost/sclerostin and Pyk2 deficiency protected against bone loss through inhibition of increases in resorption. Overall, these studies demonstrate the significant contributions of reductions in bone formation, increased osteoblast/osteocyte apoptosis, and elevations in resorption to the rapid 6-12% bone loss exhibited during the first year of glucocorticoid therapy. However, glucocorticoid excess also induces skeletal muscle weakness, which is not reversed by bisphosphonate treatment or the interventions reported here of salubrinal, Sost/sclerostin inhibition, or Pyk2 deficiency. Further, the novel finding of increased E3 ubiquitin ligase atrophy signaling induce by glucocorticoids in both bone and muscle, by tissue-specific upstream mechanisms, provides opportunities for therapeutic combination strategies. Thus, future studies are warranted to investigate the role of E3 ubiquitin ligase signaling in the deleterious glucocorticoid effects of bone and muscle.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12945号 / 論医博第2097号 / 新制||医||1010(附属図書館) / 32204 / (主査)教授 髙橋 良輔, 教授 富樫 かおり, 教授 髙橋 淳 / 学位規則第4条第2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20994号 / 医博第4340号 / 新制||医||1027(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 鈴木 茂彦, 教授 伊佐 正, 教授 大森 孝一 / 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21015号 / 医博第4361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 渡邉 大, 教授 宮本 享 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM