O camundongo mdx, modelo murino para a Distrofia Muscular de Duchenne (DMD) possui uma mutação de ponto no gene da distrofina que resulta na ausência da proteína no músculo, porém seu fenótipo é brando o que o torna um bom modelo genético e molecular, mas não um bom modelo funcional. Esperando obter um modelo para DMD que tivesse um fenótipo mais fiel ao apresentado pelos pacientes humanos, optou-se por transferir a mutação mdx para o background 129/Sv. Através de cruzamentos sucessivos foram obtidas 3 gerações de animais mdx com background 129/Sv (mdx129) e cada geração foi avaliada funcionalmente por 6 meses. Desde a primeira geração é possível observar que os animais mdx129 são mais fortes do que os mdx originais em background C57BL (mdxC57BL), sendo o oposto do esperado no início dos experimentos. O estudo então foi redirecionado para tentar entender o motivo dessa melhora. Em relação ao padrão histológico, em geral há diferenças entre o mdxC57BL e mdx129. Observa-se também que os animais mdx129 entram no processo de degeneração mais tardiamente que os animais mdxC57BL e seu processo de regeneração se estende por mais tempo. Através de estudos de microarray foi possível observar que os animais 129/Sv apresentam poucos genes diferencialmente expressos (GDEs) em relação aos animais C57BL, portanto os dois backgrounds são muito semelhantes. O mdxC57BL apresenta muito mais GDEs em relação ao seu selvagem (C57BL) do que o mdx129 em relação ao 129/Sv, entretanto, ambos os modelos apresentam mais genes superexpressos do que subexpressos, indicando que as alterações distróficas e regenerativas estão mais associadas com a ativação do que a repressão de genes. Quando os GDEs de ambos os modelos de mdx são distribuídos em categorias funcionais, há o predomínio de genes ligados ao sistema imune e quando essa categoria é omitida para melhor visualização das restantes, observa se que ambos os modelos apresentam categorias funcionais semelhantes, porém com proporções diferentes. No modelo mdx129 se destaca a diminuição da participação da categoria de rota endo/exocítica (tráfego de vesículas) e homeostase e aumento da participação das categorias de matiz extracelular e atividade enzimática. Cada modelo apresenta genes exclusivos, destacando os genes SPP1 e IL1RN na comparação 129/Sv x mdx129F3. O gene SPP1 codifica a proteína osteopontina (OPN) e o polimorfismo rs28357094 neste gene é utilizado como biomarcador de prognóstico para DMD. O papel da OPN na progressão da distrofia não é bem conhecido. Alguns estudos afirmam que a ausência dessa proteína melhora a força muscular de camundongos mdx, enquanto outros apontam que sua participação é necessária para a regeneração muscular. Assim sendo, mais estudos serão necessários para verificar qual seria a via responsável pela melhora fenotípica do modelo mdx129. Já o gene IL1RN codifica a proteína IL-1Ra, a qual é um antagonista de interleucina 1 (citocina pró-inflamatória e pró fibrótica). Portanto o aumento da expressão do gene de seu antagonista sugere que os animais mdx129F3 podem estar mais protegidos do processo inflamatório causado por essas moléculas. Quando analisadas as listas filtradas para músculo esquelético das comparações C57BL x mdxC57BL e 129/Sv x mdx129F3 para a formação de vias metabólicas, foi gerada apenas uma via em ambas as comparações com número relevante de moléculas. A via gerada pela análise da lista C57BL x mdxC57BL possui mais moléculas do que a via gerada pela analise da lista 129/Sv x mdx129F3, porém, todas as moléculas presentes nesta via, estão presentes na via C57BL x mdxC57BL, indicando que mesmo com número diferente de moléculas envolvidas, os genes participam das mesmas vias. Tanto a comparação de cada geração de mdx129 com o 129/Sv como a comparação das gerações entre si mostram que os efeitos da mudança de background estão presentes desde a primeira geração e não se alteram significativamente com os cruzamentos sucessivos. / The mdx mouse, murine model for Duchenne Muscular Dystrophy (DMD) has a point mutation in the dystrophin gene that results in the absence of the protein in the muscle, however its phenotype is mild, which makes it a good genetic and molecular model, but not a good functional model. Hoping to obtain a model for DMD with a phenotype that is more similar the patients\', it was chosen to transfer the mdx mutation to the 129/Sv background. Through successive breedings, 3 generations of mdx animals with 129/Sv background were obtained and each generation was functionally evaluated for 6 months. Since the first generation it is possible to observe that the mdx129 animals are stronger than the original mdx with C57BL background. The results were the opposite of what was expected in the beginning of the experiments, therefore the study was redirectioned to try to understand the reason of the improved phenotype. About the general histological pattern, there are differences between mdxC57BL and mdx129. It can be observed that the mdx129 animals enter the degenerative process later than the mdxC57BL animals and the regenerative process lasts longer. Through microarray studies it was possible to observe that the 129/Sv animals present few differentially expressed genes (DEGs) in comparison to the C57BL animals; therefore both backgrounds are very similar. The mdxC57BL presents many more DEGs in comparison to C57BL than mdx129 in comparison to 129/Sv, however both models present more super expressed genes than sub expressed, indicating that the dystrophic and regenerative alterations are more associated to the activation rather than the repression of genes. When the DEGs of both mdx models are distributed in functional categories, there is the predominance of genes related to the immune system and when this category is omitted for the better visualization of the remaining, it can be observed that both models present similar functional categories, but with different proportions. In the mdx129 model we can highlight the decrease in participation of the endo/exocytic pathway (vesicle traffic) and homeostasis categories, and increase in participation of the extracellular matrix and enzymatic activity categories. Each model presents exclusive genes, highlighting SPP1 and IL1RN in the comparison 129/Sv x mdx129F3. SPP1 encodes the protein osteopontina (OPN) and the polymorphism rs28357094 in this gene is used as a DMD prognostic biomarker. The role of OPN in the dystrophy progression is not well known. Some studies claim that the absence of OPN increases the muscle strength of the mdx mouse, while others indicate that its participation is necessary to muscle regeneration. More studies are needed to ascertain what pathway is responsible for the phenotypic improvement of the mdx129 model. The IL1RN gene encodes the protein IL-1Ra, and interleukin 1 antagonist, which is a pro-inflammatory and pro-fibrotic cytokine. Therefore, the increase in the expression of its antagonist suggests that the mdx129F3 animals may be more protected from the inflammatory process caused by these molecules. When the filtered lists for skeletal muscle of the comparisons C57BL x mdxC57BL e 129/Sv x mdx129F3 were analyzed for the formation of metabolic pathways, only one pathway was generated in both comparisons. The pathway generated in the analysis C57BL x mdxC57BL has more molecules that the one generated by the 129/Sv x mdx129F3 list, but all molecules present in the latter are also present in the former, indicating that even with different numbers of molecules involved, the genes participate in the same pathways. The comparisons of each generation of mdx129 with the 129/Sv and the comparison of the generations among each other show that the effects of the background change are present since the first generation and are not altered with the successive breedings.

Immune Cell Potentiating Factor (ICPF) represents a class of biological response modifiers initially only found within active caprine serum fractions. Controlled studies have since demonstrated active ICPF derived from several non-caprine mammalian sources; including equine and human. ICPF is able to increase survivability in murine gram negative induced sepsis (60%) as well as secondary infection and subsequent sepsis in canines infected within canine parvo virus 2 (36%) despite showing no innate antiviral properties. ICPF is able to initiate systemic proteomic changes within several organ systems; including serum, liver, brain, lung, and spleen. ICPF initiated an early acute phase response, specifically through the increased expression of serum amyloid A, with systemic serum levels increasing from 1.5 μg/mL to 403.0 μg/mL within 24 hours and increased to 3,400 μg/mL within 48 hours following ICPF administration. Evaluation of cytokine expression following ICPF treatment revealed the up-regulation of IL-6, INF-γ, and the chemokine CXCL1\KC in vivo as well as the expression of IL-6 and IFN-γ in vitro within 3 hours of treatment. Development of an in vitro bioassay through the expression of IL-6 and IFN-γ within whole blood and peripheral blood mononuclear cells will allow for further elucidation and testing of ICPF outside of an animal host. The early expression of proinflammatory cytokines and chemokines, an acute phase response including serum amyloid A, and ICPF’s inability to alleviate mortality in a lipopolysaccharide animal mortality model strongly indicates an active role for ICPF as an immune regulatory peptide capable of promoting an early inflammatory response to Salmonella enterica serovar Typhimurium thereby reducing the risk and mortality associated with sepsis.

京都大学 / 新制・課程博士 / 博士(社会健康医学) / 甲第24807号 / 社医博第131号 / 新制||社医||12(附属図書館) / 京都大学大学院医学研究科社会健康医学系専攻 / (主査)教授 中山 健夫, 教授 山本 洋介, 教授 髙橋 良輔 / 学位規則第4条第1項該当 / Doctor of Public Health / Kyoto University / DFAM

No description available.

No description available.

<P> The first histone acetyltransferase to be described was GCN5, from the yeast species Saccharomyces cerevisiae. To date, the GCN5-related N-acetyltransferases (GNATs) comprise one of the largest enzyme superfamilies with over 10,000 identified members in sequenced genomes. This protein is known to acetylate specific lysine residues on the amino-terminal tails of nucleosomal histones, thereby loosening their contact with the tightly packed DNA and facilitating transcription. </p> <p> In this study, I determined that GCN5 is able to shuttle between the nucleus and the cytoplasm using fluorescence recovery after photobleaching (FRAP). Mutational studies revealed that its nuclear import is regulated by a classical bipartite nuclear localization signal (NLS) that is dependent on the transporters importin a and f3. In contrast, we found that GCN5 lacks a CRM1-dependent nuclear export signal (NES), as demonstrated by mutational and leptomycin B (LMB) studies; instead, IKB, a previouslydescribed transcription inhibitor with a CRMl-dependent NES, was found to modulate the export of GCN5 from the nucleus. This was initially discovered while performing the LMB assays, for which IKB served as a positive control, and was subsequently confirmed by mutational studies and protein complementation assays (PCAs). Furthermore, while the PCAs demonstrated a physical interaction between these two proteins in vivo, GST pull-down experiments were employed to confirm their interaction in vitro. </p> <p> Furthermore, this study also revealed that over-expression of GCN5-e YFP in NIH 3T3 cells causes -10% of the transfected cells to exhibit nuclear GCN5-eYFP-associated filaments; these structures were confirmed to be F -actin filaments comprised of f3-actin through co-localization studies with both TRITC-phalloidin and a mRFP-f-actin construct. GCN5's acetyltransferase activity was shown to be responsible for the formation ofthese filaments through mutation of its catalytic residue. Moreover, a protein complementation assay (PCA) demonstrated an in vivo interaction between GCN5 and f-actin, while FRAP analysis of a single filament showed that GCN5-e YFP molecules rapidly and randomly associate with these filaments along their entire length. Together these results suggest that GCN5's acetyltransferase activity is responsible for the structural maintenance of these filaments. Finally, GCN5-eYFP-associated filaments were found to be spatially separate from both lamin A (a nuclear envelope structural protein) and DNA; however, this does not exclude the possibility of an indirect interaction between these cellular constituents, as treatment of a live cell with Hoechst DNA stain, which disrupts the structure of DNA, was shown to disturb the structural integrity of these filaments. </p> / Thesis / Master of Science (MSc)

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

The current research project investigates the influence of gender, age, and social class on the use of intensifiers and extreme adjectives as instruments to intensify spoken language. Employing a corpus-based approach, the study has collected data from the Spoken BNC2014 regarding the use of the common intensifiers so, very, real, and really modifying an adjective, as well as the use of synonymous extreme adjectives. Frequent intensifier-adjective pairs were retrieved from the corpus and thereafter translated into synonymous extreme adjectives using an online thesaurus. Following the data collection process, comparative analyses of the collected data were conducted. The study presents results that are in some regards coherent with previous research as well as most of the study’s hypotheses. Although the results suggest some general differences in use depending on the speaker’s gender, age, and social class, they are not as distinct as expected. Of all the sociolinguistic variables, the language use of the different social classes proved least predictable. While correlations between an overuse of intensifiers/extreme adjectives and an underuse of extreme adjectives/intensifiers have been observed in the data, this opposite relationship is not present in all social constellations.

Two different catalysts (bis[N-(3-tert-butylsalicylidene)-pentafluoroanilinato] titanium (IV) dichloride and [1-(8-quinolyl)indenyl] chromium (III) dichloride catalysts) activated with aluminoxane based co-catalysts (MAO, PMAO, MMAO12 and MMAO3A) have been evaluated in the polymerisation of ethylene leading to UHMWPE having a reduced number of entanglements between the chains. The effect of a co-catalyst modifier (BHT) on the catalytic systems and the resulting polymers is also addressed. Both catalysts are capable to promote the synthesis of UHMWPE having a reduced amount of entanglements in the conditions used. Uniaxial solid-state deformation of UHMWPE samples of different molar masses have been evaluated and related with the entanglement state of the polymers synthesised. A clear relationship between some mechanical properties and the molar mass/entanglement density of the polymers synthesised has been found.

Background: Medulloblastoma (MB) is the most common pediatric brain tumor. Current treatment regimes consisting of primary surgery followed by radio- and chemotherapy, achieve 5-year overall survival rates of only about 60 %. Therapy-induced endocrine and neurocognitive deficits are common late adverse effects. Thus, improved antitumor strategies are urgently needed. In this study, we combined irradiation (IR) together with epigenetic modifiers and differentiation inducers in a multimodal approach to enhance the efficiency of tumor therapy in MB and also assessed possible late adverse effects on neurogenesis. Methods: In three human MB cell lines (DAOY, MEB-Med8a, D283-Med) short-time survival (trypan blue exclusion assay), apoptosis, autophagy, cell cycle distribution, formation of gH2AX foci, and long-term reproductive survival (clonogenic assay) were analyzed after treatment with 5-aza-2′-deoxycytidine (5-azadC), valproic acid (VPA), suberanilohydroxamic acid (SAHA), abacavir (ABC), all-trans retinoic acid (ATRA) and resveratrol (RES) alone or combined with 5-aza-dC and/or IR. Effects of combinatorial treatments on neurogenesis were evaluated in cultured murine hippocampal slices from transgenic nestin-CFPnuc C57BL/J6 mice. Life imaging of nestin-positive neural stem cells was conducted at distinct time points for up to 28 days after treatment start. Results: All tested drugs showed a radiosynergistic action on overall clonogenic survival at least in two-outof-three MB cell lines. This effect was pronounced in multimodal treatments combining IR, 5-aza-dC and a second drug. Hereby, ABC and RES induced the strongest reduction of clongenic survival in all three MB cell lines and led to the induction of apoptosis (RES, ABC) and/or autophagy (ABC). Additionally, 5-aza-dC, RES, and ABC increased the S phase cell fraction and induced the formation of gH2AX foci at least in oneout-of-three cell lines. Thereby, the multimodal treatment with 5-aza-dC, IR, and RES or ABC did not change the number of normal neural progenitor cells in murine slice cultures. Conclusions: In conclusion, the radiosensitizing capacities of epigenetic and differentiation-inducing drugs presented here suggest that their adjuvant administration might improve MB therapy. Thereby, the combination of 5-aza-dC/IR with ABC and RES seemed to be the most promising to enhance tumor control without affecting the normal neural precursor cells.