京都大学 / 0048 / 新制・課程博士 / 博士(エネルギー科学) / 甲第21881号 / エネ博第382号 / 新制||エネ||74(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー応用科学専攻 / (主査)教授 馬渕 守, 教授 宅田 裕彦, 教授 土井 俊哉 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DFAM

No description available.

No description available.

Chromatin remodelers utilize the energy derived from ATP hydrolysis to mobilize nucleosomes. ISWI remodelers mobilize and evenly space nucleosomes to regulate gene expression. ISW2, an ISWI remodeler in yeast, has been shown to reposition nucleosome near promoter regions and represses both mRNA and antisense non coding RNA transcription. ISW2 is composed of four subunits and the catalytic Isw2 subunit consists of several conserved domains. The highly conserved ATPase domain is present at the N-terminus whereas the conserved HAND, SANT and SLIDE domain are towards the carboxyl terminal end of Isw2. Nucleosome mobilization by ISW2 requires both extranucleosomal DNA and the N-terminal tail of histone H4. DNA crosslinking and peptide mapping revealed that the ATPase domain contacts nucleosome two helical turns away (SHL2) from dyad to a site close to the H4 tail, whereas the HAND, SANT and SLIDE domain contact a 30bp stretch of DNA comprising the edge of nucleosome and ~20bp of extranucleosomal DNA. The ATPase domain and the C-terminal domains were investigated for their role in regulation of ISW2 activity both in-vitro and in-vivo. It appears that there are distinct modes of ISW2 regulation by these domains. Mutation of a patch of five acidic amino acids on the region of ATPase domain that contact SHL2 was found to be crucial for both ISW2 remodeling and nucleosome stimulated ATPase activity. Acidic patch mutant ISW2 was unable to mobilize nucleosome or hydrolyze ATP in absence of H4 tail. This indicates that the region of ATPase domain contacting nucleosome at SHL2 and H4 tail act in two separate and independent pathways to regulate ISW2 remodeling. Both HAND and SLIDE domain were shown to crosslink entry/exit site and linker DNA respectively. The roles of C-terminal domains were investigated either by deletion of the individual domain or mutation of conserved basic residues on the surface of these domains that are suspected to interact extranucleosomal with DNA. Deletion of HAND domain had minimal effect on in vitro ISW2 activity, however whole genome transcription analysis revealed one key role of this domain in ISW2 regulation. In absence of HAND domain, ISW2 had minimal role on repression of genes that were RPD3 (co-factor) dependent, however significantly derepressed genes that were RPD3 independent. At these loci, nucleosome positions were altered and ISW2 recruitment was reduced in absence of a functional HAND domain. Thus the HAND domain regulates recruitment and remodeling of ISW2 at those genes where ISW2 acts independent of other cofactors. The SANT domain, C-terminal to HAND domain, appears to control the "step size" of nucleosome remodeling and was found to be required for processive nucleosome remodeling by ISW2. Both H4 tail and SANT domain appear to control two distinct stages of ISW2 remodeling. A long alpha helical spacer separates SANT domain from SLIDE domain. SLIDE domain was found to be the protein-protein interaction domain that interacts with accessory Itc1 subunit to maintain ISW2 complex integrity. The two ways by which SLIDE domain regulate ISW2 is by binding or recruitment of ISW2 to promoter regions and additionally by binding independent regulation of both ATPase and remodeling activity. The remodeling mechanism of ISW2 was further compared with another ISWI type remodeler in yeast, Isw1a; using time resolved nucleosome remodeling combined with high resolution site specific histone DNA crosslinking at six different nucleosomal positions to track the movement of the nucleosomes. Nucleosome remodeled by the same remodeler showed discontinuous nucleosome movement between two tracking points indicating formation of small "bulges". One key difference in remodeling mechanism was that although both ISW2 and Isw1a moved nucleosomes towards longer linker DNA, only Isw1a remodeled nucleosomes "backtracked" ~11bp during remodeling. Backtracking of remodeling was prominently observed at nucleosomal regions in close proximity to translocase binding sites suggesting the potentially different mechanisms shared by similar remodeling complexes.

Syfte: Syftet med denna studie var att utvärdera ytkontamination (förorenade ytor) i behandlingsrum före och efter behandling på student- och specialistkliniken i Universitetstandvården Endodonti vid Malmö universitet. Material och metod: Provtagning utfördes vid Malmö Universitet, Universitetstandvården Endodonti, före och efter behandling på specialistkliniken och studentkliniken. Följande ytor utvärderades: tangentbord, mus, lamphandtag, behandlarens unit och assistentens unit. Totalt togs prover från 200 ytor. ATP-bioluminescence tillämpades för att utvärdera potentiell kontamination av mikroorganismer vid ett tröskelvärde på 250 RLU/100cm². Fluid thioglycollatemedium (FTM) tillämpades för bakterieodling.  Resultat: Tillväxtfrekvensen i behandlingsrummen var högre före behandling i förhållande till efter. Specialistkliniken hade högre tillväxt före behandling i jämförelse med studentkliniken. Efter behandling var tillväxten högre för studentkliniken. Högst frekvens av tillväxt påvisades på tangentbord och mus på båda klinikerna. ATP-bioluminescence visade en sensitivitet på 80 % och specificitet på 31 %.  Slutsats: Trots hygienrutiner och preventiva åtgärder som tillämpas förelåg det en svikt i desinfektion av ytor som tangentbord och mus då dessa hade högst frekvens av kontamination. Utifrån studien kan det konstateras att ATP-bioluminescence och FTM kan vara ett hjälpmedel vid kontroll av hygienrutiner. Det krävs fler studier inom tandvården som utvärderar ytkontamination och förekomst av vårdrelaterade infektioner för att uppnå en säkrare vårdmiljö. / Aim: The aim of this study was to evaluate surface contamination in treatment rooms before and after treatment at the student and specialist clinic in the Dental University, department of Endodontics at Malmö University. Materials and methods: Sampling was performed at Malmö University, Dental University, Endodontics, before and after treatment at the specialist clinic and student clinic. The following surfaces were evaluated: keyboard, mouse, lamphandle, the operator's unit and the assistant's unit. A total of 200 surfaces were sampled. ATP-bioluminescence was applied to evaluate potential contamination by microorganisms at a threshold value of 250 RLU/100cm². Fluid thioglycollatemedium (FTM) was used for bacterial cultivation. Results: The microbial growth rate in the treatment rooms was higher before treatment compared to after. The specialist clinic had a higher growth before treatment compared to the student clinic. After treatment, growth was higher at the student clinic. Highest frequency of growth was detected on keyboards and mouses in both clinics. ATP-bioluminescence showed a sensitivity of 80 % and specificity of 31 %. Conclusion: Despite hygiene routines and preventive measures that are applied, there is a failure in the disinfection of surfaces such as the keyboard and mouse as these had the highest frequency of contamination. From the study, it can be concluded that ATP-bioluminescence and FTM can be used as an evaluation tool in control of hygiene routines. More studies are required in dental care that evaluate surface contamination and the occurrence of healthcare-associated infections to achieve a safer healthcare environment.

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第24141号 / 医科博第142号 / 新制||医科||9(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 松田 道行, 教授 斎藤 通紀, 教授 妹尾 浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Mitochondria are found in all cells of the body except red blood cells. Mitochondria is the organelle that produces the most ATP in the body, which is the cells energy carrier. All processes in the body require energy in one way or another, hence the function of mitochondria is very important. In mitochondrial disease, there is an impairment in the function of the mitochondria, where the respiration usually is affected. Mitochondria play a big role in the regulation of glucose-stimulated insulin secretion of the pancreatic β-cells hence why diabetes often can be caused by mitochondrial dysfunction. Diabetes often requires treatment to achieve normal blood sugar levels. Metformin, rosiglitazone and troglitazone are three drugs for treatment of type 2 diabetes and have been proven to affect mitochondrial function or the body negatively, hence why it is important to be careful with which treatment that is used in case of mitochondrial diabetes. The purpose of this ex vivo model with human mitochondria were to investigate with high resolution respirometry whether the antidiabetic drugs could reduce the respiration in mitochondria and at what concentration it became significant. The results showed that metformin did not decrease the respiration at all with additions in the range of 10 µM to 100 µM, which rosiglitazone and troglitazone did. The therapeutic concentrations of the drugs are lower than the concentrations investigated in this study. However, since healthy cells are used in this study the effect that the drugs have on impaired mitochondria could be different. The conclusion of this study is that there was a significant decrease in the respiration for two of three drugs and should be carefully used. Further studies with impaired mitochondria should be performed to really see what affect these drugs have.

Mitochondria are important cytosolic organelles present in nearly all eukaryotic cells. The main function of mitochondria are to generate the vast majority of ATP through the process of oxidative phosphorylation. Mitochondria have key roles regarding other systems in the body as well, such as regulation of apoptosis, calcium homeostasis, reactive oxygen production etc. Mitochondrial diseases are caused by impaired mitochondrial function, originating from mutations in either the mitochondrial DNA or the nuclear DNA. Epilepsy is a common symptom of mitochondrial disease, especially in children. The pathophysiology behind mitochondrial epilepsy is primarily based on ATP deficit, leading to a negative effect on a range of different nervous system related functions that in the end leads to seizures. The study aimed to investigate the effect on mitochondrial respiration of two commonly used antiepileptic drugs, namely valproic acid and Topiramate, and the anesthesic drug propofol, commonly used in case of refractory status epilepticus. The three drugs were titrated in different concentrations in a high-resolution respirometer from Oroboros Instruments (n=6). Propofol seemed especially inhibiting of mitochondrial function, and both propofol and topiramate had a significant decrease in mitochondrial respiration within the clinical concentrations. The result of the study supported research stating that propofol should be used with caution in patients with a mitochondrial disease, but further research should be done regarding all three drugs in order to draw definite conclusions.

The relationship between myocardial cell contracture and injury during total metabolic inhibition (amylobarbital and iodacetic acid) and ischemia was examined, using 5-50 mm butanedione monoxime (BDM) as an inhibitor of contracture. BDM had no apparent effect on control myocytes during 180 min incubations, but inhibited contracture following anoxia or ischemia in a dose-dependent fashion, as directly quantitated by length/width ratios. Cellular ATP levels decreased at a similar rate in the absence or presence of BDM, following metabolic inhibition. BDM-mediated inhibition of contracture was associated with accelerated cell injury, as defined by: the uptake of an extracellular marker (trypan blue) by the cardiomyocytes, by direct analysis of myoglobin released into the supernatant and by ultrastructural demonstration of defects in sarcolemmal membrane integrity. Calcium was not required for BDM's enhancement of injury, in that cells incubated in calcium free-EGTA buffer showed a similar BDM-mediated acceleration of injury. In the presence or absence of calcium, enhancement of injury was more marked in cells osmotically stressed with a brief incubation in hypotonic buffer, than in cells resuspended in isotonic media. It is concluded that BDM enhances development of osmotic fragility of inhibited or ischemic cardiomyocytes and that contracture is not a necessary contributing factor to myocardial cell death.