The Constant Rate of Strain (CRS) test is currently the most widely used method for determination of consolidation parameters in Sweden. These parameters are used to calculate the probable settlements and behavior of soils. With the Swedish standard strain rate, the duration of a single the test is about 24h, but a higher strain rate would allow for more tests to be performed in the same amount of time. For all clays in Sweden, the Swedish standard for the CRS test suggests a fixed rate of strain that is independent of soil properties, while the North American standard proposes a strain rate that generates a pore pressure ratio of 3-15%. Soil properties such as water content, liquid limit, sensitivity and shear strength vary greatly between different regions of Sweden. It would be beneficial if these properties could be used to find the ideal strain rate for the CRS test. Performing the tests at a higher strain rate, and thus performing more tests within a shorter amount of time, would save both time and money. In this report, 24 CRS tests are performed on three different clays with distinctive properties. Each clay is tested with three different strain rates: the Swedish standard rate of 0.680%/h, a higher rate of 3.00%/h and a lower rate of 0.154%/h. The results are evaluated according to both standards, and are compared and analyzed to determine whether there is any indication that the strain rate can be selected based on the soil properties. The results indicate that the selection of the strain rate is dependent on the soil properties. In addition, the majority of the tests can be conducted with higher strain rates than what is required by the Swedish standard and still manage to lie within the 3-15% limit of the pore pressure ratio, which is acceptable for the North American standard. However, the preconsolidation pressure does rise with increased strain rates, which can generate problems and erroneous results when calculating the settlements. A more extensive testing program with CRS tests and full-scale field tests must be carried out before any recommendation of a higher strain rate can be made.

A systematic NMR cryo-porometry and -diffusometry study using nitrobenzene as a probe liquid is carried out in order to characterise pore structures of hierarchically-organised porous silica monolith possessing mesopores along with a 3D bicontinuous macropore network. The result obtained from NMR cryoporometry shows the presence of a relatively wide mesopore size distribution of 10-35 nm. Furthermore, NMR cryodiffusometry indicates that whilst the mesopores are highly tortuous (Tmeso ≈6), they have little influence on the overall tortuosity of the material (Tmacro ≈1.5), which is largely dominated by the macropores (Toverall ≈1.7).

Cell swelling may contribute to acute cell injury subsequent to ischemia/reperfusion. The potential role of mitochondrial uncoupling and the resultant mitochondrial swelling, due to opening of the mitochondrial permeability transition pore (MPTP), were examined in an in vitro ischemically pelleted isolated rabbit cardiomyocyte model using the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP) to uncouple mitochondria. Cyclosporin A (CsA) was employed to inhibit MPTP opening. Cell volume was determined by a cell-flotation, density-gradient assay, using bromododecane. Cell viability, subsequent to an osmotic stress, was determined by trypan blue permeability. Ischemic preconditioning (IPC) facilitated volume regulation following an osmotic stress. Ischemic-cell swelling was reduced by IPC. IPC protected ischemically pelleted cells, but CsA had no significant effects on injury or IPC protection. CCCP ischemia accelerated rates of ischemic contracture and injury, and abolished IPC protection. IPC protection was restored by CsA. In CCCP-ischemic-uncoupled cells, subjected to a reduced (170 mOsm) osmotic stress, CsA and IPC afforded independent and additive protection. Chelerythrine and 5-hydroxydecanoate (5-HD) blocked IPC, but did not reduce CsA protection. Electron microscopy confirmed that CCCP ischemia induced mitochondrial matrix swelling that was reduced by CsA. Cardioprotection by IPC and CsA was accompanied by proportional reductions in cell swelling. Morphometric analysis of the electron photomicrographs demonstrated that the mitochondrial volume fractions were significantly reduced in the CsA/CCCP (29.8 ± 2.3%, P < 0.004) and IPC/CsA/CCCP (31.5 ± 1.7%, P < 0.0008) groups as compared to the CCCP-ischemic group (40.5 ± 1.7%) The IPC/CCCP group (39.5 ± 4.2%) was not significantly different from the CCCP-ischemic group. NIM 811, a CsA analogue MPTP blocker with no calcineurin inhibitory activity, afforded protection similar to CsA. The results suggest that CsA protection may, in part, be mediated by reduction of mitochondrial swelling.

Nucleocytoplasmic transport (NCT) declines during aging and in the context of age-dependent neurodegenerative diseases. However, the mechanisms underlying NCT decline in the disease are poorly understood. FUS is an RNA binding protein that shuttles between the nucleus and cytoplasm and is actively involved in NCT. Mutations in FUS cause amyotrophic lateral sclerosis (ALS), a fatal and incurable motor neuron disorder. We sought to understand the disease mechanism underlying FUS-induced NCT decline in ALS. Here, I uncovered NCT-related defects in motor neurons derived from human induced pluripotent stem cells (iPSCs) harboring an ALS-linked FUS mutation. Importantly, these NCT defects were rescued by genetically correcting the FUS mutation in iPSCs. To gain insight into how expression of mutant FUS causes nuclear pore defects, I demonstrated an altered localization where FUS and nucleoporins (Nups) interact in situ within patient-derived human neurons. Moreover, FUS became aggregation-prone when interacting with Nup62 in vitro, and RNA further alleviated their aggregation propensity. Importantly, NCT-related defects and neuronal toxicity induced by ALS-FUS were ameliorated by modulating Nup expression in vivo. Collectively, these findings implicate aberrant Nup interactions in the pathogenic mechanism of ALS-FUS, and direct targeting the gain-of-function protein interactions could be therapeutic for multiple causes of neurodegeneration.

Nuclear pore complexes (NPCs) are embedded in the nuclear envelope (NE) and composed of proteins called nucleoporins. NPCs as such control the bidirectional traffic of proteins and RNAs between the nucleus and the cytoplasm in eukaryotic cells whereas individual nucleoporins were found to be implicated in other cellular processes such as, cell division, kinetochore assembly, gene expression and cell migration. A prime example for nucleoporin functional versatility can be seen in Nup153. Nup153 is since its discovery known to be a central player in nucleocytoplasmic transport, but additionally participates directly or indirectly, for example, in gene expression and cell cycle control. In this context, it was previously shown that altered levels of Nup153 led to mitotic abnormalities, particularly in cytokinesis and in the spindle assembly checkpoint (SAC). The SAC promotes accurate chromosome separation to ensure the faithful segregation of genetic material to daughter cells. Nup153 was found to interact with the SAC protein Mad1. In the present study, we have further dissected the interaction between Nup153 and Mad1 and investigated the function of the Nup153-Mad1 complex in human cells. By using the high resolution imaging technique “in situ proximity ligation assay”, we found that Nup153 and Mad1 interact with each other exclusively in the presence of a NE, from late mitosis to prophase. By in vitro binding assays, we have confirmed the direct interaction between Nup153 and Mad1 and furthermore identified two independent Nup153-binding sites in Mad1. We have also provided some evidence that Nup153 interacts also with SUMO-modified Mad1.It was previously shown that depletion of Nup153 had no obvious effect on Mad1 and SAC activity. In the present study, we have shown by time-lapse imaging microscopy that the depletion of Mad1 led to a delayed recruitment of Nup153 at the reforming NE during anaphase in living cells, which was often accompanied by a prolongation of anaphase. Furthermore, Mad1 depletion led to alterations in the NE architecture, which were characterized by a change of the membrane curvature at the NPC-NE interface. This was followed by an expansion of the spacing between the inner and outer membranes as seen by electron microscopic and three-dimensional structured illumination investigations. This suggests an implication of Mad1 in a mechanism related to the NE reformation and stability independent of the SAC. Mad1 depletion also resulted in redistribution of the ER network and mitochondria throughout the cell as seen by fluorescence microscopy. Nup153 depletion coincided with the NE abnormalities and alteration of these organelles similar to that seen in Mad1-depleted cells. Further, by fluorescence microscopy, we have shown that Nup153 depletion, but not of Mad1, partially affected the localization of the cytoplasmic nucleoporins in human and in mouse cells and thus the NPC integrity. In conclusion, altogether, our results suggest that Nup153 is essential for NE and NPC integrity. Nup153 has likely separable roles in this context: one in post-mitotic NE reformation with Mad1 and one in interphase in NPC assembly. Nup153-Mad1 complex has a function independent of the spindle checkpoint, but important for the establishment of an intact NE architecture. / Les pores nucléaires sont des structures enchâssées dans l’enveloppe nucléaire et composées de protéines appelées les nucléoporines. Ces pores nucléaires contrôlent le trafic bidirectionnel des protéines et des ARNs entre le noyau et le cytoplasme dans les cellules eucaryotes tandis que les nucléoporines individuelles sont également impliquées dans d’autres processus cellulaires tels que la division cellulaire, l’assemblage des kinétochores, l’expression génétique et la migration cellulaire. Un exemple primordial de la versatilité fonctionnelle des nucléoporines peut être observé à travers Nup153. Depuis sa découverte, Nup153 est connue pour être un élément clé dans le transport nucléo-cytoplasmique, mais il a également été démontré qu’elle participait directement ou indirectement à l’expression génétique et au contrôle du cycle cellulaire. Dans ce contexte, nous avons montrés précédemment que des niveaux altérés de Nup153 menaient à des anomalies mitotiques, particulièrement en cytokinèse et dans le point de contrôle de l’assemblage du fuseau mitotique (SAC). Le SAC assure la ségrégation correcte du matériel génétique entre les cellules filles. Il a été montré que Nup153 interagit avec la protéine du SAC Mad1. Dans cette étude, nous avons utilisé une technique d’imagerie de haute résolution, « in situ proximity ligation assay » pour disséquer davantage l’interaction entre Nup153 et Mad1 dans les cellules humaines. Nous avons montré que ces deux protéines interagissent exclusivement au niveau de l’enveloppe nucléaire, depuis les dernières phases de la mitose jusqu’à la prophase. Par des expériences d’interaction in vitro, nous avons également identifiés sur Mad1 deux sites de liaison indépendants pour Nup153. Nous avons également fourni des indications que Nup153 interagit aussi avec une forme SUMOylée de Mad1. La déplétion de Mad1 menait à un recrutement tardif de Nup153 au niveau de l’enveloppe nucléaire en cours de reformation en anaphase dans les cellules vivantes et à des altérations de l’architecture de l’enveloppe nucléaire, caractérisées par un changement de la courbure membranaire au niveau de l’interface pore nucléaire-enveloppe nucléaire. Suite à cela, une expansion de l’espace entre les membranes nucléaires internes et externes a été observée par microscopie électronique. Ceci suggère une implication de Mad1 dans un mécanisme lié à la stabilité de l’enveloppe nucléaire indépendant du SAC. La déplétion de Mad1 résultait également en une redistribution du RE et des mitochondries à travers la cellule. La déplétion de Nup153 coïncidait avec des anomalies similaires au niveau de l’enveloppe nucléaire et des organelles. De plus, la déplétion de Nup153 affectait partiellement la localisation des nucléoporines cytoplasmiques, contrairement à la déplétion de Mad1. Ensemble, nos résultats suggèrent que Nup153 est essentielle pour l’intégrité des pores nucléaires et de l’enveloppe nucléaire. Nup153 semble avoir deux rôles, un au niveau de la formation de l’enveloppe nucléaire en fin de mitose, en complexe avec Mad1 et un autre rôle au niveau de l’assemblage des pores nucléaires. Le complexe Nup153-Mad1 a une fonction indépendante du SAC, mais importante pour l’établissement d’une enveloppe nucléaire intacte. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

In this thesis the deployment of a methodological combination of two single molecule techniques, the planar bilayer technique and fluorescence fluctuation spectroscopy, is presented. The newly devised electro-optical setup will serve as a sophisticated model system for electrical excitable biological membranes. The expectation on a combined electro-optical setup is to be able to correlate the function of membrane channels (electrical activity) with its structural properties (fluorescence assays). The thesis is grouped into four chapters: A general introduction, providing the biological and methodological background, is followed by two studies on the application of the electro- optical setup in the field of membrane biophysics. In the first study the electrical and diffusion properties of planar bilayer membranes made of simple and ternary lipid mixtures are characterized. Additionally, the influence of temperature dependent lipid phase separation on the electrical activity of the ion channel gramicidin A is studied. The second study addresses the conformational changes of the pore-forming toxin Colicin A during membrane binding and ion channel formation. Finally, the potentials and the limitation of the presented setup are discussed.

The work deals with the development of new types of gas and waterproof tight coatings based on secondary crystallization of cement, using industrial waste as secondary raw material in the formulation of new recipes.

Subscription articles: Theses and dissertations which contain embedded PJAs as part of the formal submission can be posted publicly by the awarding institution with DOI links back to the formal publications on ScienceDirect.doi:10.1016/j.msec.2015.10.069 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19578号 / 医博第4085号 / 新制||医||1013(附属図書館) / 32614 / 京都大学大学院医学研究科医学専攻 / (主査)教授 安達 泰治, 教授 開 祐司, 教授 妻木 範行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Authors are permitted to post the MBoC PDF of their articles (and/or supplemental material) on their personal websites or in an online institutional repository provided there appears always the proper citation of the manuscript in MBoC and a link to the original publication of the manuscript in MBoC (http://www.molbiolcell.org/site/misc/ifora.xhtml) / 京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第19869号 / 生博第350号 / 新制||生||46(附属図書館) / 32905 / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 河内 孝之, 教授 藤田 尚志, 教授 永尾 雅哉 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第20920号 / 理博第4372号 / 新制||理||1627(附属図書館) / 京都大学大学院理学研究科地球惑星科学専攻 / (主査)教授 松浦 純生, 教授 林 愛明, 准教授 松四 雄騎 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM