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Cardiovascular disease, including hypertension, accounts for almost 50% of the deaths in patients with end stage renal disease (ESRD) on hemodialysis (HD) yet hypertension remains very poorly controlled in this population. The purpose of this study was to retrospectively compare control of hypertension in hemodialysis (HD) patients when extracellular volume (ECV) was assessed and managed by clinical parameters and physical assessment data alone with control of hypertension when data from blood volume monitoring (BVM) technology was also used to assess and manage ECV in a freestanding outpatient hemodialysis unit. The main cause of hypertension in the ESRD population has been identified as increased ECV most likely secondary to increased interdialytic weight gain and failure to attain and maintain patient’s dry weight. HD nurses often employ clinical parameters along with physical examination to determine a patient’s pre, intra, and post dialytic fluid status and this approach can have a high index of error. BVM technology is being used in many hemodialysis units to assist with assessment of ECV. A comparative retrospective chart review was used to collect data for this project. A descriptive, cross-sectional design was employed to answer the question:“Are hypertensive hemodialysis patients who dialyze in a freestanding dialysis unit, where BVM technology is utilized, more likely to be normotensive as defined by a pre dialysis blood pressure of less than 140/90 and post dialysis blood pressure less than 130/80”? A pilot study was conducted to determine if the patient population and data were available in existing patient records for extrapolation. Approval for the study was obtained from the University IRB. A convenience sample was obtained from the records of patients meeting the inclusion criteria. Variables were measured and analyzed using iv descriptive statistics such as sampled paired T-test to compare pre and post BVM systolic, diastolic blood pressures, intradialytic weight gain, serum Albumin and sodium levels, and hemoglobin. A p-value of 0.05 was assigned for statistical significance. Data analysis showed there were statisticaly significant differences in the pre dialysis systolic blood pressure, post BVM, and the serum sodium pre and post BVM when the two groups were compared These statistically significant findings support a correlation between reduction in the HD patient’s ECV and improved blood pressure control. The reduction of pre-dialysis SBP was significant because many patients on hemodialysis have systolic hypertension that may or may not coexist with diastolic hypertension. The findings of this study may be used to formulate a protocol to be used in the HD units where the BVM is available. The protocol would rely on accurate nursing assessment of clinical parameters, patient verbalizations of symptoms, and the routine use of the BVM in order to continuously assess the patient’s fluid status. Future research recommendations include conducting the study in a population closer to the national sample, a study where glucose readings and /or hemoglobin A1C levels are measured to assess the impact of glucose on ECV, and which antihypertensive class of medication works best with BVM technology to effectively manage hypertension in this population.

Every year millions of individuals suffer from traumatic brain injury (TBI) leading to permanent disabilities and even death. Mild TBI (mTBI) is the most common form of TBI comprising about 80-90% of all occurrences. Following a CNS insult like an mTBI, astrocytes can undergo activation resulting in the transformation into reactive astrocytes (RAs). RAs also play an important role in brain remodeling following an mTBI. Research on the mechanical complexity of the brain has important implications for understanding brain function and dysfunction, as well as for the development of new diagnostic and therapeutic tools for neurological disorders. This study aimed to develop and utilize an emph{in vitro} mTBI platform to investigate the intricate mechanical interplay between the extracellular matrix (ECM) and astrocytes following a simulated mTBI. Cellular mechanisms underlying mTBI and the contribution of mechanical forces that result in prolonged brain damage are yet to be comprehensively understood. Successfully devised mechanical characterization techniques for tissue-engineered models were developed utilizing atomic force microscopy and rheology. Astrocyte exposure to high-rate overpressure revealed altered mechanical properties of the surrounding matrix and decreased expression of laminin and collagen IV, which are critical for brain function and may contribute to pathologies associated with mTBI. The developed platform and methods provide new insights into the mechanistic complexity underlying ECM-astrocyte interactions following an mTBI. / Master of Science / Every year, millions of people suffer from traumatic brain injury (TBI), which can lead to permanent disabilities or even death. The most common form of TBI is mild TBI (mTBI), which accounts for 80-90% of all cases. After a mTBI, astrocytes, the most common cell type in the brain, can become activated and turn into reactive astrocytes (RAs). RAs play an important role in the brain's recovery following a mTBI. Understanding the mechanical complexity of the brain is crucial for developing new diagnostic and therapeutic tools for neurological disorders. This study aimed to investigate the mechanical interplay between the modeled tissue and astrocytes following a simulated mTBI using an emph{in vitro} platform. Development of mechanical characterization techniques allowed for any alterations caused by the astrocytes to their environment to be detectable. The astrocyte exposure to the simulated mTBI revealed altered mechanical properties of the surrounding environment and decreased expression of proteins laminin and collagen IV, which are critical to brain function and may contribute to pathologies associated with mTBI. This study provides new insights into the mechanistic complexity underlying the interaction between astrocytes and their environment, which could lead to the development of new treatments.

Geobacter species are δ-Proteobacteria and are often predominant in the Fe(III) reduction zone of sedimentary environments. Their abilities to remediate contaminated environments and to produce electricity have inspired extensive studies. Cell motility, biofilm formation, and type IV pili, which have been shown to be regulated by chemotaxis genes in other bacteria, all appear important for the growth of Geobacter species in changing environments and for electricity production. The genomes of Geobacter species show the presence of a significant number of chemotaxis gene homologs, suggesting important roles for them in the physiology of Geobacter species, although gene functions are not yet identified. In this study, we focus on identifying chemotaxis components and studying their functions in Geobacter species. We identified a large number of homologs of chemotaxis genes, which are arranged in six or more major clusters in the genomes of Geobacter sulfurreducens, Geobacter metallireducens, and Geobacter uraniireducens. Based on homology to known pathways, functions of some chemotaxis clusters were assigned; others appear to be unique to Geobacter species. We discuss the diversity of chemoreceptors and other signaling proteins as well the regulation of chemotaxis genes in Geobacter species. The functions of chemotaxis genes were studied in G. sulfurreducens, whose genome contains ~ 70 chemotaxis gene homologs, arranged in 6 major clusters. These chemotaxis clusters are also found in other Geobacter species with similar gene order and high level of gene identity, suggesting that our study in G. sulfurreducens could be extrapolated to other Geobacter species. We identified the function of the che5 cluster of G. sulfureducens as regulation of the biosynthesis of extracellular materials. We showed that G. sulfurreducens KN400 is chemotactic, and that this behavior is flagellumdependent. Our preliminary data indicated that G. sulfurreducens may use the che1 cluster, which is found exclusively in Geobacteraceae, to regulate chemotaxis. Our studies demonstrated important roles of chemotaxis genes in Geobacter physiology and their presence in large numbers could be one of the reasons why Geobacter species outcompete other species in bioremediation sites. Further studies are warranted for better understanding of the mechanisms of Che-like pathways and their potential use in optimization of conditions for applications of Geobacter species in bioremediation and electricity generation.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24198号 / 医博第4892号 / 新制||医||1060(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 川口 義弥, 教授 松田 秀一, 教授 小濱 和貴 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Mesenchymal stem/stromal cells (MSCs) release Extracellular vesicles (EVs) that are believed to play a major role in nerve regeneration after stroke. However, a major complication when trying to transition MSC-EVs from a pre-clinical to clinical setting is the convenient long-term storage of MSC-EVs. Therefore, we developed a strategy to freeze dry MSC-EVs to store them for more practical clinical applications. We first determined the optimal trehalose concentration for freeze drying the MSC-EVs, and we subsequently investigated the optimal freezing conditions. It was determined that 100 mM of trehalose and freezing temperature at -20°C were the optimal conditions to freeze dry the EVs. The therapeutic capabilities of the freeze-dried MSC-EVs was tested via tube formation assay and co-culturing them with neural stem/progenitor cells (NSPCs). It was found that human vein umbilical endothelial cells (HUVECs) treated with rehydrated MSCEVs promoted tube formation suggesting the trophic factors in the MSC-EVs survived the freeze-drying process. As for the NSPC co-culture, all treatments involving rehydrated MSC-EVs protected by trehalose during the freeze-drying process promoted proliferation and did not affect their ability to differentiate into oligodendrocytes, astrocytes, or neurons. Determining the optimum freezing-drying conditions allows us to stockpile a large amount of MSC-EVs at room temperature for on-demand applications.

Drosophila melanogaster has a tubular heart called the dorsal vessel, which is composed of contractile cardiomyocytes and hemolymph filtering pericardial cells. During larval development the dorsal vessel (heart) grows in size, and the luminal space inside the heart expands, however it has not been clear which cells are responsible for laying the extracellular matrix (ECM) during this expansion. Hemocytes (white blood cells), pericardial cells and cells of the fat body are candidate cell types that may secrete ECM for assembly during the growth of the heart lumen. With gene knock-down techniques we are exploring whether hemocytes participate in assembly of the heart ECM at this location. Additionally, studies of fluorescently tagged hemocytes in intact larvae reveal that hemocytes aggregate around pericardial cells of the dorsal vessel in 3rd instars. Confocal studies of dissected larval hearts indicate that hemocytes aggregate within infoldings of basement membrane associated with pericardial cells. Hemocyte-pericardial cell association could indicate that hemocytes take up proteins that are produced by pericardial cells and deliver them to other locations or that there might be a previously unidentified hematopoietic site at the Drosophila larval heart. / Thesis / Master of Science (MSc)

The cardiac extracellular matrix (ECM) is a dynamic protein scaffold that is required to support cardiac function. Regular remodelling of the matrix involves protein turnover and deposition and is a highly regulated process. In disease states the normal balance of the ECM is disrupted and aberrant protein deposition and crosslinking can occur. This process, termed fibrosis, causes stiffening of the cardiac ECM, which in turn impairs organ function. Fibrosis is a hallmark of cardiovascular disease, is a progressive condition that can contribute to adverse clinical outcomes, and currently has no available treatments. One of the leading causes of cardiovascular disease is obesity and fibrosis is known to occur in this context. In order to investigate the development of fibrotic remodelling in the context of obesity I have developed a dietary obesity model in the fruit fly Drosophila melanogaster. Additionally, I developed a genetic overgrowth model as increased cardiac load is also known to trigger fibrotic remodelling. Dietary obesity models reveal altered ECM organization, as well as impaired cardiac contractility, while overgrowth models demonstrate a remarkable ability to appropriately scale heart morphology with increased body size. The overgrowth model does have extremely elevated expression levels of the crosslinking enzyme LOXL2, suggesting a major contributor to impaired function is increased crosslinking rather than altered protein deposition. However, inhibition of crosslinking caused only minor ECM organizational defects but was able to rescue the elasticity of the overgrowth model. Overall, this thesis raises intriguing questions for treatment of cardiovascular disease, where tissue dynamics are often overlooked in a clinical setting. / Thesis / Doctor of Science (PhD) / The cardiac extracellular matrix (ECM) is a protein scaffold that supports heart function. Cardiovascular disease often involves increased levels of ECM proteins, a condition called fibrosis, which causes increased tissue stiffness and functional impairment. There is no cure for fibrosis and developing treatments requires an understanding of how the ECM responds to disease. I developed a dietary obesity model and a genetically triggered overgrowth model to examine how the ECM responds to disease states. I found that obesity causes ECM reorganization and functional defects, but that overgrowth models scale their hearts remarkably well with increased body size. Overgrowth models were found to have elevated levels of matrix crosslinking enzymes, which contributed to a stiffer matrix in these individuals. This was rescued by inhibition of crosslinking. Overall, this thesis reveals a connection between cardiac ECM organization, tissue elasticity, and heart function, and how these are altered in disease.