京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13111号 / 論医博第2129号 / 新制||医||1022(附属図書館) / (主査)教授 齊藤 博英, 教授 松原 和夫, 教授 林 康紀 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Twenty Holsteins, ten pregnant heifers and ten pregnant cows (third or greater pregnancy) were subdivided and fed either a low calcium (Ca) or Ca-supplemented ration for four weeks prepartum to determine the influence of age and prepartum Ca intake on hormonal control of peripartum Ca homeostasis. Jugular blood samples were taken on a fixed schedule from 21 days prepartum through 21 days postpartum for parathyroid hormone (PTH), calcitonin (CT), Ca, magnesium (Mg), and phosphorus (P) analysis. Heifers and cows receiving the high Ca ration prepartum tended to have higher prepartum serum Ca. Cows fed the high Ca ration prepartum (hi-Ca cows) exhibited severe hypocalcemia (6.1 mg/dl) at parturition and remained hypocalcemic for three days. Serum PTH concentration increased prepartum (-5 to -3 days) and at parturition, followed by an increase in CT, in all groups except high-Ca cows. Circulating CT was lower in high-Ca cows throughout the experiment. Serum concentrations of PTH and Mg increased from 7 to 21 days in all except high-Ca cows. Feed intake corrected for metabolic bodyweight was similar for both dietary treatments and ages. Milk production was greater for the first week in cows fed low Ca prepartum. There was no correlation between hypocalcemia and increased milk Ca concentration. In conclusion, heifers were able to achieve calcium homeostasis despite the high Ca ration, while high-Ca cows exhibited subclinical milk fever. / M.S.

The gastrointestinal (GI) tract is a crucial interface for the host, food derived antigens, the commensal microbiota and invasive pathogens. Here, the immune system must simultaneously protect against harmful pathogens and remain tolerogenic to commensal bacteria and nutrients. The intestinal mucosa of adult humans and mice is enriched for innate lymphoid cells (ILCs) that express the transcription factor RORγt (ILC3s). These cells are crucial for maintaining the delicate balance of tolerance and immunity in the GI tract. They serve protective roles in immune responses to infectious organisms, are essential for the formation of lymphoid tissues, and help maintain gut homeostasis via signaling to epithelial cells through interleukin 22 (IL-22). ILC3s in the GI tract can be further categorized into three main subsets with distinct and overlapping functional roles. These subsets can be identified by either the expression of CCR6, Nkp46, or by lacking both markers- double negative (DN), some of which also make IL-17A. Signals that mediate the development and function of the various ILC3 subsets are still an area of active investigation. Notch signaling is a highly conserved pathway that contributes to the development and function of many types of immune cells. There has been some investigation into the role Notch signaling plays in the development of ILC3, particularly in the transition from DN to Nkp46 ILC3. However, all three subsets of ILC3s express two Notch receptor isoforms (Notch1 and Notch2) the individual roles of these two receptors have not been dissected. We show signaling through Notch1 and Notch2 individually contribute to Nkp46 ILC3 development in a cell intrinsic manner. We also show Notch signaling, primarily through Notch2, reinforces the ILC3 program and suppresses the ILC1-like program in Nkp46 ILC3 by promoting the expression of RORγt, c-Maf, and IL-22, and suppressing the expression of T-bet and IFNγ. Notch signaling also supports ILC3-identity genes in CCR6 ILC3, promoting RORγt, IL-17A, and IL-22. We, therefore, identify a novel role for Notch signaling in ILC3 function. As such, Notch-deficient ILC3 fail to initiate proper immune response to enteric pathogen Citrobacter rodentium, leading to more severe infection. Our results show how a highly conserved signaling pathway contributes to ILC3 development, identity, and function. The GI tract is also enriched with helper CD4 T cells that express RORγt, IL-17A, and IL-22 (Th17), which share many phenotypic and functional features with ILC3. The relative contribution of ILC3 and Th17 cells to immune phenotypes remains poorly understood. Moreover, due to the lack of ILC3-specific depletion models, how ILC3 regulate mucosal protection in the presence of Th17 cells is not clear. Here, we examined non-redundant functions of ILC3 in intestinal immunity using novel ILC3-deficient mice that maintain endogenous T cells, Th17 cells, and secondary lymphoid organs. ILC3 depletion did not affect IL-22-production by CD4 T cells during homeostasis. However, despite the presence of IL-22-producing T cells, ILC3 and ILC3- derived IL-22 were required for maintaining homeostatic functions of the intestinal epithelium. ILC3 were dispensable for generation of Th17 and Th22 cell responses to pathogenic bacteria, though Th17 and Th22 responses were delayed in the absence of ILC3. ILC3- deficient mice were capable of pathogen clearance and survived infection with low dose Citrobacter rodentium in the presence of antigen-specific Th17 cells. However, ILC3 increased pathogen tolerance at early timepoints of infection by activating tissue-protective immune pathways. Consequently, ILC3 were indispensable for survival of high dose infection. We also assess the role of ILC3 and Th17 cell in metabolic syndrome, using our novel model. Our lab demonstrated commensal-specific Th17 cells are protective against metabolic syndrome and lost under high-fat, high-sugar diet. ILC3s drive the expansion of a commensal member, Faecalibaculum rodentium (F. rod), which displaces the Th17 cell-inducing commensal, segemented filamentous bacteria (SFB). Without ILC3s, SFB is not lost from the microbiota, commensal- specific Th17 cells are maintained and there is, therefore, no development of metabolic syndrome. Our results demonstrate crucial context- dependent roles for ILC3 in immune-sufficient animals during homeostasis, infection, and metabolic disease.

Immunoglobulins are key mediators of humoral immunity and can be appreciated in an isotype-dependent manner in autoimmune diseases, and to an extent, immune-mediated metabolic diseases. The most common isotype is Immunoglobulin G (IgG), yet there is little understanding of the role IgG plays in the pathogenesis of macrophage-driven metabolic disorders like atherosclerosis, metabolic dysfunction-associated steatotic liver disease (MASLD), and metabolic dysfunction-associated steatohepatitis (MASH). The contents of this dissertation introduce IgG as an activator of the macrophage inflammasome and its deposition in atherosclerotic plaques and fatty livers as a driver of disease progression. The presence of IgG in these depots and its accumulation is dictated by the neonatal Fc receptor (FcRn) in macrophages. IgG levels are known to be determined by recycling, especially in macrophages, rather than by production, and FcRn, encoded by the gene Fcgrt, is the sole receptor responsible. Interestingly, we uncover a myriad of roles for FcRn involved in regulating the biological properties of macrophages, such as their transcriptional and secretory profiles and their polarization amidst an immune response. Taken together, we identify FcRn as a hitherto unknown contender in the manipulation of macrophage function and regulation of IgG in the development of macrophage-associated cardiometabolic diseases using a multitude of methodologies. These findings highlight the importance of macrophage IgG recycling in metabolism and may warrant the potential to explore this phenomenon for therapeutic pursuits.

The primary purpose of newborn screening is to quickly identify children that are at risk of having a specific disorder in order to start treatment, prevent early death and reduce the chances of permanent physical or mental damage. The current and widely accepted approach used for identification of metabolism disorders involves a flow injection analysis with mass spectrometry detection of acylcarnitines and amino acids. Although this approach is widely accepted and has shown to be sufficient for identification of multiple metabolism disorders the method is not fully quantitative and results often have to be confirmed by second-tier tests. The primary focus of this research was to improve the accuracy and selectivity of this screening method by employing a high resolution chromatographic separation for the combined analysis of twelve acylcarnitines and seven amino acids. This method is an improvement over the current methodology allowing for separation of key isomers that are diagnostic for different metabolism disorders, reducing the need for multiple second-tier tests to confirm results and shortening the time to diagnosis. In order to further improve the efficiency of newborn screening we developed an in-line desorption device, which allows for direct analysis of DBS eliminating the need for punching disks from the filter paper cards. Our device was the first published paper that demonstrated the ability to directly analyze dried blood spots, without the need for any offline sample processing. Using this device, we validated a method to quantify biomarkers related to Maple Syrup Urine Disease, a disorder that requires a second-tier test for confirmation. To further improve the accuracy of dried blood spot analysis we evaluated a technique to correct the sample volume in low and high hematocrit samples. The level of hematocrit in blood spotted on filter paper cards affects the volume of sample analyzed, leading to errors in accuracy. Diffuse reflectance was used to relate differences in sample hematocrit on dried blood spots. We validated our technique with eighteen donor samples at various levels of hematocrit. Correcting sample volume for hematocrit showed improved precision and accuracy over the standard approach, ultimately reducing the potential to misidentify samples.

M. Tech. (Biotechnology, Department of Biosciences, Faculty of Computer and Applied Sciences) Vaal University of Technology / This study seeks to design and test specific short hairpin RNA (pshRNA) for their efficiency in knocking down the GALT gene RNA products thereby limiting the resultant enzyme activity. The following objectives were followed in designing the current study: 1. Designing a shorthairpin RNA (pshRNA) to target different regions of the coding sequence of the target GALT gene. 2. Propagating the pshRNAs in Escherichia coli (E.coli) and subsequently isolation of the respective plasmids for transfection. 3. Transfection of HeLa cells to test the efficiency of relevant pshRNAs in knocking down the GALT gene expression. 4. Transfection was followed by extraction of total mRNA, purification and quantification of total mRNA. 5. The GALT gene expression was qualitatively quantified against a house-keeping gene, glyceraldehyde phosphate dehydrogenase (GAPDH) to evaluate efficiency of knockdown using real time PCR. The three newly designed pshRNA (pshRNA2, pshRNA3 and pshRNA4) targeting the GALT gene expression showed a knockdown efficiency of 171 %, 48 % and 200 %, respectively. The results of this study will be useful for future evaluation of the possible long term glycosylation patterns under proper UDP glucose/UDP galactose levels compared with variable defective GALT gene levels.

A Doença de Alzheimer (DA) é a causa mais comum de demência e é caracterizada clinicamente por comprometimentos cognitivos. Histologicamente é caracterizada pela formação de placas senis e de emaranhados neurofibrilares intracelulares resultantes de alterações do metabolismo do peptídeo A e da hiperfosforilação da proteína tau, respectivamente. Essas alterações parecem, em parte, ser uma decorrência de uma deficiência na sinalização da insulina e conseqüente resistência do encéfalo a esse hormônio, sugerindo que a DA esporádica tenha uma relação com o Diabetes mellitus. A estreptozotocina tem sido utilizada como modelo de indução do Diabetes, e mais recentemente como modelo experimental da DA quando administrado intracerebroventricular. Nosso objetivo nesse estudo foi o de caracterizar o esse modelo experimental da DA induzido pela estreptozotocina, avaliando as conseqüências agudas e a longo prazo. Foram utilizados ratos Wistar machos de quatro meses de idade que receberam injeções intracerebroventriculares bilaterais de estreptozotocina ou de veículo. Os animais foram avaliados aguda e cronicamente por testes comportamentais de memória de referência e operacional utilizando o modelo do labirinto aquático de Morris que visavam avaliar o curso temporal dos prejuízos cognitivos após a injeção da droga. Em diferentes tempos após as injeções, os ratos foram sacrificados e regiões do encéfalo submetidas à técnica de immunoblotting para avaliação de proteínas indicadoras de neurodegeneração ou à técnica histoquimica pelo método de Fluoro-Jade C. A avaliação da memória operacional em períodos agudos mostrou que os prejuízos cognitivos parecem se instalar a partir de 3 horas da injeção de estreptozotocina. A avaliação crônica das memórias operacional e de referência mostrou que os ratos exibiram um prejuízo marcante no desempenho dessas tarefas ao longo dos testes, embora seja correto afirmar que esses animais ainda são capazes de adquirir informação relevante com relação à execução da tarefa, particularmente na versão de memória de referência. A análise de immunoblotting mostrou haver aumento da expressão do peptídeo beta amilóide significante em regiões como amigdala, córtex entorrinal, núcleos da base e do hipotálamo. Também foi observado um aumento significativo da fosforilação da proteína tau na amigdala, cerebelo, córtex, prosencéfalo basal e núcleos da base. Foi observada uma diminuição da enzima de síntese de acetilcolina, a colina acetil-transferase apenas na amigdala. Fibras em degeneração foram observadas no hipotálamo, na área septal e em neurônios piramidais na região CA1 após 1 dia da injeção de estreptozotocina. Já após 15 dias da injeção podemos observar marcação em neurônios do estriado e da região CA1 do hipocampo e em fibras próximas ao giro denteado. Em resumo a injeção intracerebroventricular de estreptozotocina parece produzir um bom modelo experimental da DA, pois reproduz as características cognitivas e histológicas encontradas nos pacientes com a doença / Alzheimer´s disease (AD) is the most common cause of dementia in aged humans. Recent reports have suggested a relationship between the onset of AD and an insulin-resistant brain condition. In this context, this study aimed at evaluating the effects of intracerebroventricular (ICV) injection of streptozotocin (STZ) in rats on behavior and neurodegeneration. Four month-old adult male Wistar rats were subjected to bilateral ICV injections of either STZ or vehicle and were tested for both reference and working memories in Morris water maze. After different survival times, rats were subjected to immunoblotting (to evaluate neurodegeneration markers) or to Fluoro-Jade C histochemistry. A marked disruption of performance in working memory was already observed after 3 hours of STZ injections. Immunoblotting analysis showed a significant increase of beta amyloid peptide expression in the amygdala, entorrinal cortex, basal ganglia, and hypothalamus. A significant increase of tau phosphorylation was also observed in the amygdala, cerebellum, cortex, basal forebrain and basal ganglia. Degenerating fibers were seen in the hypothalamus and septal area 1 day postinjection and in CA1 pyramidal neurons and close to the hippocampal dentate gyrus after 15 days. ICV injection of STZ seems therefore to produce an animal model of AD, as it reproduces the characteristic cognitive and histological changes of the disease

Beta-Amyloid (1-40) (Aβ40) is an aggregation prone protein, which undergoes a nucleation-dependent aggregation process causing the pathological neurodegeneration by amyloid plaque formation implicated in Alzheimer’s disease. In this thesis, we investigated the effects of small molecule modulators extracted from the marine invertebrate Pseudopterogorgia elisabethae on the Aβ40 amyloidogenic process using in- vitro ThT fluorescence assay and atomic force microscopy. We also investigated the effects of neutral and anionic phospholipid liposomes on Aβ40 aggregation. Our results show that a marine natural product Pseudopterosin-A and its derivatives can suppress and modulate the Aβ40 aggregation process. Furthermore, our results demonstrate that a neutral phospholipid liposome inhibits Aβ40 fibril formation, whereas the anionic liposomes promote it. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2015 / FAU Electronic Theses and Dissertations Collection