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Prostate cancer is consistently the most frequently diagnosed cancer in American males as well as the second leading cause of cancer-related mortality. This underscores the dire need to understand the healthy prostate and how it can transform into a diseased state. Therefore, I have sought to investigate the heterogeneity and ontogeny of the mouse and human prostate. To do this, I employed single-cell RNA-sequencing, electron microscopy, immunofluorescence, and immunohistochemical analyses to identify specific cell populations, as well as lineage tracing, organoid culture, and tissue recombination assays to assess the function and origin of these populations. I discovered a profound level of heterogeneity uniquely within the luminal epithelial compartment of the prostate, including several novel populations. These luminal populations differ in distribution between mouse prostate lobes and along the proximal-distal axis within each lobe. These populations demonstrated significant differences in progenitor behavior in both organoid culture and tissue recombination assays, as shown by their differential abilities to proliferate, generate patterned structures, and differentiate into distinct cell types. Comparisons of the mouse prostate cell populations to cells from several benign human prostate samples showed that there is also luminal heterogeneity in the human prostate, and that several mouse populations have substantial gene expression overlap with human prostate populations. The observed luminal heterogeneity as well as the functional differences were consistent across several different published studies of the mouse prostate, and cross-species transcriptional similarities between populations were maintained across additional human samples, indicating that these findings are robust. My findings suggest that the luminal compartment of the mouse prostate contains distinct populations of cells that may act as reserve progenitors, and that their distribution across the prostate lobes could be functional. Additionally, if these populations can be cells of origin for prostate cancer, then their differences in progenitor behavior could contribute to the heterogeneity observed in prostate cancer prognosis and treatment response, which could have substantial clinical implications for patients.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23813号 / 医博第4859号 / 新制||医||1059(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 中本 裕士, 教授 花川 隆, 教授 大森 孝一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Yes / Diabetic Peripheral Neuropathy (DPN) is one of the most common types of diabetes that can affect the cornea. An accurate analysis of the nerve structures can assist the early diagnosis of this disease. This paper proposes a robust, fast and fully automatic nerve segmentation and morphometric parameter quantification system for corneal confocal microscope images. The segmentation part consists of three main steps. First, a preprocessing step is applied to enhance the visibility of the nerves and remove noise using anisotropic diffusion filtering, specifically a Coherence filter followed by Gaussian filtering. Second, morphological operations are applied to remove unwanted objects in the input image such as epithelial cells and small nerve segments. Finally, an edge detection step is applied to detect all the nerves in the input image. In this step, an efficient algorithm for connecting discontinuous nerves is proposed. In the morphometric parameters quantification part, a number of features are extracted, including thickness, tortuosity and length of nerve, which may be used for the early diagnosis of diabetic polyneuropathy and when planning Laser-Assisted in situ Keratomileusis (LASIK) or Photorefractive keratectomy (PRK). The performance of the proposed segmentation system is evaluated against manually traced ground-truth images based on a database consisting of 498 corneal sub-basal nerve images (238 are normal and 260 are abnormal). In addition, the robustness and efficiency of the proposed system in extracting morphometric features with clinical utility was evaluated in 919 images taken from healthy subjects and diabetic patients with and without neuropathy. We demonstrate rapid (13 seconds/image), robust and effective automated corneal nerve quantification. The proposed system will be deployed as a useful clinical tool to support the expertise of ophthalmologists and save the clinician time in a busy clinical setting.

This study evaluated degradable nutrient supply effects on VFA concentrations, fluid flux and pool sizes, rumen epithelial metabolic and absorptive genes, and post-absorptive muscle and blood responses. Six ruminally cannulated Holstein heifers (BW=330 ± 11.3 kg) were used in a partially replicated Latin Square experiment with four treatments consisting of beet pulp or timothy hay and barley or corn grain. Periods were18 d with 3 d diet adaptation and 15 d of treatment. During each period, d 10 to 14 was used for in situ nutrient degradation assessment, d 16 to 18 was used for rumen fluid sampling, and d 18 was used for rumen papillae and skeletal muscle biopsies and blood sampling. In situ ruminal starch disappearance rate (barley 7.61 to 10.5 %/h vs corn 7.30 to 8.72%/h; P = 0.05) and extent of fiber disappearance (timothy hay 22.2 to 33.4 % DM vs beet pulp 34.4 to 38.7 % DM P=0.0007) differed significantly among diets. Acetate (P = 0.02) and isovalerate (P = 0.008) molar percentages (% mol) were increased by timothy hay, but propionate (P = 0.06) and valerate (P = 0.10) molar percentages were decreased. Corn increased propionate (P = 0.02) and valerate (P = 0.049) molar percentage, but decreased butyrate (P = 0.04) molar proportion. Fluid volume and fluid passage rate, and individual VFA pool sizes were not influenced by diet (P > 0.05). Four epithelial genes, two metabolic and two absorptive, had increased expression on timothy hay diets (P < 0.15). Blood acetate concentration was influenced by treatment (P = 0.067) but no other blood metabolites were. Skeletal muscle metabolic rate was significantly increased on corn diets (P = 0.023). The results of this study provide a whole-system snapshot of how the rumen environment changes on diets differing in nutrient degradability and how the post-absorptive system adapts in response. / Master of Science / Over the last 50 years, dairy cattle have been bred to optimize milk production to meet growing population demands for milk and dairy products. The world population continues to grow and is projected to reach 9.7 billion people by 2050. Because of this growing population, there is an overwhelming need for dairy nutritionists to optimize the conversion of human inedible fibers into human edible food. The ruminant animal accomplishes this conversion through microbial fermentation of feedstuffs into volatile fatty acids (VFA), which account for approximately 70% of total energy available for meat, milk, and fiber production. Because rumen fermentation is a complex biochemical system, it is influenced by myriad factors including the substrate provided, the pH of the environment, and the absorptive and metabolic capacity of the rumen wall, among others. Although we understand how diet influences individual aspects of rumen fermentation, few studies have concurrently evaluated how diet influences the rumen chemical environment, the epithelium, and the resulting shifts in postabsorptive metabolism. Our study sought to understand the impacts of feedstuffs with different expected ruminally available starch and fiber supplies on these aspects of ruminant physiology. Six ruminally cannulated Holstein heifers were fed four different diets which used either beet pulp (low fiber ingredient) or timothy hay (high fiber ingredient), and ground corn (low starch ingredient) or ground barley (high starch ingredient). Heifers were fed each diet for a period of 18 days. From day 10 to day 14 of the period, nutrient degradability was assessed by incubating bags of feed in the rumen and conducting feed analysis after removed from the rumen. During the last four days of each period, rumen fluid samples, blood samples, muscle biopsies, and rumen papillae biopsies were collected. Feed analysis indicated that the starch sources differed in degradation rates (i.e. the speed of degradation) and fiber sources different in extent of rumen degradation (i.e. the percentage of feed degraded). Timothy hay caused greater concentrations of Total VFA, Total branched-chain VFA, acetate isobutyrate, and isovalerate. Timothy hay caused greater molar proportions of acetate and isovalerate. Corn caused greater molar proportions of propionate and valerate when barley caused greater molar proportions of butyrate. Rumen papillae biopsies were used to evaluate gene expression. Out of 14 genes, four were impacted by diet. Two rumen transporters responsible for the absorption of VFA had greater expression when animals were fed timothy hay diets versus beet pulp diets. Two metabolic genes also had greater expression due to timothy hay. The changes of both absorptive genes and metabolic genes is likely connected to the increased presence of VFA in the rumen. Lastly, blood acetate was increased, but there was not a specific ingredient or combination that caused the change. These results provide an overall snapshot of rumen fermentation characteristics and how changes in the rumen affect other biology.

No / P-cadherin serves as a major topobiological cue in mammalian epithelium. In human hair follicles (HFs), it is prominently expressed in the inner hair matrix that harbors the HF pigmentary unit. However, the role of P-cadherin in normal human pigmentation remains unknown. As patients with mutations in the gene that encodes P-cadherin show hypotrichosis and fair hair, we explored the hypothesis that P-cadherin may control HF pigmentation. When P-cadherin was silenced in melanogenically active organ-cultured human scalp HFs, this significantly reduced HF melanogenesis and tyrosinase activity as well as gene and/or protein expression of gp100, stem cell factor, c-Kit, and microphthalmia-associated transcription factor (MITF), both in situ and in isolated human HF melanocytes. Instead, epidermal pigmentation was unaffected by P-cadherin knockdown in organ-cultured human skin. In hair matrix keratinocytes, P-cadherin silencing reduced plasma membrane β-catenin, whereas glycogen synthase kinase 3 beta (GSK3β) and phospho-β-catenin expression were significantly upregulated. This suggests that P-cadherin-GSK3β/Wnt signaling is required for maintaining the expression of MITF to sustain intrafollicular melanogenesis. Thus, P-cadherin-mediated signaling is a melanocyte subtype-specific topobiological regulator of normal human pigmentation, possibly via GSK3β-mediated canonical Wnt signaling.