Discovery and functional characterization of self-renewal regulatory factors constitute one of the central themes in the field of human hematopoietic stem and progenitor cell (HSPC) biology both for advancing the fundamental science as well as harnessing the knowledge to expand hematopoietic stem cells (HSCs) for regenerative therapeutics purposes. Musashi-2 (MSI2) is a marker and essential positive regulator of HSCs but knowledge of the transcription factors (TFs) that ensure its appropriate HSC-specific expression is lacking. I believed that uncovering the transcriptional circuitry of MSI2 could also offer opportunities for the discovery of candidate TFs with similar or superior functions in human HSPCs. To that end, initial investigations were set out to identify a conserved minimal promoter region bound by TFs upstream of the translation start site of human MSI2. In silico analysis of the minimal promoter region and series of TF binding-site mutagenesis biochemical assays identified USF2 and PLAG1 as key co-regulators of MSI2 expression and function in the model K562 cell line and cord blood HSPCs. The approach identified PLAG1 as a candidate TF whose function in HSPCs was not previously reported. I have characterized PLAG1 as an important regulator of HSPCs self-renewal using in vitro and xenotransplantation functional genomics coupled with RNA sequencing and downstream pathway analysis. In vitro, overexpression of PLAG1 (PLAG1OE) imparts sustained pro-survival advantage to progenitors and produces significantly more CD34+ cells compared to control culture. PLAG1OE enhances BFU-E and total colony forming unit (CFU) output. Complementary in vitro assays using shRNA-mediated knockdown (PLAG1KD) elucidated impaired CFU output, increased apoptosis and significantly reduced CD34+ cell counts. In vivo transplantation of PLAG1OE CD34+ cells into NSG mice at limiting doses robustly enhances the frequency and absolute number of HSCs by a magnitude of 24.1-fold compared to control. Serial transplantation of bone marrow cells into secondary recipients demonstrated enhanced self-renewal of HSCs compared to control. Mechanistically, transcriptome-wide gene-set enrichment and network analysis revealed that ribosome biogenesis and proteostasis pathways are significantly attenuated. PLAG1OE up-regulates expression of H19 and MEG3 imprinted long non-coding RNA (lncRNA)-derived miR-675 and miR-127 species respectively. In silico overlap analysis with experimentally validated targets of both miR-127 and miR-675 revealed ribosome biogenesis and proteins synthesis pathways components as prime targets. PLAG1 also represses the pan-ribosome biogenesis transcriptional regulator MYC. Down regulation of MYC demonstrates another layer of PLAG1-mediated transcriptional attenuation of protein synthesis. Additionally, we observed dampening of IGF1R/PI3K/AKT/mTOR signaling pathway. Conversely, PLAG1KD down regulated H19 and MEG3 expression and reverses global gene-set enrichment observed by PLAG1OE in reciprocal fashion. Taken together, our study presents PLAG1 as master regulator of ribosome biogenesis and protein synthesis from multiple checkpoints to enhance clonogenicity, survival and long-term self-renewal capacity of HSCs. / Thesis / Doctor of Philosophy (PhD)

The role of ROS in the hematopoietic system has been a subject that has received little investigation due to the hypoxic environment inherent in the bone marrow niche. Furthermore, it is not known whether or not oxidative damage accumulations play a role in the functional decline of HSCs associated with aging. Measuring DNA damage and ROS levels using the Fragment Length Analysis by Repair Enzyme (FLARE) assay, I show here that there are indeed significantly detectable levels of 8-oxoguanine, a lesion associated with ROS, present in both young and old murine HSCs. In an attempt to attenuate the presence of these lesions, a four-week treatment with the thiol-based antioxidant N-Acetyl-L-Cysteine was administered orally to mice. Analysis revealed significant decreases in oxidative lesions in both the young and old HSC compartment. Additionally, it was demonstrated that the NAC treatment significantly reduced number of baseline DNA breaks in old, but not young, HSCs. Together these results suggest that DNA damage accumulation is a dynamic process that changes as cells age. Further understanding of the role of ROS will help elucidate the importance of this type of DNA damage on the declining functional potential associated with aging.

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Inflammatory arthritis (IA) is a debilitating disease that is characterized by joint destruction. This destruction is caused by osteoclasts (OCLs) degrading bone within the synovium, however the exact cellular origins of these synovial OCLs is not well understood. We hypothesize that the synovial OCLs seen in IA are independently derived from two contributing cell lineages: the canonical source of OCL which are Hematopoietic Stem Cell (HSC) derived monocytes and the newly described Erythro-Myeloid Progenitor (EMP). To explore the contribution of these two lineages to synovial OCL, we used Cx3cr1CreERT2;Rosa26LSL-tdTomato mice to label EMP-derived cells, and Flt3Cre;Rosa26LSL-YFP to label HSC-derived cells. Using immunofluorescent histology, we found that synovial OCLs formed under arthritic conditions derive from both HSC and EMP-progenitors, suggesting the possibility that regulatory mechanisms unique to each developmental lineage promote OCL differentiation in arthritic joints. In support of these observations, in IA we detected two populations of mononuclear cells, as possible osteoclast precursors, that express both the OCL marker TRAP and the monocytic/macrophage marker CD68. These mononuclear TRAP+ CD68+ cell populations are found within the inflamed synovium and in bone periosteal surfaces of arthritic joints, and are mostly EMP derived, however, the HSC lineage significantly contributes to osteoclast formation, as suggested by our lineage-tracing strategy. We are currently investigating the dynamics of these cell populations during the early, peak, and resolution stages in an acute murine model of IA. To better understand regulatory differences between OCL derived from each of these lineages, we plan to isolate EMP- and HSC-derived OCLs directly from joints to study their precise phenotype, cytokine differentiation requirements, resorptive capacity and transcriptional activity, by flow cytometry, in vitro cell cultures and single cell RNA sequencing, respectively.

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京都大学 / 新制・課程博士 / 博士(医学) / 甲第23103号 / 医博第4730号 / 新制||医||1050(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 江藤 浩之, 教授 斎藤 通紀, 教授 滝田 順子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

We report the serendipitous discovery of HSC J142449-005322, a double source plane lens system in the Hyper Suprime-Cam Subaru Strategic Program. We dub the system Eye of Horus. The lens galaxy is a very massive early-type galaxy with stellar mass of similar to 7 x 10(11) M-circle dot located at z(L) = 0.795. The system exhibits two arcs/rings with clearly different colors, including several knots. We have performed spectroscopic follow-up observations of the system with FIRE on Magellan. The outer ring is confirmed at z(S2) = 1.988 with multiple emission lines, while the inner arc and counterimage is confirmed at z(S1) = 1.302. This makes it the first double source plane system with spectroscopic redshifts of both sources. Interestingly, redshifts of two of the knots embedded in the outer ring are found to be offset by Delta z = 0.002 from the other knots, suggesting that the outer ring consists of at least two distinct components in the source plane. We perform lens modeling with two independent codes and successfully reproduce the main features of the system. However, two of the lensed sources separated by similar to 0.7 arcsec cannot be reproduced by a smooth potential, and the addition of substructure to the lens potential is required to reproduce them. Higher-resolution imaging of the system will help decipher the origin of this lensing feature and potentially detect the substructure.

Master of Music (Music Education) / This thesis documents an investigation of the selection procedures relating to Australian vocal repertoire for mid-adolescent and Higher School Certificate (New South Wales) performers, as used by private singing teachers, school music teachers and singing students. It explores the similarities and differences in the criteria employed in these selections. Semi-structured interviews were the source of data and were conducted with participants from these three categories as well as two composers. The participants evinced highly disparate views. The private singing teachers believed that repertoire should be dictated by the technical ability and physiological constraints of mid-adolescent students and that their role in selecting repertoire was related to the long term vocal growth of each individual. They felt that the school music teachers vetted their repertoire choices with no useful explanation of their reasoning, while the school music teachers noted that students frequently presented repertoire that was too difficult or that was not readily communicated with the audience. The ability of mid-adolescent singers to communicate with and engage an audience was the prime concern of the school music teachers. The students wanted to impress their examiners and believed that infrequently heard repertoire was the best choice, though this was not endorsed by the teachers. There was a perception that the students would perform at their best when they chose repertoire to which they could relate emotionally. Many private singing teachers and school music teachers are not aware of the very broad range of contemporary Australian music and its divergent characteristics. However, there is a shortage of appropriate Australian repertoire that addresses the physiological and emotional needs of mid-adolescent singers.

<p>The homeostatic hematopoietic stem cell compartment is comprised of quiescent long term self renewing stem cells and cycling short term stem cells with finite renewal potential. To study the molecular mechanisms governing self renewal of hematopoietic cells we must force them to enter the cell cycle and proliferate. One approach to accomplish this goal is to damage the hematopoietic compartment with ionizing radiation or cytotoxic chemotherapy. Such injuries ablate mature blood cells and drive the primitive stem cells into cycle. I have elected to use a simple model of hematopoietic damage and regeneration to study the molecular mechanisms controlling self renewal in hematopoietic stem cells. At the beginning of this project it was unclear whether the signaling pathways which homeostatically control self renewal are utilized during injury repair. In particular, there is very little understanding of the signals required for regeneration after radiation damage. We hypothesized extracellular signal transduction pathways provided by the microenvironment are critical mediators of the stem cell repair process. To address these topics and extend the previous work generated in our laboratory, I chose to pursue a candidate approach focusing on the Wnt and Notch developmental signaling pathways.</p><p>In order to examine the activation and requirement for each signaling cascade after radiation and chemotherapy damage we used a combination of loss of function and reporter mouse models. To this end, we have conducted the majority of experiments for the Wnt project in animals deficient in beta-catenin, the key transcription factor required in the pathway. Our investigations revealed the Wnt pathway is turned on within regenerating stem cells and loss of beta-catenin impairs regeneration of the stem cell compartment after both radiation and chemotherapy injury. </p><p>Using a Transgenic Notch Reporter mouse to investigate the role of Notch signaling following hematopoietic damage we determined the Notch pathway is also activated during regeneration. Furthermore, using a live imaging approach we discovered Notch activated cells change their fate choice during regeneration. To determine if Notch gain of function provides radio-protection we infected stem cells with an active form of Notch prior to radiation and then scored self renewal potential in vitro. This led us to the conclusion that Notch gain of function can provide a self renewal benefit to irradiated hematopoietic stem cells.</p> / Dissertation