The eukaryotic cell nucleus is a highly organised organelle, with distinct specialised sub- compartments responsible for specific nuclear functions. Within the context of this functional framework, the genome is organised, allowing contact between specific genomic regions and sub-compartments. Previous work has shown that genes in both cis and trans can make specific contacts with each other. I hypothesise that such a preferred juxtaposition may impact the propensity for specific cancerinitiating chromosomal translocations to occur. In this thesis, I describe how I have extended and developed a ligation based proximity assay known as enriched 4C. I have coupled this technique with high throughput sequencing to determine genomic regions that spatially co-associate with the proto-oncogenes MLL, ABL1 and BCR. In addition to further developing the laboratory protocol, I have created bioinformatics tools used in the analysis of the sequencing data. I find that the association profiles of the three genes show strong correlation to the binding profile of RNA polymerase II and other active marks, suggesting that transcribed genes have a propensity to associate with other transcribed regions of the genome. Each gene also exhibits a unique repertoire of preferred associations with specific regions of the genome. Significantly, I find that the most frequent trans association of BCR is telomeric chromosome 9, encompassing its recurrent translocation partner gene ABL1. Interestingly, ABL1 is not at the maximum point of interaction. I use DNA-fluorescence in-situ hybridisation to validate the e4C association. My data supports a hypothesis that gene transcription has a direct role on genome organisation. I suggest that preferred co-associations of genes at transcription factories may promote the occurrence of specific chromosomal translocations.

Bone circulation in Hemorrhagic Shock was studied in 35 male mongrel dogs. The term hemorrhagic shock is defined in this thesis as persistent profound hypotensive syndrome, due to acute hemorrhage of more than one third of blood volume. The method of induction of shock consisted of removal of one third of estimated blood volume (8% of body weight) at a rate of 25 - 50 ml/min, and subsequently dropping the systemic blood pressure in a stepwise manner until the maintaining level of 30 - 35 mmHg is reached. The central venous pressure, pulse and respiratory rates were also recorded. Bone circulation was studied by (1) recording the blood flow through a cannula inserted into the tibial nutrient vein or artery and (2) recording the intramedullary pressure of tibia. When one third of estimated blood volume was removed, the bone blood flow through the nutrient vessel decreased to 22.5 ± 3.4% of control level. The decreased bone blood flow persisted as long as the hemorrhagic shock was maintained for 4-18 hours. The decreased bone blood flow was also evidenced by a profound and persistent fall of the intramedullary pressure of bone. Reinfusion into the animal of lost blood within fifteen minutes to six hours after hemorrhage resulted in a complete or partial recovery of the control systemic blood pressure as well as the control rate of bone blood flow and the control level of intramedullary pressure of bone. The curve showing relationship between the changes in bone blood flow and the systemic blood pressure is an exponential one with concavity towards the flow axis. This indicates that bone has a vasomotor control mechanism of increasing peripheral resistance during hemorrhagic shock. This was substantiated by the following observations: (1) The severity of decrease in bone blood flow on the side of lumbar sympathectomy was much milder (16% less) compared to the side of the intact sympathetic nerve; (2) Dibenzyline (phenoxybenzamine) a sympatholytic drug or alpha-receptor blocking agent alters the pressure-flow curve of bone circulation in chock to a linear pattern which indicates that the drug blocks the bone vasoconstricting mechanism(s). It is concluded that bone blood flow decreases in hemorrhagic shock and is not merely due to a decrease in circulatory blood volume, but also due to sympathetic and catecholamine hormonal vasoconstrictor mechanisms. / Surgery, Department of / Medicine, Faculty of / Graduate

Although the key role played by stem cells in maintaining hemopoiesis is well recognized, mechanisms that determine stem cell behaviour (e.g. self-renewal) have remained poorly defined. Historical precedent has illustrated the usefulness of in viitro colony assays in facilitating the investigation of various primitive hemopoietic progenitor classes with restricted differentiation and proliferative potential. In particular, such assays have made possible the definition of the sequential nature of a series of events that early erythropoietic cells undergo and suggested properties that might be anticipated to characterize colonies derived from stem cells proliferating and differentiating in vitro. The present studies were undertaken to test the hypothesis that very large, late maturing erythroid colonies previously noted on occasion in routine erythroid colony assays were, in fact, derived from progenitors with the self-renewal and multiple myeloid differentiation properties associated with stem cells. Initial experiments showed that cells capable of yielding macroscopic sized erythroid colonies were present at low frequency in normal marrow but became the predominant erythropoietic cell type after 2 to 3 weeks in flask culture. Macroscopic erythroid colony formation in assays of cells from either source were shown to have: 1) identical very late maturation kinetics (onset of hemoglobinization after 1 week of initial colony growth), 2) the same high erythropoietin requirements, and 3) similar responsiveness to factors present in media conditioned by mitogen stimulated spleen cells. Optimization of these 2 classes of stimulant yielded an assay that was linear down to very low cell concentrations (less than 5 x 103 cells/ml). This made possible the cytological analysis of macroscopic erythroid colonies under conditions where overlap with other colony types was minimal. Such studies revealed that macroscopic colonies contained, in addition to erythroid cells, megakaryocytes ( > 90% of colonies) and granulocyte cells (30% of colonies) including cells of the eosinophil lineage. Such colonies were also found to contain cells capable of macroscopic spleen colony formation in irradiated mice, the conventional assay for mouse hemopoietic stem cells (on average 1 CFU-S per colony of flask culture origin, and 0.3 CFU-S per colony in assays of fresh marrow). Direct evidence for self-renewal was obtained from replating experiments using irradiated feeders to optimize plating efficiency. Mixed colonies of macroscopic size were regularly demonstrable in replating assays after 1, and even 2, generations of mixed colony formation indicating up to 6 self-renewal divisions during colony formation. By comparison to flask culture cells, the extent of self-renewal exhibited by cells in fresh marrow yielding macroscopic erythroid colonies was found to be 5-fold lower. Finally, the in vitro expression of stem cell self-renewal behaviour was investigated in individual colonies. The number of stem cells generated, when assessed either by CFU-S or replating assays was found to vary markedly. This variation was not accounted for by errors of detection or by variations in colony size. Such findings are similar to previous data on the CFU-S content of individual spleen colonies and provide the first evidence that the type of variation in stem cell self-renewal observed in vivo also occurs in vitro where microenvironmental factors are unlikely to be contributing factors. These results are consistent with a model of stem cell self-renewal in which intrinsic cellular factors play the key role in influencing the decision of individual cells to self-renew. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Successful bone marrow (BM) transplantation requires the homing of the transplanted hematopoietic stem/progenitor cells (HSPCs) to their bone marrow niche, where they undergo differentiation to form mature cells that are eventually released into the peripheral blood. However, the survival rate of patients receiving BM transplants is poor since many of the transplanted HSPCs do not make it to their BM niches in the recipient’s body. Since the availability of HSPCs from traditional sources is limited, transplanting more number of HSPCs is not a solution to this problem. This study aims to characterize the adhesion molecules mediating cell migration in order to better understand the adhesion mechanisms of HSCs with the bone marrow endothelium. This will aid in developing future tools to improve the clinical transplantation of HSPCs. This study also aims to understand the factors that influence HSPC proliferation in the bone marrow niche. E-selectin plays an important role in the process of homing; however, its ligands on HSPCs are not well characterized. We used western blotting and immunoprecipitation to show that endomucin is expressed on HSPCs and plays a role in the binding of HSPCs to E-selectin. We also studied the effect of recombinant E-selectin on the expression of a newly characterized E-selectin ligand in our lab, CD34, in HSPCs. This will provide us insight into novel roles for endomucin and E-selectin and help us to understand the factors influencing HSPC migration to BM endothelium.

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For over 50 years, bone marrow transplants have used CD34 to select stem cells. Recent research suggests that the most primitive hematopoietic stem cells (HSCs), long-term HSCs (LT-HSCs), are found in the CD34-negative portion of murine and human bone marrow cells. LT-HSCs are rare and cannot be isolated directly, making them difficult to study. During a bone marrow transplant, these stem cells must find their way to the bone marrow niche and engraft to become blood cells. Several cell adhesion molecules on the stem cell engage with their ligands on the endothelial cells lining the bone marrow vasculature to control this migration. Human LT-HSCs cells do not migrate and engraft well when infused in vivo, which may be due to a lack of adhesion molecules. Thus, the goal of this study was to determine whether this population of HSCs lacked adhesion systems (proteins and carbohydrate modifications) and, if so, to improve their migration and engraft ability by modifying key mechanistic steps in the adhesion cascade. Therefore, we investigated how distinct hematopoietic stem cell populations migrate to the bone marrow using adhesion mechanisms. This study represents the first direct analysis of adhesion molecules expression in LT-HSC and will potentially shed light on methods to optimally use these very valuable cells in the clinical bone marrow and cord blood transplants worldwide.

Indiana University-Purdue University Indianapolis (IUPUI) / Hematopoietic stem and progenitor cells (HSCs/HPCs) transplantation is a curative treatment for a variety of hematologic and non-hematologic diseases. Successful HSC transplantation requires infusing patients with a sufficient number of long-term engrafting HSCs. As a result, research efforts have focused on optimizing the collection process. Previous work established that harvesting mouse bone marrow HSCs under low oxygen tension similar to that reported for the bone marrow niche in situ (physioxia), results in enhanced HSC recovery and function. However, collecting bone marrow cells under physioxia is not a clinically viable approach. Here, I demonstrated that the collection and processing of peripheral blood mobilized with G-CSF alone or G-CSF and Plerixafor under physioxia resulted in a greater number of phenotypically defined long-term engrafting HSCs. Using high-resolution single cell sequencing to explore the molecular programs governing HSCs under physioxia, I identified increased expression of genes involved in HSC self-renewal and maintenance. In contrast, HSCs under ambient air upregulated genes implicated in HSC differentiation, apoptosis, and inflammatory pathways. Furthermore, wild-type HSCs under physioxia revealed a significant reduction in gene expression and activity of the epigenetic modifier Tet2. Consequently, I evaluated the phenotyping, engraftment potential and gene expression of preleukemic Tet2-/- bone marrow cells under physioxia and ambient air. Unlike wild-type HSCs, Tet2-/- HSCs/HPCs were unresponsive to changes in oxygen tension. Notably, we observed similar phenotypes, functions, and self-renewal and quiescence gene expression in wild-type HSCs under physioxia and Tet2- /- HSCs under physioxia or ambient air. These findings imply that the preserved stemness and enhanced engraftment of HSCs under physioxia may in part be a result of Tet2 downregulation. Understanding the mechanisms regulating wild-type and preleukemic HSCs under physioxia will have therapeutic implications for optimizing HSC transplantation and mitigating the growth advantage of preleukemic stem cells. / 2022-12-15

Hematopoietic stem cells (HSCs) transplantations are widely used for the treatment of hematological and non-hematological disorders in clinic. Successful transplantation requires sufficient number and efficient homing of HSCs. Many studies have focused on developing an effective strategy to expand functional HSC population. Some regulatory molecules have been recently shown great promise for controlling the amplification of HSCs. In these dissertation studies, I first aim to identify gene(s) and their allelic variants contributing to strain-specific difference in HSC numbers between C57BL/6 (B6, low) and DBA/2 (D2, high) mice by using a classic forward genetic approach. Firstly, 3 quantitative trait loci (QTL) on chromosome (Chr) 3,5 and 18 were mapped by linkage analyses and confirmed in congenic mice. Secondly, Chr.3 QTL affected several HSC number-related biological processes. The D2 allele increased cycling and self-renewal whereas it decreased apoptotic rates of HSCs. Both actions conspired to increase HSC population size. Lastly, a small number of differentially-expressed genes was identified in Chr.3 congenic HSCs by a microarray-based candidate gene method, and the differential expression of one candidate, latexin, was found to relate to HSC number variations. Our studies report the strong evidence for the potential functions of latexin in HSC number regulation, and they are important for understanding molecular mechanisms of stem cell regulation and developing effective stem cell expansion strategies for clinical applications. In the second part of my studies, I studied homing and engraftment capabilities of HSCs. By using functional assays for progenitor and stem cells, I first reported the absolute homing efficiencies of murine young or old donor cells into young or old recipient mice. The results indicated that homing of primitive hematopoietic cells was not efficient and significantly decreased by aging of donors and recipients. The proliferation and differentiation states of HSCs were also impaired by homing itself, as well as by donors' and recipients age. Moreover, the hematopoietic reconstitution dynamics following transplantation were also affected by aging. Together, these findings will provide useful information for clinical applications especially when older individuals increasing serve as stem cell donors for elderly patients.

"Human umbilical cord blood (CB) hematopoietic stem cells (HSCs) have well established applications for cellular therapy. Current protocols for isolating HSCs from bone marrow or cord select for CD34 + cells, however some CD34 - populations have recently been shown to also contain strong HSC activity. Thus the positive selection of HSCs based on cell surface markers remains controversial. However, it is clear from the literature that differentiated hematopoietic cells (lineage positive, Lin + ), representing the vast majority (>90%) of most blood populations, contain no long-term reconstitution potential. Thus Viacell Inc. (Worcester, MA) expands and enriches its populations of cells containing HSCs by removing only those Lin + cells known not to contain HSCs. This is accomplished on two separation columns (post-sep-1, and post-sep-2) (separated by 7 days of cell growth) that contain a variety of antibodies to known differentiation surface markers. Although this process strongly enriches functional HCSs, these primitive cell populations remain biochemically uncharacterized. Because HSC populations containing long chromosomal telomeres and high telomerase activity (which helps maintain telomeres) have been shown to display the strongest long-term reconstitution potential, the purpose of this thesis was to investigate these two parameters in selected samples of Viacell’s ex vivo amplification procedure. Two specific hypotheses were tested: 1. the removal of Lin + cells will appear to increase the telomerase activity and telomere lengths in the remaining cell population, and 2. these two parameters will decrease upon hematopoietic cell differentiation and proliferation. Telomerase activity was assayed using a telomeric repeat amplication protocol (TRAP), and normalized relative to a cancer cell line positive control. Relative to fresh cord blood, telomerase activity was found to increase significantly in post-sep-1 (from 8.5 ± 1.5% to 76.2 ± 4.9%, p = 0.0001, n = 5) and post-sep-2 (8.5 ± 1.5% to 111.3 ± 4.9%, p = 0.0001, n = 5) fractions following the removal of Lin + cells. This increase was found to be highly reproducible, showing very low intra-cord and inter-cord variability. Telomere lengths were assayed using a telomere length assay (TLA). Relative to fresh cord blood, telomere lengths increased significantly in post-sep-1 (from 10 to 12 kb, n = 2) and post-sep- 2 (from 10 to 14 kb, p = 0.001, n = 2) fractions. These apparent increases likely result from the direct removal of cells low in telomerase activity with short telomeres since the Lin + cells from the post-sep-1 column were found to contain relatively low telomerase activity (32.1 ± 15%, p = 0.001, n = 2) and short telomeres (7.5 kb, p = 0.001), which supports our first hypothesis. Finally, we show that telomerase activity and telomere lengths decreased in Day-14 cells (expanded and differentiated 14 days) relative to post-sep-2 (from 111.8 ± 19.6% to 54 ± 21.2%, p = 0.001, n = 3 for the TRAP, and from 14 kb to 9 kb, p = 0.0001, n = 2 for the TLA). Those two parameters also decreased in pre-sep-3 cells (terminally differentiated by treatment with All Trans Retinoic Acid for 14 days) relative to post-sep-2 (from 111.3 ± 4.9% to 14.8 ± 1.7%, p = 0.0001, n = 6 for the TRAP, and from 14 kb to 7.5 kb, p = 0.001 for the TLA), supporting our second hypothesis. Telomerase activity was found to not directly correlate with CD34 + CD38 - content, supporting recent observations that a significant portion of HSCs reside outside this population."

Opportunistic infection is always a potentially life threatening complication in pediatric oncology patients and hematopoietic stem cell transplant recipients. With the advances in various disease treatment protocols, the overall and event-free survivals of this high risk population improve significantly. In this thesis, the author reported a number of original studies to discuss different strategies in prevention of this serious complication. Firstly, the author demonstrates that pediatric oncology patients are still vulnerable to various vaccine-preventable infectious diseases up to 18 months after stopping chemotherapy. For those vaccine-preventable infectious diseases, pediatric oncology patients can mount a significant and persistent immune response to common inactivated vaccine (namely diphtheria-tetanus-pertussis vaccine). For non-vaccine preventable infectious diseases, regular monitoring of plasma viral load and strategic use of antiviral agents as pre-emptive or prophylactic agent is an effective approach to prevent infection. In hematopoietic stem cell transplant setting, adoptive transfer of acquired immunity from donor to recipient and incorporation of this parameter in donor selection process can be considered. The findings of the studies can be applied to clinical setting. The future direction of our studies includes the immune responses of other common vaccines namely pneumococcal vaccine and pandemic influenza vaccine in high risk population. The role of transfer of donor's varicella zoster immunity in prevention of herpes zoster infection in transplant recipient can be further explored. With the advances in supportive care of our vulnerable patients, the survival rate is expected to be further improved in the future. / by Frankie Wai Tsoi, Cheng. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (M.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 193-208). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.