VictorJuiz/thesis huggingface dataset:
Controlled Generation of Hematopoietic Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) provide an exciting source for regenerative therapy because they have the potential to differentiate towards specialized tissues. However, in vitro generation of hPSC-derived cell lineages, such as definitive hematopoietic cells, remains challenging and typically only generates primitive blood cells. In the developing embryo, blood cell emergence is coordinated by spatial and temporal cues that include autocrine/paracrine signaling, oxygen tension, local cell density, and immobilized growth factors. We hypothesize that engineering aspects of the native definitive blood niche in vitro will enable robust generation of adult-like blood progenitor cells. To this end, we have investigated whether hPSC-derived hemogenic endothelial (HE) cells seeded into engineered niches of controlled size, distribution and composition could be optimized to promote differentiation towards phenotypical CD45+ blood cells, including definitive blood precursors. First, we used microwells to initiate hPSC differentiation as size-controlled cell aggregates in serum-free conditions to promote HE induction. These cells display multi-lineage differentiation capabilities (myeloid and lymphoid) and short-term engraftment. HE cells served as our starting population to study the effects of lithography-based micropatterned (MP) controlled culture parameters such as colony size, spacing and clustering on the endothelial-to-hematopoietic transition. MP treatments yield 5.5-fold greater enhancement in CD45+ progenitors compared to unpatterned treatments and demonstrate endogenous inhibitors at play during hematopoietic differentiation. We show that deficiencies in hematopoietic induction can be overcome via MP niches and link the induced interferon gamma protein (IP-10)/p-38 MAPK signaling pathway as a mechanism for hematopoietic inhibition. Using this platform, we identified interferon-gamma (IFN-Ę ), interleukin-3 (IL-3), VX-702 (p38 MAPK inhibitor) and Fasudil HCl (Rho-kinase inhibitor) as key modulators for definitive hematopoietic induction from hPSC-derived sources that confer long-term splenic engraftment in immunocompromised mice. This demonstrates that in vitro niche engineering can mimic in vivo embryonic development and provides a model to investigate blood cell emergence.

Keywords: Hematopoiesis
Hematopoietic Stem Cells
Human Embryonic Stem Cells
Micropatterning
Niche Engineering
T-cells
Advisor: Zandstra, Peter W
The role of NKR-P1:Clr family interactions in murine cytomegalovirus infection

Natural killer (NK) cells play a key role in innate immunity by detecting alterations in self and non-self ligands via paired NK cell receptors (NKR). Here, an MHC-I-independent NK cell recognition mechanism that involves modulation of NKR-P1:Clr receptor-ligand function upon mouse cytomegalovirus (MCMV) infection is characterized. Expression of host “self” Clr-b ligand is rapidly lost on MCMV-infected cells, invoking “missing-self” NK recognition. Cross-species viral infections using rodent fibroblasts suggest this response is evolutionarily conserved. Active viral infection is necessary for Clr-b loss, as inactivated virus and innate signalling agonists fail to elicit Clr-b downregulation. Clr-b loss is partially blocked by cycloheximide, suggesting early viral or nascent host proteins are required for Clr-b downregulation. Further investigation using CD3/NKR-P1 reporter cell assays identified a novel Clr-b-independent MCMV-encoded decoy immunoevasin, m12, that directly engages the paired NKR-P1A/B/C receptor clade. Thus, MCMV m12 is the first physiological ligand for the prototypical NK1.1 orphan receptors (NKR-P1B/C). MCMV m12 restrains NK effector function by directly engaging the inhibitory NKR-P1B receptor. However, m12 also interacts with the activating NKR-P1A/C receptors to counterbalance m12 decoy function. Independent structural analyses reveal that m12 is IgV-like and sequesters a large surface area of the C-type lectin-like NKR-P1 protein via a "polar claw" docking mechanism. Polymorphisms in, and ablation of, the viral m12 protein and host NKR-P1B/C alleles impact NK cell responses in vivo. This work identifies the long-sought foreign ligand for this key immunoregulatory NKR family and reveal how it controls the evolutionary balance of immune recognition during host-pathogen interplay. Additional work provides evidence that the MCMV m153 immunoevasin stabilizes host Clr-b surface levels. In contrast, infections using m153-deficient MCMV mutants show exacerbated Clr-b downregulation. Importantly, enhanced Clr-b loss by MCMV m153-mutant infection is reversed by exogenous m153 complementation. Intracellular m153 localization suggests the Clr-b stabilization effect may be indirect. This is confirmed by m153 tetramer staining of dendritic cells, and in vivo analyses showing that m153 MCMV exhibits enhanced virulence, in an Nkrp1b/Clrb-independent manner. Thus, m153 may bind an activating immunoreceptor. Together, these data demonstrate that the NKR-P1:Clr recognition axis is subverted by multiple MCMV immune evasion strategies.

Keywords: Cytomegalovirus
Innate immunity
Missing-self
Natural killer cells
NK1.1
Nkrp1-Clr
Advisor: Carlyle, James R.
The Paromomyidae (Primates, Mammalia): Systematics, Evolution, and Ecology

Plesiadapiforms represent the first radiation of Primates, appearing near the Cretaceous-Paleogene boundary. Eleven families of plesiadapiforms are recognized, including the Paromomyidae. Questions surrounding this family explored in this thesis include its pattern of extinction, its phylogeny and migration, and its dietary ecology. Firstly, there is a record of misclassifying small-sized omomyoid euprimates as late-occurring paromomyid plesiadapiforms. Here, a new omomyoid from the Uintan of California is described. This material was previously thought to pertain to a paromomyid, similar to previously named supposed paromomyids Phenacolemur shifrae and Ignacius mcgrewi. The new Californian species, Ph. shifrae, and I. mcgrewi are transferred to Trogolemurini, a tribe of omomyoids. The implications of this taxonomic change are that no paromomyids are found between the early Bridgerian and the Chadronian, suggesting that the group suffered near-extinction during a period of particularly warm climate. Secondly, migration of paromomyids between North America and Europe is poorly understood. The European material (genus Arcius) is taxonomically reassessed, including emended diagnoses for all four previously named species, and description of two new Arcius species. A cladistic analysis of the European paromomyids resolves Arcius as monophyletic, implying that the European radiation of paromomyids was a product of a single migration event from North America. Finally, paromomyid diet has only been assessed qualitatively in previous literature. Dental topographic metrics are used to assess diet in paromomyids and observe patterns of evolutionary change in the P4. The results for P4 are consistent with previous literature for inferences about changes within given lineages, but comparisons across different lineages is challenging. The results for M2 suggest a more varied diet that previously thought for paromomyids. The results presented here show a general trend from more insect intake in primitive members to a more frugivorous diet in more derived and/or later occurring members of various lineages.

Keywords: Ecology
Evolution
Mammalia
Paromomyidae
Primates
Systematics
Advisor: Silcox, Mary T
Balancing Tensions in Regulatory Reform: Changes to Regulation of Health Professions in Australia, the United Kingdom, and Ontario, Canada

Regulation of health professions is a dynamic field that continues to cause strife among policy, government, and regulatory actors. At the heart of these debates is the question of how to balance the potentially conflicting policy tensions in the regulatory agenda: professional versus public interests, transparency versus privacy, and accountability versus autonomy. In recent years, the balance of these tensions has shifted in many jurisdictions through legislative reform, impacting the relationship between governments and health profession regulators. This changing balance and the factors and forces behind it were examined using a qualitative multiple case study research design of Australia, the United Kingdom, and Ontario, Canada. In Australia, the struggle between striving for national uniformity while still achieving adequate accountability was dominant after the enactment of a nationally coordinated regulatory scheme and subsequent state-based deviations. In the United Kingdom, the balance between autonomy for regulators and legislated consistency was contentious following the creation of a powerful meta-regulator and the elimination of elected professional majorities on regulatory bodies. Reform in Ontario was more incremental, with continued adherence to elements of traditional self-regulation, including professional majorities on governing councils of regulators and considerable delegated power for these professional regulators. Despite this, the nature of health profession self-regulation in Ontario has been altered through enhanced government oversight powers, heightened accountability and transparency requirements, and constrained discretion for regulatory bodies. The level of autonomy and freedom from state intrusion that self-regulated health professions previously enjoyed are unlikely to be seen in Ontario again. It is clear there is no panacea for perceived problems in regulation of the health professions. Reform agendas were coloured by the modern political, legal, and social context of each jurisdiction. These findings help elucidate the different policy tensions that have informed the reform. Certain values are gaining greater traction in each regulatory framework, including the importance of transparency and collaboration, the need to balance regulatory force to ensure economic viability, the utility of articulating regulatory principles, and the necessity of sustaining governmental and societal confidence in the ability to do what regulation of health professions is supposed to: protect the public.

Keywords: case study
health law
health professions
legislation
policy
regulation
Advisor: Nelson, Sioban
The Influence of Attachment Phenomena on Alcohol and Tobacco Use

Alcohol and tobacco use are leading causes of preventable death. Studying novel determinants of substance use may inform prevention and cessation. Attachment insecurity (attachment avoidance and attachment anxiety) describes interactions between adults and their attachment figures, which may influence substance use. This thesis assessed how attachment may be relevant in understanding and addressing substance use. Study 1, a cross-sectional survey study of 348 adult primary care patients, tested hypothesized associations between childhood adversity, attachment insecurity and substance use; if attachment insecurity could mediate between childhood adversity and substance use; and hypothesized sex differences in these associations. In Study 2, 38 smokers participated in experiments designed to assess the influence of cigarette cues and an attachment figure photo on craving and affect. In Study 1, 61% of participants reported childhood adversity, 11% reported harmful drinking and 20% reported current smoking. Attachment anxiety was associated with harmful drinking in women and men. Attachment anxiety was a statistical mediator between childhood adversity and harmful drinking (95% CI 0.09 to 0.30). Sex did not moderate the relationship between attachment anxiety and hazardous drinking in this mediation model. In women, attachment anxiety was associated with both childhood adversity (p=.002) and current smoking (p=.04), and was a statistical mediator between childhood adversity and smoking (95% CI 0.003 to 0.236). In Study 2, 56% of participants reported decreased craving after seeing an attachment figure photo (Neutral photo = 50.9 (SD 29.8), Attachment figure photo = 38.2 (SD 31.6), t(35)= -2.661, p=0.01). Cigarette cues (d=.50) and the attachment figure photo (d=.42) had similar effect sizes. Cigarette cue reactivity and attachment figure smoking status did not influence responses to photo exposures. Attachment anxiety was associated with alcohol and tobacco use, and may mediate between childhood adversity and substance use. Viewing an attachment figure photo decreased craving in smokers, with an effect that is comparable to the increase caused by cigarette cues. These studies indicate that attachment phenomena may play a role in substance use. Childhood adversity, attachment anxiety, and the presence of an attachment figure are factors to consider in the design of prevention and cessation strategies.

Keywords: alcohol
attachment security
child abuse
tobacco
Advisor: Maunder, Robert G
Functional Divergence of Glioblastoma Stem Cells

Genetic heterogeneity in cancer is well-established using genomic sequencing, while functional assays of prospectively isolated cancer subsets have demonstrated the existence of developmental programs in many hematological and solid malignancies. CD133 expressing cells have a unique propensity for initiating and sustaining hierarchical growth in human glioblastoma, a property analogous to that of neural stem cells. However, it remains unclear whether putative cancer stem cells exhibit further intratumoral variations in functional properties as a consequence of genetic or epigenetic heterogeneity. Here, we describe two complementary approaches to characterize functional intratumoral heterogeneity of human glioblastoma within the glioma stem cell (GSC) compartment at single cell resolution. In the first approach, we generated cohorts of single-cell derived stem cell cultures from primary glioblastoma and subjected them to in depth functional analyses. We demonstrate that multiple clones derived from the same GBM are capable of tumour initiation, yet differ in key functional properties such as proliferation, differentiation, and response to temozolomide chemotherapy. Genetic mutations unique to each clone could also be applied to reconstruct tumour phylogeny, indicating that the self-renewing compartment is reflective of complex tumour evolutionary histories. In the second approach, we labelled primary GBM cells with permanent genetic barcodes for the characterization of their functional properties in vivo. Using this approach, we demonstrated that self-renewal is restricted to a subset of GBM xenograft forming cells thereby confirming the maintenance of a stable developmental hierarchy within human GBMs at initial presentation. We also show that functional divergence of GBM clones emerges over time as a small subset of clones become more proliferative, and that intrinsically chemotherapy resistant clones may be selected for by frontline therapies. Furthermore, we show differential targeting of proliferative and relatively non-proliferative clones within the same tumour using epigenetic compounds. Our findings demonstrate the relevance of clonal heterogeneity in GBM pathogenesis, and suggest that complete elimination of all GSCs is possible using a combination of epigenetic compounds.

Keywords: Cancer Heterogeneity
Cancer Stem Cell
Glioblastoma
Advisor: Dirks, Peter B
On-Chip Jitter Measurement and Mitigation Techniques for Clock and Data Recovery Circuits

This thesis describes three contributions in the area of on-chip jitter measurement and characterization, which can be used to help optimize the performance of wireline transceivers. Two on-chip jitter measurement techniques are developed and demonstrated, along with an adaptive loop gain CDR, which characterizes jitter on-chip to optimize its jitter tolerance. In the first measurement technique, the absolute jitter of random data is measured on-chip by correlating the phase detector outputs of two 10Gb/s CDRs locked to the same data. This technique allows the jitter's autocorrelation function to be estimated, from which the jitter's RMS value and power spectral density are extracted without using any external reference clock. Correlating the phase detectors in the CDRs with a third phase detector, which measures the phase difference between the clocks recovered by the two CDRs, allows measurement of the absolute recovered clock jitter. A test chip fabricated in 65nm CMOS demonstrates that this scheme can measure RMS jitter with sub-picosecond accuracy. To demonstrate the usefulness of on-chip jitter characterization in improving CDR performance, a loop gain adaptation strategy is proposed, which optimizes the jitter tolerance of a 28Gb/s PI-based CDR. The technique increases the CDR's loop gain to suppress the most jitter while monitoring the autocorrelation function of the bang-bang PD output to prevent the CDR from becoming too underdamped. The proposed technique requires no prior knowledge of the CDR's latency or jitter characteristics and can also be extended to operate in the presence of sinusoidal jitter. The concept is demonstrated in a test chip fabricated in 28nm CMOS. Lastly, a second jitter measurement technique is proposed, which estimates the relative jitter between the input data and recovered clock of a 28Gb/s half-rate digital PI-based CDR without using an eye monitor. Square wave jitter with a known amplitude is injected into the CDR, by adding a corresponding signal to the CDR's PI code. By measuring the effect of the injected jitter on the autocorrelation function of the CDR's bang-bang PD output, the RMS relative jitter is estimated with sub-picosecond accuracy. The scheme is demonstrated in the same 28nm test chip described above.

Keywords: adaptive cdr
cdr
clock and data recovery
jitter
jitter measurement
wireline
Advisor: Sheikholeslami, Ali
Hypoxia Promotes Stem Cell Phenotypes, Resistance to Therapy and Poor Prognosis through Epigenetic Regulation of DICER

Hypoxia is a common characteristic of solid tumors, associated with aggressive disease and poor prognosis in multiple sites, including breast and cervix cancer. These properties are attributed to the influence of hypoxia on treatment resistance to both radiotherapy and chemotherapy as well as biological responses that promote altered tumor biology. Extensive research has led to a good understanding of the mechanisms of cellular adaptation to hypoxia, and regulation of metabolic and angiogenic phenotypes. However, considerably less is known regarding mechanisms responsible for the influence of hypoxia on metastasis and stemness. In a search to identify novel genes that contribute to the cellular response during hypoxia, we discovered that hypoxia represses DICER, a key enzyme required for generation of mature miRNA. DICER is a known haplo-insufficient tumor suppressor that can stimulate cancer development through a reduction in miRNA generation. We demonstrate that tumor hypoxia is a major regulator of DICER expression in large cohorts of breast cancer patients and that DICER expression is suppressed by hypoxia to levels similar to that in tumors with monoallelic DICER loss. We also demonstrate that DICER repression occurs through a novel epigenetic mechanism requiring inhibition of the oxygen-dependent H3K27me3 demethylases KDM6A/B. Hypoxic suppression of DICER creates a miRNA processing defect and results in selective reduction of mature levels of the miR-200 family. Consequently, hypoxia leads to derepression of the miR-200 target ZEB1, stimulates the epithelial to mesenchymal transition and results in acquisition of stem cell phenotypes in human mammary epithelial cells. Finally, we demonstrate the importance of reduced DICER in mediating therapeutic response to fractionated radiation treatment in vivo. Our work suggests that in breast cancer the acquisition of EMT and stem cell properties, through epigenetic repression of DICER and loss of miR-200, may drive the known association of hypoxia and poor clinical outcome.

Keywords: DICER
EMT/stemness
epigenetics
hypoxia
miRNA
tumor microenvironment
Advisor: Wouters, Bradly G
Surface Modification of Commonly used Polymer Plastics for Increased Hemocompatibility in Medical Equipment

Exposure of synthetic materials to body fluids may result in undesirable protein−material interactions, this can potentially trigger deleterious biological processes such as thrombosis. The result is increased chances of heart attack, stroke or other cardiovascular complication after extravascular blood procedure. For our experiment, we have chosen a wide variety of materials used in making perfusion equipment components: polyethylene terephthalate (PET), polycarbonate (PC), polyvinyl chloride (PVC), polypropylene (PP), poly (methyl methacrylate) (PMMA), polyurethane (PU), polysulfone (PS). All materials had MEG-OH adlayer formed and characterized with x-ray photoelectron spectroscopy, attenuated total internal reflection and contact angle goniometry. Several of the materials (PVC, PC, and PS) were tested directly for their compatibility with whole human blood. Antithrombogenicity was assessed after 2,5,10 and 60 min, 3 hours, 6 hours, and 3 days exposure to whole human blood dispensed at a shear rate of 1, 300, 900, 1000 and 1500 s−1. Most MEG-OH covered plastics had >90% less platelet adhesion than bare plastics, the result was repeated on all exposure times and shear rates. Surface protein composition was analyzed by SDS–PAGE. First trails using MEG-OH on PVC as an implant material for cranial windows on rats decreased the amount of fouling comparable to bare PVC. Overall the MEG-OH proved to be robust on a wide variety of materials with very high potential in blood compatibility.

Keywords: Biomedical
Blood
Coating
Plastics
Polymer
Surface Chemistry
Advisor: Thompson, Michael