The occurrence of D. weischeri Chizhov, Borisov & Subbotin, a newly described stem nematode species of creeping thistle (Cirsium arvense L.), and D. dipsaci (Kühn) Filipjev, a pest of garlic and quarantine parasitic species of many crops, has been reported in Canada. This research was conducted to determine if D. weischeri is a pest of agricultural crops, especially yellow pea (Pisum sativum L.) in the Canadian Prairies. Significant (P < 0.05) slight reproduction (1 < ratio of final to initial population < 2) of D. weischeri occurred on two (Agassiz and Golden) of five varieties of yellow pea examined. Other annual pulse and non-pulse crops, including common bean, chickpea, lentil, spring wheat, canola, and garlic were non-hosts for D. weischeri. Conversely, a range of reproduction responses to D. dipsaci was observed with all pulse crops being a host of the nematode. Ditylenchus weischeri was not a seed-borne parasite of yellow pea, unlike, D. dipsaci which was recovered from seed. Conversely, D. weischeri and not D. dipsaci was recovered from creeping thistle seeds. In callused carrot disks, with no addition of medium, an increase of 54 and 244 times the addition density of 80 nematodes was obtained for D. weischeri and D. dipsaci, respectively, after 90 days. Temperature had a significant influence on the development of D. weischeri and D. dipsaci in yellow pea. Development of D. weischeri did not proceed past adult stage at 17 and 22°C whereas a minimum generation time of 30 days was apparent at 27°C with the associated accumulated growing degree-days of 720 degree-days (above a base temperature of 3°C). The minimum generation time for D. dipsaci was 24, 18 and 22 days with 336, 342 and 528 degree-days at 17, 22 and 27°C, respectively. In field microplots, grain yield of yellow pea were not significantly affected by addition density of D. weischeri. At harvest, the total number of recovered nematodes per plant was not significantly different than the added at the start. The results of these studies confirm that D. weischeri is unlikely to be a pest of yellow pea for weather conditions of the Canadian Prairies. / October 2016

In allogeneic stem cell mobilization, peripheral blood stem cell mobilization with filgrastim can be considered standard of care. Poor mobilizers may be at risk for inadequate stem cell collection during apheresis. He we present a successful case of salvage plerixafor use with filgrastim in a haploidentical identical transplant patient.

Latinos are the largest minority group in the U.S.; however despite the continuous growth of the Hispanic population, Latinos are severely underrepresented in STEM fields. One of the reasons that might explain why Latinos do not major in STEM is the way they encounter science curriculum in primary school. Students' limited proficiency in English may constrain their science achievement when instruction is delivered exclusively in English. A quantitative analysis with graduation rates in STEM from 2009 to 2014 at the University of North Texas was conducted, finding that there is a significant difference (p<0.05) in the number of bachelor's degrees in STEM between Hispanic, White, African American and other student populations. Interviews with teachers, librarians and publishing companies were performed to describe the limited science literature in Spanish at the Dallas ISD schools. Improving science literacy by teaching according to ELLs' linguistic skills and culture may lead to a better understanding of science curriculum throughout their education, which may translate into higher college graduation rates by Hispanic recipients in STEM.

Multiple sclerosis (MS) is a leading cause of neurological disability in young adults. Although current treatments can reduce symptomology and relapse rate, they are unable to prevent the chronic neurodegeneration that occurs at later stages. MS pathology is mediated by complex interactions between invading immune cells, neurons, glia, and endogenous stores of neural progenitor cells (NPCs). Factors critical to NPC/immune cell communication as well as the survival, differentiation, and proliferation of NPCs are not well defined. Elucidation of these factors will allow for the advancement of NPC transplantation therapies as well as the identification of novel pharmacological targets. Fas – a member of the tumor necrosis superfamily of death receptors – has diverse, cell-specific functions and is a major modulator of autoregulation within the immune system. Although Fas is expressed by NPCs, its exact role in this cell type was previously unknown. To contribute to this body of knowledge, the experiments in this dissertation examined the role of the Fas receptor (Fas) and Fas ligand (FasL) in NPC survival, differentiation, and T-cell cross-talk in vitro and in vivo in experimental autoimmune encephalomyelitis (EAE; a well-established animal model of MS). Activation of Fas via FasL increased NPC survival by decreasing apoptosis (as opposed to increasing proliferation) in vitro. This decreased apoptosis correlates with upregulation of the inhibitor of apoptosis protein (IAP) Birc3. Further investigation into the importance of Fas in NPCs was accomplished by comparing wild-type and Fas-deficient (lpr) NPCs. Lpr NPCs exhibited decreased apoptosis, decreased proliferation, and increased differentiation to oligoprogenitor and neuronal lineages. These studies suggest the Fas system plays multifaceted roles in NPCs and that its exact functions are dependent on both functional Fas expression and presence or absence of FasL. To determine the role of Fas/FasL in neuroimmune cross-talk, co-cultures of wild-type or lpr NPCs with different T-cell subtypes (Th1, Th2, and Th17 cells) were performed. Th1 cells were the only subtype capable of inducing NPC apoptosis. Th1-mediated death was dose-dependent and was not mediated via Fas. On the other hand, NPCs were able to induce significant apoptosis in pro-inflammatory Th1 and Th17 cells without affecting anti-inflammatory Th2 cells. NPC-induced Th17 cell death was mediated via Fas. These data suggest NPCs can specifically target pro-inflammatory T-cells and can promote neuroprotection by inducing death of these proencephalogenic cells. Finally, intravenous injection of wild-type or lpr NPCs into EAE mice reduced clinical symptoms and CNS immune infiltrate to the same extent. Few NPCs enter the CNS, where they remain undifferentiated. This suggests the main mechanism through which NPCs produce beneficial results in EAE is via peripheral immunoregulation, which is not dependent on Fas expression. Overall, this dissertation elucidates the Fas system as an important modulator of NPC cell-fate and immunoregulatory capacity.

Understanding the reasoning behind the low enrollment and retention rates of Underrepresented Minority (URM) students (African Americans, Hispanic Americans, and Native Americans) in the disciplines of science, technology, engineering, and mathematics (STEM) has concerned many researchers for decades. Numerous studies have used traditional statistical methods to identify factors that affect and predict student retention. Recently, researchers have relied on using data mining techniques for modeling student retention in higher education [1]. This research has used neural networks for performance modeling in order to obtain an adequate understanding of factors related to first year academic success and retention of URM at Virginia Commonwealth University. This research used feed forward back-propagation architecture for modeling. The student retention model was developed based on fall to fall retention in STEM majors. The overall freshman year GPA was used to model student academic success. Each model was built in two different ways: the first was built using all available student inputs, and the second using an optimized subset of student inputs. The optimized subset of the most relevant features that comes with the student, such as demographic attributes, high school rank, and SAT test scores was formed using genetic algorithms. A further step towards understanding the retention of URM groups in STEM fields was taken by conducting a series of focus groups with participants of an intervention program at VCU. Focus groups were designed to elicit responses from participants for identifying factors that affect their retention the most and provide more knowledge about their first year experiences, academically and socially. Results of the genetic algorithm and focus groups were incorporated into building a hybrid model using the most relevant student inputs. The developed hybrid model is shown to be a valuable tool in analyzing and predicting student academic success and retention. In particular, we have shown that identifying the most relevant student inputs from the student’s perspective can be incorporated with quantitative methodologies to build a tool that can be used and interpreted effectively by people who are related to the field of STEM retention and education. Further, the hybrid model performed comparable to the model developed using the optimized set of inputs that resulted from the genetic algorithm. The GPA prediction hybrid model was tested to determine how well it would predict the GPA for all students, majority students and URM students. The root mean squared error (RMSE) on a 4.0 scale was 0.45 for all students, 0.47 for majority students, and 0.45 for URM students. The hybrid retention model was able to predict student retention correctly for 74% of all students, 79% of majority students and 60% of URM students. The hybrid model’s accuracy was increased 3% compared to the model which used the optimized set of inputs.

In human pluripotent stem cell (hPSC) research and applications, the need for a culture system devoid of non-human components is crucial. Such a system should exhibit characteristics observed in conventional culture systems that have used mouse embryonic fibroblast feeders for hPSC self renewal without the requirement of excessive supplementation with growth factors. To achieve this, we focused on the identification and characterization of extracellular matrix (ECM) substrates for hPSC propagation. ECM substrates derived from mouse and human fibroblasts were assessed for their ability to support self-renewal of hPSCs. Characterization of hPSCs on ECM-based substrates demonstrated maintenance of pluripotent characteristics based on a) high nuclear-cytoplasmic ratio b) immunocytochemical analyses for pluripotent markers (Alkaline phosphatase, AP, Octamer Binding Transcription Factor-4, OCT4 and Specific surface embryonic antigen-4, SSEA4) c) in vitro differentiation potential by embryoid body formation d) Real time RT-PCR analysis for pluripotent and germ-layer specific markers and e) karyotype analysis for chromosome number. Compositional characterization of the ECM substrates using proteomic analysis identified some of the major constituents of the matrix that might contribute to hPSC self-renewal. Based on results from the proteomic analysis, combinatorial ECM substrates were formulated using commercially available proteins and evaluated for applicability in hPSC propagation. Extensive characterization of hPSC propagated on the ECM substrates suggest that a combination of heparan sulfate proteoglycan and fibronectin was sufficient for the promoting hPSC sef-renewal. Finally, an in-direct co-culture system utilizing microporous membranes coated with acellular substrates and a physically separated feeder layer was developed as a microenvironment for hPSC propagation. Real time conditioning of the growth medium and an ECM-based substrate for hPSC adhesion provides a synergy of the biochemical and biophysical cues necessary for hPSC self-renewal. hPSCs cultured in this system demonstrated equivalent pluripotent characteristics as those propagated in conventional culture systems, and provided opportunities for scale up without cell mixing. Overall, these studies could prove to be useful in the development of humanized propagation systems for the production of stable hPSCs and its derivatives for research and therapeutic applications.

Melanoma is the most aggressive skin cancer with high recurrence and low survival rate. In addition to genetic mechanisms, resistance also arises from phenotypic heterogeneity in which a proportion of cells, the so-called melanoma stem or initiating cells, survive therapy. Due to a lack of reliable markers, however, there is still debate about the existence of these cells in melanoma. Consistent with phenotypic heterogeneity, previous observations in our laboratory have demonstrated that cells in melanoma can reversibly segregate in vivo into different subpopulations with different properties, such as differentiation or increased invasive capacity (potentially attributed to the existence of de-differentiated stem-like cells). To characterise these cells, a dual reporter lentiviral system was engineered, expressing fluorescent proteins under cell stage/phenotype-specific promoters. The promoters for the transcription factors POU3F2 (Brn2) (to mark de-differentiated cells) and the microphthalmia-associated transcription factor (MITF) (to mark proliferating and differentiated cells) were chosen. Lentivirally-transduced cells were used to screen a library of kinase inhibitors for their potential to affect promoter activity in vitro. The RhoA/ROCK pathway, known to contribute to invasion and metastases, was identified to play a role in Brn2 promoter activity and exhibited differential effects on both the MITF and Brn2 promoters in 501mel and SKmel28 cell lines. Through investigation of other signalling pathways involved in melanoma metastasis, we also identified the co-activator Mastermind-like 1 (MAML1), previously reported to act in the Notch pathway, as an activator of the Brn2 promoter via the transcription factor TCF3, and the MITF promoter through the lymphoid-enhancer binding factor 1 (LEF1). The effects of MAML1 on Brn2 and MITF promoter activity were potentiated by &beta;-catenin. These findings provide new opportunities for the identification of therapeutic targets to prevent metastases formation in melanoma.

The cost of drug development is high with many drugs failing during toxicity testing. This is a particular problem in veterinary medicine where the pharmaceutical market size is so small that it may not be economically viable to develop drugs. The liver and specifically hepatocytes have a crucial role in drug metabolism via oxidation by cytochrome enzymes (CYP), conjugation and excretion into the biliary system. This drug metabolism is unpredictable between species as each has unique CYP profiles. Furthermore there is breed variation of CYP profiles within the canine species. The ability to produce an in vitro source of canine hepatocytes to model drug metabolism in this species and in different breeds would greatly reduce the expense of candidate drug testing. If an unlimited supply could be produced in vitro this would reduce the number of animals required in pre-clinical testing. The aim of this thesis was to produce an in vitro supply of canine hepatocyte-like cells from stem cell sources, namely hepatic progenitor cells (HPC), mesenchymal stem cells (MSC) or induced pluripotent stem cells (iPSC). Cultures of canine primary hepatocytes were produced to use as a gold standard, but also to develop and refine tests of hepatocyte characterisation and function. A panel of primers was developed for use in real time polymerase chain reaction (PCR) as well as optimising tests for low density lipoprotein (LDL) and indocyanine green uptake, albumin production, periodic acid- Schiff staining for glycogen and CYP activity using a luciferase-based system. As primary hepatocytes rapidly lost their defining characteristics and function in vitro, methods of maintaining function using CYP inducers and culture substrates were assessed. Isodensity centrifugation and magnetic-activated cell sorting was employed to isolate HPCs. Selection of cells from the non-parenchymal cell fraction with stem cell marker Prominin 1 demonstrated that these were keratin 7 positive, a HPC marker. Cells morphologically consistent with HPC appeared and expanded in culture after 2 weeks. On passaging, these cells failed to continue expanding, despite plating onto collagen, laminin, SNL feeder cells or using Kubota’s medium (known to allow rapid expansion of rodent and human HPCs). Canine adipose (Ad-MSC) and bone marrow-derived mesenchymal stromal cells (BM-MSC) were isolated post mortem. These were characterised as CD45, 105 and STRO-1 positive, CD11b, 19 and 45 negative cells which could be differentiated into adipocytes, chondrocytes and osteocytes based on staining characteristics and relative gene expression. Protocols published for other species were used to differentiate both Ad-MSC and BM-MSC towards a hepatocyte phenotype. Although a dramatic change in morphology and a reduction in vimentin gene expression were noted, suggesting a loss of mesenchymal phenotype, these protocols did not induce a hepatocyte phenotype. Pre-treatment with 5-Aza-2′-deoxycytidine to cause DNA demethylation and valproic acid to inhibit histone deacetylation also failed to allow transdifferentiation. A polycistronic vector containing Oct-4, c-Myc, Sox2 and Klf4 was successfully transfected into canine epidermal keratinocyte progenitor cells which became alkaline phosphatase positive and assumed a morphology consistent with iPSC. After colony selection and expansion, PCR evidence of plasmid presence was lost, colony morphology changed, and alkaline phosphatase activity reduced, consistent with vector expression factor and pluripotency loss. Canine iPSCs produced by lentiviral method were then differentiated towards hepatocyte phenotype using a published protocol for mouse and human iPSC. These cells were then assessed for hepatocyte characteristics using the developed reagents and primers. These cells demonstrated increased gene expression and morphology consistent with differentiation towards a hepatocyte-like phenotype. This thesis demonstrates successful culture of canine primary hepatocytes and validation of tests of hepatocyte phenotype. This provides a basis for optimising primary hepatocyte function in vitro and assessment of the success of differentiation protocols on stem cell sources. Canine mesenchymal stromal cells do not appear to transdifferentiate towards a hepatocyte-like phenotype using published protocols for other species. Canine iPSC are a promising candidate for an in-vitro source of hepatocyte-like cells.

This thesis describes for the first time, how the membrane capacitance of pluripotent human embryonic stem cells (H1, H9, RCM1) increases with their differentiation (H1-MSC, H9-MSC, RCM1-trophoblast) based on the literature review. The method used to determine membrane capacitance was dielectrophoresis (DEP), which is an electrokinetic technique capable of characterising and sorting cells without the need for antibody-based cell surface markers, magnetic beads, or other chemical tags. This finding has potential biomedical importance because human embryonic stem cell (hESCs) isolated from early blastocyst-stage embryos and differentiated progeny have been identified to be of possible use in drug screening and regenerative cell based therapeutic treatment. Current cell sorting methods require membrane surface markers that limit their applicability in stem cell therapeutics, a limitation that is either removed or reduced if DEP-based sorting was used. The work described in this thesis consists of the design, fabrication and testing of DEP based microfluidic devices for characterization and separation of human embryonic stem cells. The cells studied were human undifferentiated hESC lines (H1, H9, RCM1, RH1, and T8) and their differentiated progeny (H1-MSC, H9-MSC, RCM1-trophoblast, hES-MP). The cell membrane capacitance (Cm) of the cells was determined by measuring a parameter known as the DEP cross-over frequency (fxo), where the electrical polarisability of a cell equals that of its suspending electrolyte and so experiences no DEP force. The studies of hESC lines cultured from different sources indicate, on the basis of their similar Cm values, that they have similar membrane morphologies. The change in calculated Cm value upon differentiation of these hESCs indicates that changes occur in their membrane morphology, texturing and possibly of their membrane thickness. Subsequent enrichment of these hESCs from human dermal fibroblasts (hDFs) has been achieved based on fxo measurements. The results presented in this thesis confirm the existence, previously indicated in the literature, of distinctive parameters for undifferentiated and differentiating cells on which future application of DEP in hESC manufacturing can be based.

Pluripotent embryonic stem (ES) cells are heterogeneous mixtures of naïve and lineage-primed states defined by distinct transcription factor expression profiles. However, the events that prime pluripotent cells for differentiation are not well understood. Id proteins, which are inhibitors of basic helix-loop-helix (bHLH) transcription factors, contribute to pluripotency by blocking differentiation. Using Yeast-Two-Hybrid screening, our lab identified Tcf15 as an Id-regulated transcription factor. In this study, I first examined the expression of Tcf15 during differentiation in vitro and during early development in vivo in the mouse. Tcf15 expression is higher in primed pluripotent embryonic stem (ES) cells than in naïve ES cells or epiblast stem cells (EpiSCs). In addition, Tcf15 is expressed heterogeneously in ES cells and is also detected in the inner cell mass (ICM) of E4.5 mouse embryos. Expression of Tcf15 was upregulated during early stages of differentiation and downregulated before cells committed to any specific lineage. Using Tcf15-Venus reporter cells, I found that expression of Tcf15 is specifically associated with a novel subpopulation of ES cells primed for somatic lineages. Gain of function and loss of function studies were then performed to perturb Tcf15 expression in ES cells in order to assess the function of Tcf15 in self-renewal and during differentiation. An inducible Id-resistant form of Tcf15 accelerates somatic lineage commitment by maturating naïve pluripotent ES cells transit toward primed epiblast and later on epiblastderived somatic lineages whilst suppressing differentiation towards extraembryonic endoderm. Preliminary loss of function studies also suggest that down-regulation of Tcf15 may promote a naïve state within pluripotent cells. I investigated the mechanism by which Tcf15 expression becomes associated with the epiblast-primed state by identifying the upstream regulators and downstream targets of Tcf15. Tcf15 expression is dependent on FGF signalling. Microarray analysis identified that Tcf15 downregulates the naïve pluripotency determinant Nanog and upregulates the epiblast determinant Otx2. Taken together, our results suggest that Tcf15 acts in opposition to the pluripotency network to prime pluripotent cells towards differentiation.