VictorJuiz/thesis huggingface dataset:
Role of the Src-like Adaptor Proteins, SLAP and SLAP-2, in Dendritic Cell Development and SIgnaling

Dendritic cells (DC) are critical to an immune response by functioning as sensors of foreign antigen and presenting antigen to direct T cell responses. Under the influence of Flt3 ligand this heterogeneous group of cells originate from hematopoietic stem cells (HSCs) in the bone marrow and takes up residence in lymphoid organs, skin as well as mucosal surfaces where they are most likely to encounter pathogens. GM-CSF is another cytokine involved in DC development that is specifically involved in the maintenance of skin resident DC. Together Flt3L and GM-CSF as well as their respective receptors, Flt3 and GM-CSFR, are the most important factors identified to date for maintenance of DC homeostasis. The Src-like adaptor proteins, SLAP and SLAP-2, are negative regulators of antigen receptor signaling. SLAP and SLAP2 contain SH3 and SH2 protein interaction modules that facilitate interaction with proline-rich sequences and phosphotyrosine motifs, respectively. Through a unique C-terminal region, SLAP and SLAP2 also interact with the E3 ubiquitin ligase, c-Cbl. SLAP and SLAP2 enhance c-Cbl-mediated ubiquitylation and down-regulation of antigen receptors by binding both activated receptors and c-Cbl. SLAP and SLAP2 have also been shown to function as negative regulators of receptor tyrosine kinases (RTKs) and our group has shown that SLAP and SLAP2 bind to and inhibit the colony-stimulating factor-1 receptor (CSF-1R). In the work presented here, we identify a novel role for SLAP in regulation of the GM-CSF receptor of bone marrow (BM)-derived DC. We show that potentiated GM-CSF signaling, in the absence of SLAP and SLAP2, impairs BM-DC maturation such that these cells express minimal MHCII, secrete low amounts of IL-12 and are functionally impaired in their ability to stimulate T cell responses. SLAP and SLAP2 deficiency also has an effect on Flt3L-derived BM-DC development. For example, SLAP/SLAP2-/- BM-DC numbers are reduced in the presence of Flt3L as compared to wild-type BM-DC. To investigate the mechanism of reduced DC numbers, we examined splenic DC and found that DC numbers were similar in wild-type and SLAP/SLAP2-/- mice. In fact, SLAP/SLAP2-/- mice had proportionally more CD8α+ splenic DC in vivo than wild-type mice. Thus there may be cytokines affected by SLAP/SLAP2-deficiency in our cultures that are either dispensable or compensated for in vivo DC development. The work presented in this thesis has implications for the role of SLAP and SLAP2 in immune response to infections by the regulation of GM-CSFR and Flt3 in maintaining dendritic cell homeostasis.

Keywords: adaptor protein
dendritic cells
signal transduction
Advisor: McGlade, Jane
Transnational Landscapes of Opportunity? Post-graduation Settlement and Career Strategies of International Students in Toronto, Canada and London, UK

This thesis explores the emerging issue of cities and countries competing for international students as part of market and talent-based economic development strategies. Based on case studies in London, UK and Toronto, Canada, this research draws on interviews with senior policy-makers as well as international students completing their overseas studies to examine three issues. First, this thesis investigates the process by which similar policies to attract and retain greater numbers of international students have been developed and introduced in both countries. Arguing that these policies are “mobile,” this thesis demonstrates how the competitive interconnectedness of policy-making leads to the transfer of policy ideas from one jurisdiction to another, while also recognizing the mediating role of institutions for contributing to continued geographic differences in the policy landscape regarding international education. Second, it examines the decision-making process for international graduate students upon the moment of graduation with regard to their settlement and employment strategies. Through a comparison of international students finishing advanced degrees in science and engineering in both sites, it reveals the extent to which students’ plans involve the complex intermingling of personal, professional and (im)migration regulation factors. The confluence of these factors tend to pull students in different geographic directions, indicating that the conventional ‘stay or return’ construct is too simplistic as a framework for understanding students’ future movements. Moreover, the comparison of students’ strategies in the two sites illustrates the differential effect of multi-scalar institutional frameworks in constructing certain types of migrant subjects. Third, this thesis investigates how career development strategies of international students differ according to broad disciplinary differences. Contrasting the career plans of graduating students in science, engineering, and art and design programs, this research finds that there are key differences in the socio-spatial career strategies held by international students in line with the differentiated knowledge bases literature.

Keywords: international student mobility
economic geography
Advisor: Gertler, Meric S.
DNA Hybridization on Walls of Electrokinetically Controlled Microfluidic Channels

The use of microfluidic tools to develop two novel approaches to surface-based oligonucleotide hybridization assays has been explored. In one of these approaches, immobilized oligonucleotide probes on a glass surface of a microfluidic channel were able to quantitatively hybridize with oligonucleotide targets that were electrokinetically injected into the channel. Quantitative oligonucleotide analysis was achieved in seconds, with nM detection limits and a dynamic range of 3 orders of magnitude. Hybridization was detected by the use of fluorescently labeled target. The fluorescence intensity profile evolved as a gradient that could be related to concentration, and was a function of many factors including hybridization reaction rate, convective delivery speed, target concentration and target diffusion coefficient. It was possible to acquire kinetic information from the static fluorescence intensity profile to distinguish target concentration, and the length and base-pair mismatches of target sequences. Numerical simulations were conducted for the system, and fit well with the experimental data. In a second approach, a solid-phase nucleic acid assay was developed using immobilized Quantum Dot (QD) bioprobes. Hybridization was used to immobilize QDs that had been coated with oligonucleotides having two different sequences. The hybridization of one oligonucleotide sequence conjugated to a QD (a linker sequence) with a complementary sequence that was covalently attached to a glass substrate of a microfluidic channel was shown to be an immobilization strategy that offered flexibility in assay design, with intrinsic potential for quantitative replacement of the sensing chemistry by control of stringency. A second oligonucleotide sequence conjugated to the immobilized QDs provided for the selective detection of target nucleic acids. The microfluidic environment offered the ability to manipulate flow conditions for control of stringency and increasing the speed of analytical signal by introduction of convective delivery of target sequences to the immobilized QDs. This work introduces a stable and adaptable immobilization strategy that facilitates solid-phase QD-bioprobe assays in microfluidic platforms.

Keywords: DNA
Hybridization
Microfluidics
Quantum dots
Fluorescence resonance energy transfer (FRET)
Biosensor
Solid-phase
Numerical simulation
Advisor: Krull, Ulrich Jorg
Pathology of Calcific Aortic Valve Disease: The Role of Mechanical and Biochemical Stimuli in Modulating the Phenotype of and Calcification by Valvular Interstitial Cells

Calcific aortic valve disease (CAVD) occurs through multiple mutually non-exclusive mechanisms that are mediated by valvular interstitial cells (VICs). VICs undergo pathological differentiation during the progression of valve calcification; however the factors that regulate cellular differentiation are not well defined. Most commonly recognized are biochemical factors that induce pathological differentiation, but little is known regarding the biochemical factors that may suppress this process. Further, the contribution of matrix mechanics in valve pathology has been overlooked, despite increasing evidence of close relationships between changes in tissue mechanics, disease progression and the regulation of cellular response. In this thesis, the effect of matrix stiffness on the differentiation of and calcification by VICs in response to pro-calcific and anti-calcific biochemical factors was investigated. Matrix stiffness modulated the response of VICs to pro-calcific factors, leading to two distinct calcification processes. VICs cultured on the more compliant matrices underwent calcification via osteoblast differentiation, whereas those cultured on the stiffer matrices were prone to myofibroblast differentiation. The transition of fibroblastic VICs to myofibroblasts increased cellular contractility, which led to contraction-mediated, apoptosis-dependent calcification. In addition, C-type natriuretic peptide (CNP), a putative protective molecule against CAVD, was identified. CNP supressed myofibroblast and osteoblast differentiation of VICs, and thereby inhibited calcification in vitro. Matrix stiffness modulated the expression of CNP-regulated transcripts, with only a small number of CNP-regulated transcripts not being sensitive to matrix mechanics. These data demonstrate the combined effects of mechanical and biochemical cues in defining VIC phenotype and responses, with implications for the interpretation of in vitro models of VIC calcification and possibly disease devleopment. The findings from this thesis emphasize the necessity to consider both biochemical and mechanical factors in order to improve fundamental understanding of VIC biology.

Keywords: Calcific aortic valve disease
Valvular interstitial cells
Aortic valve
Matrix stiffness
C-type natriuretic peptide
Microarray
Advisor: Simmons, Craig Alexander
Sensorimotor Recovery, Functional and Structural Brain Plasticity, and the Development of Chronic Pain Following Upper Limb Peripheral Nerve Transection and Microsurgical Repair

Following peripheral nerve transection and microsurgical repair (PNIr) most patients retain significant sensorimotor impairments, a proportion of which also develop chronic neuropathic pain. Individual psychological factors may contribute to the development, intensity and duration of chronic pain. Furthermore, a large body of evidence has indentified beneficial and maladaptive cortical plasticity following disease or injury. The general aim of this thesis was to determine the extent of sensory and motor recovery, functional and structural brain changes, and the impact of chronic neuropathic pain on sensorimotor outcomes following upper limb PNIr. Towards this main aim a sensorimotor psychophysical assessment (that included psychological assessments), nerve conduction testing, and an MRI session that examined brain function and structure was performed in patients with peripipheral nerve injury induced neuropathic pain (PNI-P) and those with no neuropathic pain (PNI-NP). Nerve conduction testing demonstrated that all patients had incomplete peripheral nerve regeneration, and that PNI-P patients had worse sensory nerve regeneration. Psychophysical assessment confirmed that all PNIr patients had significant sensorimotor deficits. Additionally, deficits on tests of vibration detection, sensorimotor integration, and fine dexterity were significantly greater in PNI-P patients. Psychological measures clearly distinguished PNI-P from PNI-NP and healthy controls (HC). Vibrotactile stimulation of the deafferented territory in PNI-NP patients results in reduced BOLD activation within the primary and secondary somatosensory cortices. Interestingly, the regions of reduced BOLD corresponded with gray matter thinning which was negatively correlated with behavioural measures of sensory recovery. Structural abnormalities were also identified in the right insula. PNI-P patients had thinning within the right middle insula and a corresponding decrease in white matter pathways projecting into/out of that region. PNI-P patients also had white matter abnormalities in pathways feeding into/out of the contralesional primary somatosensory cortex and thalamus. In conclusion, PNIr is clearly associated with sensorimotor impairments and brain plasticity. Furthermore, neuropathic pain is associated with worse peripheral nerve regeneration, sensorimotor deficits, different psychological profiles, and structural alterations in brain regions involved in pain perception and somatosensation. These results provide insight into peripheral regeneration, the development of chronic pain, brain plasticity and structure-function-behavioural relationships following nerve injury and have important therapeutic implications.

Keywords: peripheral nerve injury
plasticity
functional MRI
diffusion tensor imaging
cortical thickness analysis
psychophysics
chronic neuropathic pain
Advisor: Davis, Karen
Clinical and Molecular Characterization of Psychosis in 22q11 Deletion Syndrome

The past two decades have witnessed an accelerated effort to understand the nature of schizophrenia and related psychotic disorders, but no causative gene(s) has been discovered yet. Family, twin, and adoption studies indicate that genetic factors are clearly implicated in the etiology of these disorders (Cardno and Gottesman, 2000; Cardno et al., 2002; McGuffin et al., 2003; Weinberger, 2005). Several aspects of 22q11 Deletion Syndrome (22qDS) - the most common chromosomal microdeletion found in humans - create a unique opportunity for susceptibility gene identification. For instance, the reported risk of psychotic disorders in 22qDS is 25-fold higher than in the general population (Murphy et al., 1999) and genome-wide linkage studies in families with schizophrenia without 22qDS indicate that the 22q11.2 region is a strong susceptibility locus for psychosis (Badner and Gershon, 2002; Lewis et al., 2003). This thesis aims to identify genetic factors associated with the development of psychosis in 22qDS by i) investigating the relationship between the length of the 22q11.2 deletions and the presence of a psychotic disorder in patients with 22qDS; ii) studying diagnostic molecular methods that improve detection of 22q11.2 deletions and duplications; and iii) exploring the relationship between 22qDS-psychotic phenotype and gene expression patterns. The central hypothesis was that psychosis in 22qDS would not be associated with haploinsufficiency (having one copy of the gene), but rather, it would be associated with distinct 22q11.2 gene expression profiles. Chapter 2 showed that 22q11.2 deletion size did not appear to be associated with the development of psychosis in adults with 22qDS. In Chapter 3, a molecular method that detects and size 22q11.2 deletions and duplications of various sizes was shown to be superior to the traditional molecular diagnostic technique used for molecular diagnostic of 22qDS. Finally, in Chapter 4, decreased gene expression of three genes located in the 22q11.2 region (SNAP29, COMT and BID) was significantly associated with psychosis in adults with 22qDS. Focusing on genes located in the 22q11.2 region has helped revealing genetic alterations associated with the frequent development of psychosis in 22qDS. Future studies focusing on investigating the heterogeneity of the psychotic presentation in 22qDS and further elucidating potential genetic mechanisms likely to explain the gene expression changes in the 22q11.2 region demonstrated here will help advance the scientific understanding of the etiology of psychosis.

Keywords: psychosis
human chromosome band 22q11
22q11 Deletion Syndrome
Advisor: Siminovitch, Katherine A.
Inhibition of Hsp90 and its Client Kinase FAK has Therapeutic Potential in Squamous Cell Carcinomas of the Uterine Cervix and Oral Cavity

Heat shock protein 90 (Hsp90) is an essential and conserved chaperone, required for the conformational maturation and stability of many signaling kinases. We hypothesized that the functional pleiotropism of Hsp90 can be exploited during pharmacological inhibition causing simultaneous restraint of tumor growth as well as suppression of distant spread. Recognizing the lack of therapeutic options in advanced and metastatic squamous cell carcinomas (SCC) of the uterine cervix as well as the oral cavity, this dual concept was tested in corresponding cell lines and xenografts, and correlated with clinical data on client protein expression. Examination of the cell cycle response to Hsp90 inhibition revealed a G2/M-arrest in a panel of four cervical cancer cell lines and a contribution of abnormal mitosis to apoptosis induction in vitro. Although limited to intraperitoneal application, in vivo evidence of biological activity including heat shock response and decreased client kinase phosphorylation was seen with the geldanamycin derivative 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG). Importantly, focal adhesion kinase (FAK) signaling and associated functional parameters were inhibited by the drug treatment. Functional significance of FAK as a client was confirmed using a molecular model based on FAK-related non-kinase (FRNK) expression. Dependency on FAK appeared to be a requirement for full response to FRNK as well as 17-DMAG, and was observed in the mesenchymal-like cervical cell line SiHa. FAK expression and E-cadherin loss were features found in both cervical and oral malignancies, but absent from normal mucosa of either anatomic site. Particularly high FAK expression was noted in oral SCC with sarcomatoid features. Thus, we conclude that Hsp90 inhibition has potential in the treatment of advanced and metastatic SCC of cervical and oral origin. The further examination of novel Hsp90-targeting compounds as well as strategies focused on other components of the Hsp90 chaperone complex seems warranted.

Keywords: Hsp90
focal adhesion kinase
cancer
metastasis
uterine cervix
oral cavity
Advisor: Hedley, David
Locomotion Synthesis Methods for Humanoid Characters

This thesis introduces locomotion synthesis methods for humanoid characters. Motion synthesis is an under-constrained problem that requires additional constraints beyond user inputs. Two main approaches to introducing additional constraints are physics-based and data-driven. Despite significant progress in the past 20 years, major difficulties still exist for both approaches. In general, building animation systems that are flexible to user requirements while keeping the synthesized motions plausible remain a challenging task. The methods introduced in this thesis, presented in two-parts, aim to allow animation systems to be more flexible to user demands without radically violating constraints that are important for maintaining plausibility. In the first part of the thesis, we address an important subproblem in physics-based animation --- controller synthesis for humanoid characters. We describe a method for optimizing the parameters of a physics-based controller for full-body, 3D walking. The objective function includes terms for power minimization, angular momentum minimization, and minimal head motion, among others. Together these terms produce a number of important features of natural walking, including active toe-off, near-passive knee swing, and leg extension during swing. We then extend the algorithm to optimize for robustness to uncertainty. Many unknown factors, such as external forces, control torques, and user control inputs, cannot be known in advance and must be treated as uncertain. Controller optimization entails optimizing the expected value of the objective function, which is computed by Monte Carlo methods. We demonstrate examples with a variety of sources of uncertainty and task constraints. The second part of this thesis deals with the data-driven approach and the problem of motion modeling. Defining suitable models for human motion data is non-trivial. Simple linear models are not expressive enough, while more complex models require setting many parameters and are difficult to learn with limited data. Using Bayesian methods, we demonstrate how the Gaussian process prior can be used to derive a kernelized version of multilinear models. The result is a locomotion model that takes advantage of training data addressed by multiple indices to improve generalization to unseen motions.

Keywords: physics-based animation
controller synthesis
human motion
optimization
Advisor: Hertzmann, Aaron
Fleet, David J.
Determinants of Working Memory Performance

This dissertation investigated different factors contributing to age differences in working memory (WM) performance. Younger and older adults participated in five experiments, four on visuospatial WM (VSWM) and one on verbal WM. All addressed methodological issues that may differentially lower older adults’ performance. Experiments 1a and 1b manipulated the administration of a VSWM span task, with participants performing the task under either an ascending format (shortest sets presented first), or an interference-reducing descending format (longest sets presented first). Older adults’ performed significantly better in the descending compared to ascending format, consistent with an age-related susceptibility to proactive interference (PI). By contrast, younger adults did better in the ascending compared to descending condition, possibly due to their ability to benefit from practice and strategy use when easier trials are presented first. Experiment 2 considered how the similarity of task material influenced the build-up of PI and whether or not the combination of two PI-reducing manipulations (i.e., descending format and distinct trials) would further improve older adults’ performance. Distinctiveness helped older adults on the ascending format; however, combining distinct trials and a descending format provided no additional benefit. Experiment 3 considered whether or not synchronizing a VSWM task with an individual’s circadian arousal pattern would moderate interference effects, with the task administered at a peak or off-peak time of day. Peak-time administration improved older adults’ performance on the descending, but not the ascending, condition. Experiment 4 investigated the possibility that the serial order requirement of many WM tasks contributes to age differences in performance. Younger and older adults participated in a verbal WM span task – Operation Span - under either serial order or free recall instructions. Typical age differences were found when order but not free recall was required. Further analyses of the order condition data revealed that older adults were, in fact, recalling the items just as well as young adults, only not in the correct order. Taken together, the findings strongly suggest that age differences found on typical WM span tasks are influenced by numerous factors, such as task presentation, individual circadian arousal patterns, material similarity, and recall instructions.

Keywords: working memory
age differences
interference
practice effects
Advisor: Hasher, Lynn