VictorJuiz/thesis huggingface dataset:
Regulation of Biological Timekeeping and Secretory Trafficking by the N-recognin UBR4/POE
Keywords: Central pacemaker
Circadian rhythms
Golgi
Neuropeptide
UBR4
Vesicular trafficking

Most organisms possess circadian clocks that allow them to anticipate and accordingly respond to daily cycles in the natural and ecological environment. This endogenous timekeeping machinery is driven by numerous clock proteins, whose interaction generates daily rhythms in gene expression. At the core, ubiquitin ligase enzymes contribute immensely to the molecular clock by controlling the degradation of core clock proteins, which in turn serves to govern the speed and resetting properties of the central circadian clock. However, few studies have addressed their potential to regulate other cellular events within clock neurons beyond clock protein turnover. Here, I report that the ubiquitin ligase, UBR4/POE, strengthens the central pacemaker by facilitating neuropeptide trafficking in clock neurons and promoting network synchrony. Ubr4-deficient mice are resistant to jet lag and prone to constant light-induced arrhythmicity, whereas poe knockdown flies become arrhythmic under constant dark conditions. These behaviors are reflective of the reduced axonal trafficking of circadian neuropeptides induced by the loss of ubr4/poe in clock neurons. In addition to impaired neuropeptide transport, the loss of ubr4/poe also perturbs the expression of the core clock protein, PERIOD in both mouse and fly clock neurons. However, it appears that the in vivo behaviors are best explained by the neuropeptide transport deficit, which led me to pursue this phenotype further. The biosynthetic secretory pathway is responsible for the synthesis and trafficking of membrane and secretory proteins, including neuropeptides. Within this pathway, I found that UBR4 ablation leads to altered Golgi morphology and impedes the export of secreted proteins from the Golgi apparatus by reducing the expression of Coronin 7, a protein essential for the budding of Golgi-derived transport vesicles. In summary, this work reveals a conserved and unexpected role of UBR4/POE in vesicular trafficking of neuropeptides, a function that has important implications for circadian clock synchrony and circuit-level signal processing.
The Multifaceted Roles of Fragile X Mental Retardation Protein in Developmental Plasticity at a Central Synapse
Keywords: AMPA-R
developmental plasticity
Fragile X Mental Retardation Protein
Fragile X Syndrom
NMDA-R
synapse

During their critical period of sensory development, postsynaptic glutamatergic receptors, namely AMPA receptors (AMPARs) and NMDA receptors (NMDARs), undergo rapid remodeling in abundance and composition to enable fast neurotransmission. Dysregulation of this process can lead to neurodevelopmental disorders such as Fragile X Syndrome (FXS) which shares several mental retardation-associated features of Autism Spectrum Disorders (ASD). This is caused by a loss of Fragile X Mental Retardation Protein (FMRP), a critical translational repressor at the synapse recently implicated in ion channel modulation. To investigate the roles of FMRP in synaptic remodeling of glutamatergic receptors, I have established developmental profiles at the calyx of Held-MNTB synapse in the auditory brainstem by measuring whole-cell electrophysiological synaptic responses from pre-ear canal opening (post-natal day 6-9) and after-hearing onset (post-natal day 16-19) synapses in wild-type and the FMR1-/- mouse model of human FXS. My results indicate the loss of FMRP causes early maturation of AMPARs by accelerating the developmental switch from the slower-gating GluA1-dominant to faster-gating GluA4-dominant subunit composition of AMPARs, consistent with the canonical role of FMRP as an mRNA translational repressor. In contrast, developmental downregulation of NMDARs is not only stunted in FMR1-/- synapses, but also resistant to activity-dependent downregulation. This defect can be acutely rescued by intracellular delivery of the protein binding N-terminus fragment of FMRP during whole-cell patch clamp recordings, suggesting FMRPN-terminal is sufficient for mediating activity-dependent plasticity of NMDARs. Importantly, pulldown of the NMDAR subunit GluN1 and mGluR5 by FMRP in co-immunoprecipitation experiments provides evidence of FMRP binding to mGluR5-NMDAR complexes. This raises the possibility that a protein-protein interaction involving FMRP is engaged in the developmental downregulation of NMDARs. Based on these novel findings, I propose a model whereby activity induced increases in intracellular calcium triggers a two-pronged biochemical cascade through which FMRP differentially regulates two glutamatergic receptors within the same cell type. Altogether, FMRP functions as a canonical translational repressor to regulate AMPAR subunit expression, as well as as a non-canonical signaling molecule to mediate activity-dependent downregulation of NMDARs. These deficits in developmental synaptic remodeling provide mechanistic insights into the sensory hypersensitivity and cognitive impairments in FXS and ASD.
The Brain Complex: Indeterminacy in a neuroscience laboratory
Keywords: Complexity
Indeterminacy

This dissertation is an ethnographic study of how notions of the human and scientific practices in a neuroscience laboratory transform under the contemporary condition of complexity. I focus on a group of researchers working in a neuroscience laboratory in Ottawa, Canada, and who have close ties with researchers in China and Taiwan. Rejecting especially deterministic and reductionist approaches, these researchers take up a notion of the human brain—the “most complex” scientific object, that is embodied, embedded in the surrounds, and emplaced in time, but also active on its own, and thus what they see as a living brain, an especially lively and active one. Captivated by the lively activity of the brain, the scientists bring dynamic, temporalized, noisy, and processual matters and methods into their scientific practices of imaging, experimenting, measuring, and modelling brains, what I term the brain complex. With the brain complex, the scientists revision the brain less as a thing, and more as a verb, a process or a doing, and captured in the term ‘activity’ that was the focus of study and materialized through dynamic interactivity. This dissertation argues that, with this revision, the scientists’ efforts to study the lively brain involve incorporating indeterminacy in their scientific practices and doing so challenges notions of science as reductive, deterministic and as a fixed practice itself, studying static scientific objects with stabilized technical apparatuses. Indeterminacy in practice instead acknowledges the change and transformation of this science, as well as the complex anthropological notions with which the brain is conjoined, namely the human, the body, and life. By following how scientific practices take up indeterminacy in this neuroscience laboratory, this dissertation challenges taken for granted understandings of the neuroscientific human as not just fixed but also dynamic, and, even further, how this dynamic human, still latent in anthropology as well, can (re)produce an uneven politics of knowledge.
SHP2 Inhibition as a Therapeutic Strategy to Target High-risk Neuroblastoma
Keywords: combination therapy
neuroblastoma
NRAS
pediatric cancer
RAS-MAPK pathway
SHP2 inhibitor

Survival for high-risk neuroblastoma (NB) remains poor and treatment for relapsed disease rarely leads to long-term cures. Large sequencing studies of NB tumors from diagnosis have not identified common targetable driver mutations other than the 10% of tumors that harbor mutations in the anaplastic lymphoma kinase (ALK) gene. However, at NB recurrence more frequent mutations in genes in the RAS-MAPK pathway have been detected. The PTPN11-encoded tyrosine phosphatase SHP2 is an activator of the RAS pathway, and we and others have shown that pharmacologic inhibition of SHP2 suppresses the growth of various tumor types harboring KRAS mutations such as pancreatic and lung cancers. Here, we report inhibition of growth and downstream RAS-MAPK signaling in NB cells in response to treatment with the SHP2 inhibitors SHP099, II-B08, NSC-87877, and RMC-4550. Furthermore, our findings provide a rationale for the molecular and clinical potential of SHP2 inhibition in the treatment of ALK mutated NB, the most commonly mutated gene in NB tumors. Conversely, we showed that NB cell lines harboring endogenous NRASQ61K mutation (which is commonly detected at relapse) or isogenic NB cells engineered to overexpress NRASQ61K are distinctly resistant to SHP2 inhibitors. We further show that combinations of SHP2 inhibitors with other RAS-MAPK pathway inhibitors, such as the MEK inhibitor trametinib, RAF inhibitor vemurafenib and ERK inhibitor ulixertinib, are synergistic and reverse NRASQ61K-associated resistance to SHP2 inhibition in NB. Moreover, we demonstrate that combinatorial treatment with SHP2 and ALK inhibitors sensitize ALK-mutant and other MAPK-activated NB cells to ALK tyrosine kinase inhibitors and synergize in vitro. Finally, our pre-clinical studies in NB xenografts have provided evidence of the synergistic anti-tumor effects of SHP099 plus trametinib or ulixertinib treatments and showed that anti-SHP2/MAPK combinations led to an overall reduction in RAS signaling and alleviation of RTK feedback signaling in vivo. Together, these results suggest for the first time that combination therapies targeting SHP2 and other components of the RAS-MAPK pathway may be effective against conventional therapy-resistant relapsed NB including those that have acquired NRAS mutations.
Cross-species Multi-platform Screens to Characterize Novel Targetable Pathways in Glioblastoma Multiforme
Keywords: brain tumours
C elegans
cancer therapy
glioblastoma
RNAi screens
SOX2

Glioblastoma multiforme (GBM) is an invariably fatal brain tumour. Despite advances in surgical techniques, radiation therapy, and chemotherapy, the prognosis of patients with glioblastoma remains grim. The median survival in adults following diagnosis with GBM is only 14 months, and most patients succumb to their disease within two years. Our failure to offer a meaningful therapy to these patients has driven scientific studies towards increasing our understanding of the biology of these tumours, and in the process, identifying new targets for therapy. We propose that an integrated therapeutic approach which targets the pathways involved in regulating the hyperproliferative signaling, replicative immortality and cancer stem cell subpopulation, and the migration and invasion potential of the tumour cells is needed to achieve remission and prevent recurrence of GBM. Here, we performed multiplatform in silico and in vivo cross-species screens to identify novel therapeutic targets in GBM. Using these approaches, we have identified 25 different targets from the KRAS/PTEN, Rho-GTPase, and SOX2 signaling pathways. These studies may prove helpful in understanding the utility of targeting tumours in preclinical models, and potentially lead to novel therapeutic avenues for treating patients with GBM.
Biophysical Techniques and Assay Design for the Development of Small Molecule Inhibitors
Keywords: Assay development
HDAC
High-throughput screening
Prenylation
STAT
UBA5

One of the most important pillars in preclinical research involves the biophysical characterization of protein-compound interactions. This characterization, describing the in vitro potency and binding properties of the interaction, is measured through techniques such as fluorescence polarization assays, thermal shift assays, isothermal titration calorimetry experiments, and activity-based assays. A broad biophysical profile of each compound against the target of interest allows for fine-tuning of the molecule through structure-activity relationship-guided design. Biophysical profiling can enable accurate prediction of the interaction within a biological setting and thus provide insight into the mechanism-of-action observed in a cellular setting. This thesis details the recombinant protein expression and purification as well as the biophysical assay development of several attractive therapeutic targets for cancer treatment including STAT3, STAT5B, UBA5, HDAC6, and HDAC8. To aid in STAT inhibitor discovery, exploration of assay development to confirm STAT-phosphorylation inhibition was described. This thesis reports a rapid fluorescence-based screening method to identify SH2 domain binders followed by a phosphorylation-inhibition assay to validate mechanism of action for molecules determined from the high-throughput screening assay. Additionally, the development and optimization of a fluorescence polarization (FP) assay for binding affinity determination of DNA binding domain inhibitors for STAT3 was also described. Similarly, FP assays and thermal shift assays were also validated for drug discovery program geared towards identification of highly selective and potent HDAC6 and HDAC8 inhibitors. Fluorescence-based activity assays and in cellulo nanoBRET assays were also established for HDAC proteins to confirm the results obtained from preliminary biophysical assays. Exploration of high-throughput virtual screening was also employed to identify novel UBA5 inhibitor scaffolds from a diverse natural product and natural product derivative libraries. This experiment yielded a lead candidate with potential for refinement through structure-activity relationship (SAR) to improve drug-like qualities of the molecule. Lastly, the hijacking of the upregulated prenylation pathway in cancer for artificial protein-membrane anchorage was investigated for utility as an alternative protein inhibition mechanism. Collectively, these studies demonstrate the progress in the assay development area for the targeting of multiple proteins to aid the drug discovery process.
Large-scale Network Models of Mild Traumatic Brain Injury and Repetitive Head Trauma
Keywords: brain network models
mild traumatic brain injury
neuroimaging
sports concussion

Mild traumatic brain injury (mTBI) may be understood as a multi-scale system deficit where adverse clinical outcomes emerge due to interacting brain changes that span spatial scales. At the micro-scale a neurometabolic cascade affects neurotransmission, while on the macro-scale diffuse axonal injury disrupts long-range connections. Large-scale brain network modeling allows us to make insights across these spatial scales; integrating neuroimaging data with biophysically based models to predict whole-brain dynamics. In this thesis, I used brain network models to study the long-term effects of mTBI and repetitive head trauma. Study 1 found mTBI patients experiencing active post-concussion syndrome symptoms had lower regional inhibitory connection dynamics relative to comparison participants. Study 2 expanded these findings to a sample of mTBI patients recruited from hospital emergency rooms in the semi-acute phase (1-2 weeks) by showing regional inhibitory dynamics were related to chronic TBI outcomes. Finally, in Study 3 I linked lower regional inhibitory connection dynamics with higher concussion exposure, lower cognitive performance, and higher psychosocial complaints in a sample of retired professional hockey players. Together, the work offers converging evidence that lower regional inhibitory dynamics are related to concussion exposure and poorer clinical outcomes following mTBI. The work exemplifies how large-scale network modeling may be used to make cross-scale inferences otherwise inaccessible with conventional neuroimaging.
Addressing Vaccine Hesitancy in Canada: Paediatricians’ Perspectives and Social Work Opportunities
Keywords: interprofessional practice
paediatricians
paediatrics
social work
vaccination
vaccine hesitancy

The objectives of this three-paper thesis are to: (1) explore the prevalence and impact of parental vaccine hesitancy on paediatricians in Canada; and, (2) use a social work lens to examine vaccine hesitancy and identify novel approaches involving social work to enhance vaccine uptake for young children in Canada. This research is guided by four theoretical perspectives: The Health Belief Model, The Theory of Planned Behaviour, Ecological Systems Theory and The Transtheoretical Theory of Change Model. The first and second papers are based on survey data collected through the Canadian Paediatric Surveillance Program (CPSP). The first paper examined the frequency with which paediatricians encounter vaccine hesitancy in clinical practice, challenges and impacts of hesitancy on practice and factors associated with vaccine-compliance among parents with concerns about vaccination. The second paper examined factors predicting physician-reported parental intention to vaccinate following the diagnosis of a paediatric vaccine-preventable disease. The third paper explores how social workers could contribute to addressing vaccine hesitancy with respect to early childhood vaccines in Canada. The findings of this dissertation indicate that paediatricians encounter parents with concerns about vaccination frequently in clinical practice. The most significant predictor of vaccine compliance among parents of young children with concerns about vaccination was receiving a strong personal recommendation from their primary care physician. The only significant predictor of physician-reported parental intention to vaccination following the diagnosis of a paediatric vaccine-preventable disease was whether the parent previously refused all vaccines for the child. Vaccine hesitancy is a complex phenomenon which has traditionally remained within the purview of physicians. Social workers have the training and skills to effectively address vaccine hesitancy, particularly among families facing structural barriers to accessing vaccination. This dissertation concludes with a discussion of key findings, implications for social work and healthcare and suggestions for future research, policy and practice efforts.
Spinoza’s Metaphysics of the Finite: Composition, Essence, Modality
Keywords: composition
contingent
essence
necessitarian
Spinoza
universal

This dissertation is about Spinoza’s metaphysics of ordinary objects and organisms. It focuses on three topics: mereological composition, essence and universals, and the nature of contingency. Chapter one argues that Spinoza’s thinking about parts and wholes provides a powerful alternative to so-called ‘fictionalist’ accounts of mereological composition. I show that even though Spinoza agrees with fictionalists that part-whole reasoning doesn’t aim at literal truth, at the same time he considers it instrumental in elucidating our limited understanding of causal processes. I conclude that Spinoza is an anti-realist about composition because he is a perspectivalist. That is, for Spinoza, expressions such as ‘x is a part of y’ or ‘x is a whole composed of some ys’ convey abstract judgments from a limited perspective. Chapter two is about Spinoza’s account of universal natures, that is, essences shared between members of a certain natural kind. I show that evidence for commitment to universal natures pervade Spinoza’s metaphysics, epistemology, and political philosophy. However, at the same he provides an error theory explaining how extant realist accounts of universals go wrong. On my reading, Spinoza’s seemingly contradictory statements about shared essences in fact constitute a coherent, modest realist account of the genuine qualitative similarity of entities belonging to a certain natural kind or species. Lastly, chapter three makes a novel case for interpreting Spinoza as a necessitarian, which is arguably one of the most important commitments in his metaphysics. I argue that even though Spinoza holds necessitarianism, he allows for contingency, although in a different sense than it is standardly taken to be in contemporary discussions. His definition of contingency is closer in meaning to the familiar use of the word ‘contingent’ according to which something is contingent if it depends on an external cause. On my reading, this means that Spinoza holds modal compatibilism, that is, on his account, something can be both contingent since it depends on an external cause, and necessarily exist in with regard to its causal history.
Protecting Rights in the digital Age: The Application of the Charter in Cyberspace
Keywords: Charter of Rights and Freedoms
cyberspace
espionage
extraterritoriality
human rights
national security law

Since the swift passage of the Anti-Terrorism Act (Bill C-51) in 2015, the Canadian Security Intelligence Service (CSIS) has had the unprecedented and highly controversial authority to take “reasonable and proportionate” measures — often referred to as “threat reduction measures”—to reduce threats to Canadian security. While there are some limits to the types of measures CSIS can employ, the Canadian Security Intelligence Service Act permits the use of measures that would otherwise contravene the laws of Canada or limit a right protected by the Canadian Charter of Rights and Freedoms (Charter) so long as they are judicially authorized by a judge of the Federal Court of Canada. As new threats and threat actors proliferate online, it is anticipated that CSIS will increasingly carry out its threat reduction mandate in the digital world. Yet, in June 2018, CSIS’s former review body, the Security Intelligence Review Committee, reported that CSIS had never sought a threat reduction warrant from the Federal Court. What remains unclear is why. Is it possible that CSIS has never employed a measure that might violate someone’s freedom of association, expression, or mobility rights, or their right to liberty and personal security protected under the Charter? Or has CSIS concluded that the Charter does not govern actions carried out in cyberspace and, as such, that their online conduct falls beyond the reach and oversight of the Federal Court? Unfortunately, Canadian Courts have never been asked, and legal academics have never answered whether the Charter constrains online state action. The Department of Justice and CSIS have also never publicly enunciated their legal position on this subject. To date, the government’s interpretation of the Charter’s application to state cyber operations remains classified. This dissertation dissects these issues for the first time and ultimately concludes that the Charter governs state conduct in cyberspace.