VictorJuiz/thesis huggingface dataset:
Studying the Regulation and Function of Biomolecular Condensates with In vitro Reconstitution
Keywords: biomolecular condensates
CARPIN1
FMRP
Intrinsically disordered protein regions
phase separation

A central dogma in biochemistry is that a protein’s function depends on its fixed three-dimensional structure. However, bioinformatics analysis predicts that the majority of human proteins (~58%) contain both folded protein domains along with protein regions that are entirely devoid of any tertiary protein structures, referred to as intrinsically disordered protein regions (IDRs). Despite the strong association of proteins with significant IDRs to numerous diseases, most studies tend to overlook the potential function of IDRs and focus solely on their folded domain portions. Moreover, mutations within IDRs are hard to functionally interpret and become frequently neglected or annotated as variants of unknown significance. Given these observations, we need to understand better the function and biological role of IDRs in health and disease. Recently, IDRs have been shown to drive membrane-less RNA-protein granule formation. Similar to the separation of oil and water, specific proteins and nucleic acids can phase separate and form distinct membrane-less compartments (also referred to as condensates). While we have some understanding of the phase separation process, the biological regulation of phase separation and the functional consequences of condensates remains enigmatic. Here, I use biochemistry and biophysical approaches to study the regulation and function of IDR phase separation and extend these findings to disease implications. Beginning in chapter 3, I studied the phase separation of the C-terminal IDR of the fragile X mental retardation protein (FMRP). I demonstrated that post-translational modifications, including phosphorylation and methylation, regulate FMRP phase separation which correlates with in vitro translational inhibition. In chapter 4, I studied the protein interactions and co-phase separation properties between two interacting IDRs, from FMRP and CAPRIN1. I found that different phosphorylation patterns on each IDR can regulate their co-phase separation properties and modulate their impact on deadenylation and translational activity. In chapter 5, I showed that the solvent environment of a CAPRIN1 condensate can regulate deadenylation activity by CNOT7 within the condensate. Together, these results support the functional importance of IDR phase separation and also provide new methods to study condensates. Importantly, my findings provide a platform to understand how disease mutations within IDRs may perturb phase separation leading to disruption of biological function.
Uncovering the Complexities in Writing from Sources from an Activity Theory Perspective: A Cross-Case Analysis of Chinese International Graduate Students in Education
Keywords: Activity Theory
Chinese students
English for acadmic purposes
Literacies development
Writer identities
Writing from sources

With a skyrocketing international student population from Asia at Canadian universities in recent decades, second language (L2) students’ abilities to adapt to the (inter)textual practices of their prospective discourse communities have received increased attention in L2 writing research. This thesis study aimed to uncover the complexities and heterogeneity inherent in L2 student writers’ uses of textual sources. Through a multi-case study design (Stake, 2006), I examined the textual borrowing practices of 3 first-year international graduate students from China studying Education at an English-medium university in Canada. Drawing on Engeström’s (2001) activity theory and Ivanič’s (1998, 2005) analytic framework of writers’ construction of identity, I answered 2 research questions: (1) What activity systems do students from China experience while learning to read and write in China and writing assigned papers for master’s courses in Education in Canada? and (2) What challenges do these students encounter while writing papers from sources and how do the students address these challenges, with what consequences in their writing and identities? The instruments for this multi-case study were learning history and text-based interviews with each student along with data from their written assignments and certain source texts, a learner profile questionnaire, and a reading-writing questionnaire. My analysis revealed tensions and contradictions within and between the students’ former literacy learning activity systems in China and their current writing-from-sources activity systems in Canada. These tensions and contradictions posed challenges for the students to (a) effectively process source information and accurately and appropriately present that information to demonstrate comprehension and critical thinking, evident in their patchwriting, direct copying, inappropriately quoting, transposing from Chinese sources, and paraphrasing abstracts as purported summaries of their own; (b) cultivate a genuine interest in what they wrote and manifest deep or extended learning in their writing, evident in their tendencies to cite secondary sources without reading the originals of those sources; and (c) take ownership of the knowledge co-constructed through dialogic interactions between the source authors and the students or accept their roles in shaping the double-voiced discourse in the process of writing despite their (imprecise) uses of quotations, paraphrases, summaries, and syntheses to do so.
Circadian Regulation of the RNA-binding Protein Fxr1 and its Role in Regulating Core Clock Genes
Keywords: Circadian Rhythm
Fragile X Protein
Fxr1

The fragile X autosomal homolog 1 (Fxr1) is an RNA-binding protein that controls mRNA translation and has been associated to mood regulation, schizophrenia, and bipolar disorders. These neurological and psychiatric disorders are known for presenting disrupted circadian rhythm and sleep problems. However, due to the fact that Fxr1 knockout mice die soon after birth, it is increasingly difficult to study its role. The circadian clock coordinates physiology with environmental cues through the regulation of daily oscillations of gene expression. It is not known whether Fxr1 is regulated in a circadian-dependent manner, or if the absence of Fxr1 is able to affect genes in the transcription-translation feedback loop (TTFL). Here, we examined the contribution of the circadian clock to the regulation of Fxr1 as well as the effect this RNA-binding protein may have on the TTFL. Our findings indicate that Fxr1 mRNA levels lose their oscillation in clockΔ19 mice model. We also show that Fxr1 has e-boxes in its promoter region that are regulated via CLOCK/BMAL1 and NPAS2/BMAL1 heterodimers. Furthermore, we observed that CLOCK/BMAL1 and NPAS2/BMAL1 activation via Fxr1 was repressed by GSK3β. Our results also demonstrate that Fxr1 is required for the presence of rhythmic oscillation of several circadian genes. Moreover, Fxr1 is able to regulate its circadian gene targets by binding to their 3’UTR and coding sequences, thus regulating their translation. These results show that Fxr1 may have an essential role in regulating the circadian behavior in mammals and suggests that this role is potentially relevant to sleep and other behavioral alterations observed in several neurological and psychiatric disorders.
Hope Amidst Violence: Youths’ Agency and Learning Experiences to Transform Social Conflicts in Iran
Keywords: conflict education
Middle East
peacebuilding education
youth agency

This study foregrounded youths’ voices and lived experiences of social conflicts in greater Tehran, Iran, a non-democratic and understudied context in the Middle East. Understanding and addressing various (direct, cultural, and social-structural) dimensions involved in conflicts is necessary to build just and sustainable peace. The study examined selected youths’ socially constructed understandings of the conflicts and violence affecting their lives, their perceptions of their roles and agency in non-violently addressing those conflicts, and the learning experiences (inside and outside school) available to them that might have contributed to forming their understandings and agency. The study’s intersectional gender- and social class-sensitive perspective and design enabled understanding how social-structural, cultural, and political dynamics, and participating Iranian young people’s unequally positioned social identities, interacted with their experiences of social conflicts and their peacebuilding citizenship agency . The main evidence in this thesis was collected through multimodal participatory activities in a series of focus group sessions with youths in each of four contrasting high schools, segregated by gender and social class. This knowledge was contextualized through ethnographic observations of the participating students’ natural settings in their schools and communities, and interviews with school teachers and administrators and nonformal community educators. The findings, from this rich diversity of sites and participant populations, call for conflict sensitive and contextually relevant approaches to education to enhance students’ opportunities to develop key aspects of peacebuilding agency, even in nondemocratic settings where citizenship and peace education are absent from the explicit curriculum inside and outside schools. Despite the disconnections between the participating students’ lived experiences of conflicts and their learning opportunities at school, the findings point to spaces of feasibility for schools in the Iranian context to provide students with learning experiences relevant to democratic citizenship and peacebuilding. Such learning opportunities seem especially crucial in marginalized girls’ schools, where intersecting gender and socioeconomic status had narrowed the girls’ access to multiple sources of information and spaces to represent their voices without fear of punishment, limiting their repertoires of possible conflict handling options to the existing non-democratic and discriminatory alternatives they had already experienced.
Structural Brain Networks and Complex Behaviors in Healthy and Brain-injured Children
Keywords: Brain Tumors
Connectivity
Network Neuroscience
Neurodevelopment
Radiation
White Matter

A paradigm shift in our understanding of brain-behavior relationships suggests that behavior emerges from interactions between distributed brain regions. These interactions are facilitated by the structural connectome, the dense, integrative network of white matter connections linking the entire brain. Recent techniques aimed at capturing the complexities of whole-brain connectivity have enabled a better understanding of the relations between the structural connectome and behavior. Evidence suggests that the development of the structural connectome is closely aligned with the acquisition of new skills and abilities in children. Children with compromise to the structural connectome demonstrate learning and behavioral challenges. Intellectually gifted children possess structural connectomes that are suited for integrative processing of information. Despite these advances, data on disruptions to the structural connectome in the context of a childhood brain injury is limited. Moreover, the ways in which the structural connectome influences behavior is less well understood. Novel to the field, results from this dissertation showed that organizational properties of the structural connectome are vulnerable to the effects of brain injury. Altered connectome properties were indirectly associated with worse intelligence in brain-injured children; this relationship was mediated by the impact of brain injury on the speed of information processing. These findings were derived from three separate studies: Study I established the impact of childhood brain injury on the microstructure of short-range white matter connections. These connections are an integral part of the structural connectome but are less well understood than long-range white matter connections. Study II explored injury-related alterations to the structural connectome by conducting a thorough analysis of the different properties of the connectome in children with varying subtypes and intensities of brain injury. Study III examined the relationships between the structural connectome and intelligence in a cohort of typically developing and brain-injured children. The results of this dissertation improve our understanding of brain-behavior relationships by extending previous work showing that the structural connectome is vulnerable to brain injury. It also characterized, for the first time, a path from connectome organization to complex behaviors in children.
Contribution of Endothelial Heterogenity and Borrelia burgdorferi Adhesins to Vascular Interaction Mechanisms
Keywords: adhesin
Borrelia burgdorferi
endothelial cells
flow chamber
tissue tropism
vascular interaction

Systemic dissemination of microbes is crucial in infectious disease progression and is associated with most mortality due to bacterial infection. A critical step in dissemination is microbial adhesion to endothelial cells lining blood vessels, which is challenging due to shear stress generated by blood flow. Lyme disease, caused by Borreliella burgdorferi, is the most common vector-borne infection in the northern hemisphere. B. burgdorferi transmitted to humans by infected ticks disseminates via the bloodstream to multiple tissues. Different B. burgdorferi species and strains exhibit distinct tissue dissemination patterns, suggesting that these bacteria have a diverse repertoire of dissemination mechanisms; however, the mechanistic basis for tissue-specific dissemination is largely unknown. It is challenging to interpreting these studies because many microbial molecules “multitask” and (may) contribute to both immune evasion and microbe-vascular interactions and extravasation. At the start of my Ph.D., there was an urgent need for methods to systematically investigate microbial dissemination mechanisms that controlled for potentially confounding effects of immune clearance. In this thesis I report the development of tissue-specific flow chamber models of bacterial-endothelial cell interactions and flow chamber-validated methods for accurately measuring immune clearance in vivo and applied these methods to define the mechanistic basis for tissue-specific tropism patterns of diverse B. burgdorferi strains, and to begin the process of identifying microbial and host molecules and mechanisms supporting tissue-specific dissemination. For example, these approaches identified an unknown factor encoded/controlled by B. burgdorferi plasmid lp25 that was crucial for interaction with human brain endothelial cells and dissemination to brain in mice, and several candidate adhesins that may contribute to vascular dissemination to bladder, brain, joint and skin. These methods and model systems (flow chamber and short-term intravenous studies) are invaluable for identifying novel dissemination mechanisms and discovering novel adhesins of B. burgdorferi and other blood borne pathogens. This work might be useful for characterizing tissue tropism and vascular dissemination mechanisms of other blood-borne microbes.
Functional Reserve: How Everyday Experience Enhances Resiliency to Functional Decline and a Novel Measure of this Construct
Keywords: Cognitive Reserve
Functional Reserve
Instrumental Activities of Daily Living
Mild Cognitive Impairment
Neuropsychology

Reserve is a hypothetical construct that buffers the relationship between pathology and the clinical expression of aging and brain-based disorders, with cognitive reserve being the most widely studied. While cognitive reserve is associated with neuropsychological and neuroimaging outcomes, relatively little research has examined how measures of cognitive reserve might relate to real-world functioning, or instrumental activities of daily living (IADLs). This program of research was first interested in exploring whether cognitive reserve differentially relates to neuropsychological functioning versus IADL functioning. This is crucial for identifying possible treatment targets that may support functional independence. Random-effects meta-analyses were conducted using studies of participants diagnosed with mild cognitive impairment (k=58; n=7,871; 53% female). Common proxies of cognitive reserve (i.e., educationand occupational attainment) appeared to have limited benefits with respect to functional outcomes. Instead, a cognitive reserve proxy that better approximates IADLs (i.e., leisure activity) appeared to be more strongly associated IADL functioning. The results of this meta-analysis lead one to question whether participation in IADLs may demonstrate even stronger effects in terms of functional decline, perhaps through a functional reserve. To investigate the functional reserve hypothesis, this research program next developed the first instrument to quantify IADL experience – the Scale for Activity of Daily Living Experience (SADLE). Participants were recruited through Mechanical Turk at baseline (n=287) and 3-month follow-up (n=54). The final scale consisted of 39-items and two subscales. Test-retest reliability was excellent to good. Overall SADLE scores and participation across specific activities were associated with age, sex, domestic partnership, and gender-role identity, with effects ranging from weak to modest. The development and validation of the SADLE is an important first step in studying the functional reserve hypothesis, with further research needed in clinical samples. Finally, this research program proposed mechanisms that may underlie functional reserve – namely, cognitive, physical, psychological, and social mechanisms. In doing so, numerous future directions were provided to further validate the functional reserve hypothesis. Research that builds on this body of work may allow for the identification of individuals at risk for IADL impairment and may inform the development of interventions that are more efficacious in targeting functional decline.
Care Redistribution: Reassembling Task Shifting with Naloxone Distribution and First Aid
Keywords: Conceptual Frameworks
First Aid
Harm Reduction
Health Equity
Public Health
Task shifting

This dissertation develops the conceptual connection between first aid and task shifting and task sharing (TS/S), and advances a conceptual framework for the redistribution of health care services, especially where those practices involve lay providers. The dissertation uses overdose education and naloxone distribution as a leading example of an intervention at the intersection of first aid and TS/S to problematize the World Health Organization’s dominant definition and conceptualization of TS/S. The first study is a systematic review on the health effects of first aid and TS/S with laypeople in underserved populations and low-resource settings. The review concludes that lay responders may improve patient morbidity and mortality, and build community capacity to manage health emergencies including trauma, burns, cardiac arrest, opioid poisoning, malaria, paediatric communicable diseases and malnutrition. The second study is a mixed methods study to establish the feasibility of recruitment and retention strategies for a randomized trial on overdose education and naloxone distribution involving participants who are likely to witness opioid overdose. The study met its feasibility goals by recruiting 30 participants over 24 days and retaining 21 participants (retention rate 70%, 95% CI 56.7% – 100%). Study procedures were acceptable to participants, and the randomized trial deemed feasible for implementation. In the third paper, a Delphi process was used to achieve consensus on the purpose of TS/S and characteristics of contexts amenable to TS/S, and then develop a revised conceptual framework for TS/S programmes. The framework offers a refined definition of TS/S and a general purpose statement to guide TS/S programmes. The framework also proposes five opportunities for health system improvement arising from TS/S programmes depending on the implementation context, and enumerates eight necessary conditions and important considerations for implementing TS/S programmes. The dissertation contends that a broader, more inclusive conceptual assembly is required to capture the diversity of redistributive programmes and the history, politics, actors, and goals involved in the redistribution of care. The dissertation proposes “care redistribution” as a more suitable assembly. Care redistribution occurs when communities deliberately reallocate or reassign heath care practices, functions, and relationships to enhance care, improve health, or redress inequities.
Serotonin CEST: From In Vitro Characterization to In Vivo Detection
Keywords: CEST
Convection
MRI
MT
Serotonin
Tryptophan

Serotonin is a neurotransmitter involved in nearly all major brain functions; however, when deficient can lead to severe neurological disease and psychiatric disorder. Currently, no quantitative technique exists to detect or measure serotonin, suggesting that the diagnosis and treatment evaluation of serotonin-related pathology is inconsistent and unreliable. Chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) is a non-invasive contrast mechanism which can indirectly evaluate brain metabolites with exchangeable protons and offers a 100 – 1000 times increase in signal sensitivity compared to proton spectroscopy. The purpose of this thesis was to assess the feasibility of using CEST MRI to detect and quantify in vivo serotonin with sufficient sensitivity to aid in the diagnosis and treatment evaluation of serotonin-related pathology. The feasibility of using CEST MRI to non-invasively detect, image, and quantify in vivo serotonin was investigated by pursuing the following four specific aims: 1) design an experimental setup capable of imaging in vitro samples under stable physiological conditions, 2) prepare in vitro samples of serotonin and other similarly structured metabolites to characterize their magnetic resonance properties using CEST, 3) measure a change in the CEST signal following serotonin modulation in a rodent model, and 4) determine whether a unique CEST signal at serotonin’s resonance frequency can be detected in healthy human volunteers at clinical fields strengths. A methodology was developed to eliminate convection currents in warm liquid phantoms to accurately image in vitro samples under stable physiological temperature conditions. Serotonin and its precursor molecules (L-tryptophan and 5-HTP) exhibit significant and similar CEST contrast at both a pH of 5.5 (Δω = 2.64 – 2.71 ppm) and 7.4 (Δω = 5.29 – 5.47 ppm). A broad increase in CEST contrast was measured between 1.5 – 6 ppm in the rodent brain following the neurochemical modulation of serotonin. As expected, a unique CEST signal was measured between 4.75 – 5.75 ppm, which encompassed serotonin’s resonance frequency offset, in the midbrain raphe nuclei of healthy human volunteers. Serotonin CEST was successfully characterized in vitro and in vivo detection experiments show promise, however more work is required to improve the biological interpretation and validation of the results.