VictorJuiz/thesis huggingface dataset:
Functional Annotation of Uncharacterized Enzymes in Saccharomyces cerevisiae
Keywords: Enzyme
Functional Genomics
Metabolism
Metabolomics
Saccharomyces cerevisiae

In the post-genomic era, clinicians and scientists are increasingly reliant on interpretation of variants in metabolic genes for determining pathogenicity. These interpretations depend on functional annotation of the roles genes provide in metabolism, annotation which is far from complete. I embarked on a journey of enzyme discovery to fill gaps in our knowledge of metabolism in the budding yeast, Saccharomyces cerevisiae. I carried out a genetic and metabolomic screen of 120 uncharacterized candidate enzyme encoding genes that comprised my MSc. This dissertation describes my work in ascribing function to two distinct enzymes, Das2 and Tda5. Throughout my study I have found that Das2 is a novel uridine/cytidine kinase that functions in concert with a second minor uridine kinase, Urk1. These two enzymes are interdependent and in turn depend on a third enzyme, the major uracil phosphoribosyl transferase, Fur1 for stability. These three enzymes form a complex that is essential to wild-type pyrimidine salvage. As I aimed to elucidate the function of Tda5, I discovered that this uncharacterized enzyme is essential to growth. Loss of function mutations in TDA5 are alleviated by de-repression of its sporulation specific paralog, Ydl114w, and when YDL114W is deleted, tda5Δ is rescued by hypomorphic mutations in the ergosterol biosynthetic pathway. Analysis of metabolite levels in hand with nutritional sensitivities and genetic interaction data support the essential role of Tda5 in lipid homeostasis. Together, these studies assign new functions to previously uncharacterized genes and reveal functional interdependence of enzymes that sheds light on metabolic interpretations of genetic data.
Exosomal microRNAs in Cerebrospinal Fluid as Biomarkers for Amyotrophic Lateral Sclerosis and Frontotemporal Dementia
Keywords: Amyotrophic lateral sclerosis
Frontotemporal dementia
microRNA

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by loss of motor neurons from the brain, brain stem, and spinal cord, resulting in progressive paralysis and death. Patients with frontotemporal dementia (FTD) usually present with either behavioural or language impairment secondary to atrophy of the frontal and/or temporal lobes. Recent evidence suggests that about 15% of patients with ALS develop FTD and about 15% of patients with FTD develop ALS. ALS and FTD are typically diagnosed months to years after symptom onset. Hence, there is an unmet need to enhance the diagnostic process. The overlap in clinical presentation and the recent discovery of shared genetic and pathological features have changed our view of ALS and FTD, which are now often thought of diseases on a spectrum, rather than two distinct disease entities. Aberrant RNA processing causing dysfunction of vital cellular processes has emerged as a common theme in ALS and FTD research. There is increasing evidence that alterations in the expression of non-coding RNAs, such as microRNAs (miRNA or miR-), occur in ALS and FTD. In human biofluids, miRNAs have been found enriched in small vesicles termed exosomes, providing an opportunity for miRNA biomarker profiling in ALS and FTD with the goal to identify miRNAs that have diagnostic value. Studies presented here have identified dysregulated exosomal miR-204-5p and miR-632 in cerebrospinal fluid (CSF) as diagnostic biomarker candidates for genetic FTD and miR-632 also for sporadic FTD. For ALS, we provide evidence for altered exosomal 16-5p, miR-376c-3p, miR-451a, miR- 632, and miR-2110 expression in CSF. In silico analysis uncovered targets of these miRNAs in biological pathways associated with ALS and FTD. TAR DNA-Binding Protein 43 (TDP-43), a hallmark protein of ALS and FTD pathology, has been shown to be implicated in different steps of miRNA biogenesis and data presented here further supports the role of TDP-43 in the dysregulation of exosomal miRNA. As such, exosomal miRNAs may represent disease-relevant biomarkers with potential to improve the diagnostic process resulting in earlier and more accurate diagnosis of ALS and FTD.
Investigation of the Tail Fibres and Tail Fibre Assembly Proteins of Contractile-tailed Phages
Keywords: Bacteriophage
Phage
Phage receptor-binding proteins
Phage tail
Tail fibre assembly
Tail fibres

The tail fibres of long-tailed phages are complex elongated protein assemblies capable of specific recognition of bacteria during the first step of viral infection. Correct assembly of these structures is crucial for successful phage propagation and often requires a phage-encoded tail fibre assembly (Tfa) protein. In Escherichia coli phage T4, the formation of the distal portion of tail fibres requires a Tfa (product of gene 38) which is absent from mature particles. Despite the essential role of Tfa proteins for many diverse phages, only a few studies have addressed their functional mechanisms. Here, I investigate the tail fibre and Tfa of E. coli phage Mu. I demonstrate that Tfa forms a stable complex with the tail fibre, and I present a 2.5 Å resolution X-ray crystal structure of this complex. The Tfa:tail fibre complex is shown to bind the phage baseplate wedge and lipopolysaccharide, the outer membrane receptor for this phage. My in vitro and bioinformatics studies reveal that Tfa proteins are comprised of two domains: a non-conserved N-terminal domain that binds to the fibre and a conserved C-terminal domain that likely mediates fibre oligomerization and assembly. I propose that the key role of Tfa proteins is to initiate multimerization and assembly at the C-terminus of fibres, ensuring that their intertwined and repetitive structural elements come together in the correct sequence. Surpringly, I also detected Mu Tfa in the mature particles of Mu. In addition, I discovered that the Tfa proteins of phages Mu and E. coli phage P2 bound to lipopolysaccharide. Despite sharing 93% sequence identity these Tfa proteins displayed different LPS-binding preferences, consistent with those of full phage particles. In summary, I conclude that Mu Tfa is required for tail fibre folding and oligomerization as was seen for phage T4. In contrast to T4, however, Mu Tfa binds stably to the tail fibre and stays bound even in the mature particle. This observation coupled to the demonstrated LPS-binding activity of Mu and P2 Tfa suggests that Tfa proteins may play a role in determining host specificity. Thus, the function of “chaperone” may not apply to many Tfa proteins.
The Role of the Transcription Factor FUSCA3 in Reproductive Development of Arabidopsis thaliana
Keywords: FUSCA3
Reproduction
Seed Development

Reproduction is a fundamental stage in the cycle of life. In flowering plants, this begins with flowering and ends with seed setting. Although extensive work has been done on these complex processes, there are many gaps in our understanding of the underlying mechanisms and genetic networks. The B3 transcription factor FUSCA3 (FUS3) plays a key role in controlling seed maturation processes and developmental phase transitions through the modulation of hormone levels. Many downstream targets have been identified through transcriptomic and chromatin immunoprecipitation studies, however, the regulation of FUS3 activity is poorly understood. Recently, it was shown that FUS3 interacts with and is phosphorylated by AKIN10, a kinase subunit of the Sucrose non fermenting 1 (Snf1)-RELATED KINASE 1 (SnRK1) complex. SnRK1 is a central regulator of carbon levels and stress response that is conserved among eukaryotes. This opens the possibility that stress signals working through SnRK1 may regulate seed development through FUS3 phosphorylation. In this study, I investigated the function of FUS3 phosphorylation in Arabidopsis thaliana. The SnRK1 phosphorylation site of FUS3 is embedded in the basic B2 domain, which is strongly conserved among eudicot species. Characterization of the loss-of-function fus3-3 mutant, as well as transgenic fus3-3 lines carrying phosphonull and phosphomimic FUS3 variants, showed that FUS3 phosphorylation does not affect seed maturation, but regulates reproductive and early seed development, uncovering novel roles for FUS3. FUS3 was found to regulate pollen viability and the developmental rate of the endosperm, integument and embryo. Fertility was reduced in fus3-3 as a result of both pollen defects and maternally-derived post-fertilization seed abortion. Intriguingly, pollen and seed abortion in fus3-3 were higher under heat stress compared to the wild type. Except for pollen defects, all these novel fus3-3 phenotypes were rescued by wild type and phosphomimic FUS3 variants, while the phosphonull FUS3 variant enhanced them. Together, these findings demonstrate that FUS3 phosphorylation plays an important role in reproductive and early seed development, and that FUS3 regulates adaptive reproductive responses by acting downstream of stress signals in Arabidopsis thaliana.
Shared Decision Making in Youth with Brachial Plexus Birth Injury and Their Families
Keywords: Adolescent
Brachial Plexus
Children
Decision Making
Decision Support Techniques
Elbow Contracture

Introduction: This dissertation used an overarching interpretivist qualitative framework in the development of a patient decision aid (PtDA) for youth with brachial plexus birth palsy (BPBI) and their parents when making preference-sensitive decisions regarding rehabilitation and surgical treatment options for an elbow flexion contracture. The objective was to develop a tool containing evidence-based information and a values clarification method to help these youth (ages 8 and 18 years) and their families discern what they value most related to their options. Methods: PtDA development was guided by a conceptual framework built on existing PtDA models and standards. A philosophical hermeneutic approach combined with arts-based methods was used to conduct a decisional needs assessment based on two qualitative sources: in-depth interviews with 5 young adults and 14 youth-parent dyads, and 15 participant observation sessions of families and clinicians in the clinic setting. These qualitative findings were integrated with evidence from knowledge synthesis methods on elbow flexion contractures and its treatment options to develop the PtDA prototype, which was in turn field-tested with 17 youth via cognitive-based interviewing. Results: Several substantive contributions to the fields of BPBI and pediatric shared decision making were achieved through the process of developing this PtDA. A PtDA for youth with BPBI was created and an arts-based qualitative approach provided a unique way to interpret and understand the family-team clinic experience. Overall, the shared decision making in youth with BPBI was influenced more by a family’s appearance-related concerns and the strength of their trust relationship with clinicians, and less by their activity and participation concerns. Although decisional conflict in these families seemed rare, once rehabilitation treatment was initiated, decision regret was sometimes expressed. Regret was associated with youth-parent decision discord that was influenced by parental emotions, youth’s response to external pressures, and experiences with treatment adherence. Conclusions: By revealing the complexities of shared decision making in youth with BPBI, this research reflects the real-world concerns of these families in the design of this PtDA. The insights gained offer guidance for future PtDA implementation that optimize its potential to promote positive health outcomes in youth with BPBI and their families.
Perceptions of Change Among Emergency Department Staff in a University Teaching Hospital in the United Kingdom
Keywords: change management
continuing education of health professionals
hospitals
organizational change
organizational learning
staff attitudes

University teaching hospitals provide acute and emergency care in most healthcare systems. One of the priority areas in hospitals are emergency departments (ED) where practice changes are the norm due to the unpredictable and open “public good access” nature of service demands. At present changes to EDs are implemented to improve efficiency and reduce costs in the United Kingdom (UK) and other developed countries. Also, at present, there is limited evidence on how or why change is successful in some instances and not in others. The objective of this research was to study staff perceptions of change and the factors influencing the implementation of changes at a university teaching hospital in the UK. A modified grounded theory methodology was used to perform a secondary analysis of 41 interview transcripts to answer the research questions. Interview transcripts from participants consisted of 20 physicians, 13 nurses, two support workers and six managers involved in paediatric emergency care at the hospital. Data analysis provided answers to the first research question of how changes happen in the emergency department by identifying triggers and barriers to both planned and emergent changes. The second research question of what factors impact change identified three themes; communication, education and leadership. The concept that permeated all identified themes was that change provokes emotional responses at individual, team, and organizational levels. Good communication leading to staff involvement, education about new processes and credible trusted leadership, encouraged positive emotions and reduced negative emotions. In this study, negative emotions stemmed from a perceived inability of healthcare professionals to function effectively and efficiently to ensure safe patient care with changes. Professional values dictated the actions or inactions that transpired either as a result of these emotions or despite these emotions in health professionals. The study findings illustrate an emerging concept of emotions in change and the importance of acknowledging the reasons for negative emotions and fostering positive emotions in individuals and groups through communication, education and good leadership. I hope this concept will ultimately advance the theoretical understanding and practical strategies to help the process of change management in healthcare organizations.
Investigating the functional roles of NBEAL2 in megakaryocytes and platelets
Keywords: Granules
Gray Platelet Syndrome
Hematology
Megakaryocytes
NBEAL2
Platelets

Platelets are small anucleate blood cells that co-ordinate blood clotting in response to vascular injury. In additional to their hemostatic role, platelets are involved in a diverse range of physiological and pathological processes. Platelets contain abundant secretory vesicles associated with their functions, which are generally classed as α-granules, δ-granules and lysosomes. These vesicles originate in megakaryocytes (MK), hematopoietic cells that produce platelets in the bone marrow. The inherited bleeding disorder Gray Platelet Syndrome (GPS) is characterized by platelet α-granule deficiency arising from loss of function of neurobeachin-like 2 (NBEAL2). The goal of my study was to investigate the roles and functions of NBEAL2 in MKs and platelets. Using biochemical assays, it was found that NBEAL2 is a cytosolic protein capable of self-association and adopts two distinct oligomeric states in MKs. Immunoprecipitation-mass spectrometry was used to identify several potential NBEAL2-interacting proteins, providing insights into its cellular functions. Among these interactors, SEC22B was shown to be essential for the formation of α-granules, where loss of SEC22B expression resulted in an α-granule deficiency similar to that caused by the loss of NBEAL2. Forms of NBEAL2 expressing the GPS-associated mutations E1833K or R1839C failed to interact with SEC22B, indicating a functional significance for NBEAL2-SEC22B interactions in α-granule biogenesis. A Nbeal2-/- mouse strain was generated to model GPS. Studies of Nbeal2-/- mice revealed that NBEAL2 is required for normal platelet activation and aggregation, and for normal development and proplatelet-platelet production by MKs. Studies of the uptake and intracellular trafficking of the α-granule cargo protein fibrinogen (FGN) revealed that NBEAL2 is not essential for FGN endocytosis by MKs, nor its passage through endocytic compartments associated with RAB5, RAB7 and the α-granule membrane protein P-selectin. The latter compartment retains both endocytosed and MK-synthesized cargo proteins when NBEAL2 is present, but in cells lacking NBEAL2 cargo proteins are lost from this compartment and exit MKs via a RAB11-mediated secretory pathway. Intracellular protein colocalization and co-immunoprecipitation studies revealed a potential interaction between NBEAL2 and P-selectin in MKs and platelets. Thus we propose that NBEAL2 is required for the stabilization of α-granules during cargo loading, maturation and circulation within platelets.
Writing Rights, Writing Violence: The Bureaucracy of Palestinian Testimonies in Israeli Human Rights NGOs
Keywords: Bureaucracy
Human Rights
Israel/Palestine
NGOs
Violence
Witnessing

This dissertation is an ethnography of Israeli non-governmental organizations (NGOs), that document the violations of Palestinians’ human rights (HR) perpetrated by Israel’s occupation of Palestinian territories. Israeli anti-occupation NGOs rely on a rigid bureaucratic logic in their processing of testimony – a written-down narration of a subjective experience of violence into a coherent narrative in document-form: violence is translated into a written text that can be used for legal-action purposes, research, media circulation, and is eventually archived. By anthropologically examining bureaucratic lugubriousness in NGOs, I theorize HR as a project of documentation and archiving that legitimates itself as a subversive bureaucracy. Simultaneously, this project’s own critical authority is undermined since it relies on colonial modes of knowledge production and representation. Based on 18 months of participant-observation in four NGOs, I trace the trajectory of Palestinians’ testimonies as NGOs’ main source of data and as HR’ dominant genre of representation. NGO practices are considered against the backdrop of growing disdain in Israel/Palestine towards HR, not only in the populist rhetoric and efforts of anti-liberal politicians, but also amongst activists, critical scholars, Palestinians living under the occupation, and within NGOs’ own staff. The main recurring critique is that HR fail to protect Palestinians or curb Israel’s settler-colonialism. Some detractors claim that NGOs are complicit with Israel’s violence. This study explores why Palestinians continue to testify for Israeli NGOs that, in turn, persist in documenting Palestinians’ testimonies. I suggest that HR are a genre of anti-colonial historiography that is itself based on colonial reason, mainly genealogies of surveying and bureaucratic writing: NGOs rely on what I term as “frames of in/validation” - phases of incessant verification and adaptation of Palestinian experiences of violence into simplified narrative structures, that conform to legal-moral discourses and definitions of HR. While the strictures of documentation and writing problematically limit the moral claims and political clout of NGOs, HR paradoxically remain relevant through the relational foundation affirmed by frames of in/validation, namely: between a violent past written as a bureaucratic document, and a distant future in which imagined publics make moral-political judgments based on NGOs’ archives.
Exploring Latent Profiles of Writing in Linguistically Diverse Students in the Middle School Grades
Keywords: English Learners
gender
literacy
narrative text writing
predictors of at-risk writing

This thesis consists of two studies that investigate longitudinally narrative writing development and factors that lead to low performance in writing in the middle school grades with native English speakers (EL1) and English Learners. The first study consists of two parts. Part A examined writing profiles in Grade 4 and 6 and the resulting relationships to reading outcome measures as well as the effect of language status and gender. Three distinct writing profiles were found in Grade 4 and 6 (low performing, average, and strong writers) which corresponded to similar ability levels on the word reading and reading comprehension outcome measures. There was no EL-EL1 effect on the writing profiles, however there was a gender effect showing that boys were more at risk for writing difficulties than girls. Part B investigated the transition patterns of these writing profiles between Grades 4 to 6. The writing profiles were fairly stable across the middle school grades with a high proportion of students remaining in the same latent profile across time (76.4%). Differences were noted in the transition profiles of boys and girls with more boys remaining in the low performing group over time.The second study investigated the contribution of oral language (vocabulary and syntactic skills) to low performing writing status, beyond that of a base set of predictors, that included rapid naming, working memory, and spelling. In Grade 4, both vocabulary and syntactic skills contributed independently to low performing writing status beyond the base measures. By Grade 6, only syntactic skills were found to be a unique predictor of low performing writing status beyond the base measures. Overall, this thesis provides support that distinct and stable writing profiles can be recognized by the middle school grades in EL and EL1 students and offers insight into the characteristics of low performing writers. The implications of these findings for assessment and intervention and directions for future research are discussed.