VictorJuiz/thesis huggingface dataset:
Uncovering the Functional Implications of Mu- and Delta-opioid Receptor Heteromerization in the Brain
Keywords: mu opioid receptor
delta opioid receptor
heteromer
depression
anxiety
brain
chronic morphine
G protein coupled receptor
mood
binding
internalization
interaction

Opioid Receptors (ORs) are involved in the pathophysiology of several neuropsychiatric conditions yet remain an untapped therapeutic resource. Although only mu-, delta-, and kappa-OR types have been cloned, additional subtypes result from complexes generated by direct receptor-receptor interactions. Mu- and delta-ORs form a heteromeric receptor complex with unique pharmacological and signalling properties distinct from those of mu- and delta-OR homomers. In these studies, we sought to characterize the ligand binding pocket and agonist-induced internalization profile of the mu-delta heteromer, to investigate mu-delta heteromer-specific signalling in brain, and to interrogate the contribution of this receptor complex to opioid-mediated behavioural effects. In competition radioligand binding studies, delta-agonists displaced high affinity mu-agonist binding from the mu-delta heteromer but not the muOR homomer, suggestive of delta-agonists occupying or allosterically modulating the muOR ligand binding pocket within the heteromer. Delta-agonists induced internalization of the mu-delta heteromer in a dose-dependent, pertussis toxin resistant, and muOR- and deltaOR-dependent manner from the cell surface via the clathrin and dynamin endocytic machinery. Agonist-induced internalization of the mu-delta heteromer persisted following chronic morphine treatment conditions which desensitized the muOR homomer. Using Galpha-specific GTPgammaS binding assays, we demonstrated that mu-delta heteromer signalling previously characterized in cell lines was present in the striatum and hippocampus, and did not desensitize following prolonged morphine treatment conditions which desensitized muOR homomer-mediated signalling. Since delta-agonists which also target the mu-delta heteromer possess antidepressant-like and anxiolytic-like properties, we investigated the role of this receptor complex in mood regulation. We devised a strategy to selectively analyze the effects of the mu-delta heteromer by dissociating it using a specific interfering peptide aimed at a sequence implicated in mu-delta heteromerization. The interfering peptide abolished the unique pharmacological and trafficking properties of delta-agonists at the mu-delta heteromer and dissociated this receptor complex in vitro. Intra-accumbens administration of the interfering peptide disrupted the mu-delta interaction in vivo and allowed for isolation of the mu-delta heteromer contribution to the mood-regulatory effects of a delta-agonist with activity at the heteromer. Activation of the mu-delta heteromer in the nucleus accumbens produced antidepressant-like and anxiolytic-like actions in animal models of depression and anxiety.
Femtosecond Laser Microfabrication of Optofluidic Lab-on-a-chip with Selective Chemical Etching
Keywords: microfluidics
femtosecond laser
laser micromachining
optical waveguide
chemical etching
optofluidic
electrophoresis
cytometry

The three-dimensional (3-D) writing capability of a high repetition rate (1 MHz) fiber-amplified femtosecond laser with a wavelength of 522 nm was harnessed together with wet-chemical etching for laser-patterning of 3-D optofluidic microsystems in fused silica glass, by the method of Femtosecond Laser Irradiation followed by Chemical Etching (FLICE). Selective chemical etching of laser irradiated glass with dilute hydrofluoric acid (HF) enabled micro-fabrication of high aspect-ratio embedded micro-channels and fine-period 3-D glass meshes in a 3-D inverted woodpile (IWP) arrangement that permitted high density lab-on-a-chip (LOC) integration of flow channels, reservoirs, glass chromatography columns, and optical circuit devices. Optical waveguides, reservoirs, micro-channels, and IWP structures were first laser patterned and followed by selective wet etching controlled by the polarization orientation of the writing laser. With the laser polarization perpendicular to the scanning direction, the volume nanogratings were aligned perpendicular to glass surfaces to facilitate HF etching and thus created designer shaped micro-channels with the smoothest sidewall surfaces measured at present and terminated with open reservoirs. An array of vertical access holes spaced periodically apart facilitated etching of continuous and highly uniform buried channels of unrestricted length in the glass to interconnect flow channels and reservoirs. Alternatively, laser polarization parallel to the scan direction provided low-loss optical waveguides with nanograting walls resisting the acid etching, providing a convenient one-step laser scanning process of optofluidic microsystems prior to wet etching. For the first time, dual-channel capillary electrophoresis was demonstrated by simultaneous fluorescent detection of separating dyes in a 3-D microsystem having over- and under-passing crossed channels in fused silica. In addition, an on-chip particle counting device based on capillary force to drive analytes through an embedded micro-channel into a calibrated reservoir for particle counting was designed and demonstrated. Further, a new type of glass mesh structure is presented where a 3-D IWP micro-channel array with diamond-like symmetry was integrated inside a micro-channel for capillary electrophoretic chromatography. The FLICE technique thus enables rapid prototyping of fully integrated 3-D optofluidic systems in bulk fused silica glasses for numerous applications, and these provide the groundwork and open new 3-D design approaches for advanced microsystems in the future.
Proper Language, Proper Citizen: Standard Linguistic Practice and Identity in Macedonian Primary Education
Keywords: Macedonian
identity
Macedonian language
education
textbooks
primary education
linguistic anthropology
post-socialist
Yugoslavia
Macedonia
anthropology
linguistics
illustration
linguistic identity
ethnic idenity
language and culture

This dissertation analyzes how the concept of the ideal citizen is shaped linguistically and visually in Macedonian textbooks and how this concept changes over time and in concert with changes in society. It is focused particularly on the role of primary education in the transmission of language, identity, and culture as part of the nation-building process. It is concerned with how schools construct linguistic norms in association with the construction of citizenship. The linguistic practices represented in textbooks depict “good language” and thus index also “good citizen.” Textbooks function as part of the broader sets of resources and practices with which education sets out to make citizens and thus they have an important role in shaping young people’s knowledge and feelings about the nation and nation-state, as well as language ideologies and practices. By analyzing the “ideal” citizen represented in a textbook we can begin to discern the goals of the government and society. To this end, I conduct a diachronic analysis of the Macedonian language used in elementary readers at several points from 1945 to 2000 using a combination of qualitative and quantitative methods. I catalogue and chart the frequency of certain linguistic forms and changes in their usage over time and contextualize these choices and changes within the greater changes of the narratives in the books. I conduct a similar analysis of the visual depictions of identity in these textbooks and the content of the textbooks with respect to notions of identity, nationalism, and other cultural factors.
Molecular Markers of Sensitivity to the Anticancer Effects of Different Statins in Human Tumour Cell Lines
Keywords: statin
P-glycoprotein
breast cancer
drug resistance
HMG-CoA reductase
mevalonate

Statins, common cholesterol control drugs, are appreciated to have promising anticancer activity through inhibition of the mevalonate pathway. Several lines of evidence suggest that certain tumours are susceptible to statins, but the underlying molecular features arbitrating this sensitivity remain unknown. We hypothesize that (i) not all statins will behave equivalently in the context of anticancer therapy, and (ii) a molecularly-defined subset of tumours are intrinsically sensitive to statins. My objectives have therefore been to further our understanding of functional differences between statins influencing their anticancer effects, and to investigate molecular features associated with statin sensitivity in breast cancer. Specifically, this thesis addresses two aims: (i) to characterize differential interactions between four statins and the xenobiotic transporter P-glycoprotein (P-gp; also known as ABCB1), and (ii) to identify molecular features associated with fluvastatin and lovastatin sensitivity in breast tumour cell lines. We first characterized the interactions of statins with P-gp in vitro and in multidrug-resistant (MDR) tumour cells. While lovastatin could directly bind to P-gp and modulate MDR, no significant interactions were observed with fluvastatin. Fluvastatin may therefore be appropriate for use in unselected patients, to avoid adverse drug interactions with coadministered P-gp substrate chemotherapeutics. Fluvastatin has also shown promise in breast cancer treatment, where molecular features predictive of statin sensitivity would be particularly valuable. A panel of 19 immortalized breast cell lines was therefore characterized for sensitivity to fluvastatin and lovastatin. Relatively statin-sensitive cells underwent apoptosis upon statin treatment, and were more likely to have an estrogen receptor alpha (ERα)-negative, basal-like phenotype. By mining available baseline gene expression data, a candidate 10-gene signature predictive of fluvastatin sensitivity was also generated. Taken together, this research provides insight into molecular markers of statin sensitivity that may facilitate fast-tracking of these drugs to clinical trials in subsets of cancer patients most likely to respond.
An Investigation of the Predictors of Emergent Literacy Skills in Junior Kindergarten
Keywords: Emergent literacy
Social Behaviour

Purpose: The primary aim of this thesis was to examine two aspects of early child development, namely emergent literacy skills and social-behavioural development. An examination of these two aspects of development occurs at the within child-level factor and the within-family level factor. Specifically, we seek to examine the effects of the family literacy environment in predicting 4-year-old children’s emergent literacy skills at entry to Junior Kindergarten. Additionally we seek to examine the interrelationships between children’s social behaviours and phonological awareness skills across the academic year. The final aim was to examine the relationship between teacher report and direct observations of children’s social behaviours. Method: One hundred and two children (52 boys, 50 girls) were recruited from 11 schools serving low-income neighbourhoods in a large metropolitan city and were assessed at the beginning and end of the Junior Kindergarten year. Family literacy questionnaires were collected at the beginning of the year. All children completed standardized assessments of their expressive vocabulary, non-verbal IQ, and phonological awareness skills. In addition, teachers completed behavioral ratings for all children. Results: The results of hierarchical multiple regression analyses revealed that direct teaching of letters/sounds in the home contributed 8% of the variance in children’s alphabet knowledge. The results also revealed that being excluded by peers at entry to Junior Kindergarten contributed up to 3% of the variance in negatively predicting phonological awareness outcomes at the end of the year. Further, the results revealed that poorer phonological awareness skills at the beginning of the year contributed up to 5% of the variance in predicting children’s preference for solitary behaviours at the end of the year. Finally, significant relationships were found between teacher report and direct observations for certain classroom behaviours. Conclusions: The results of this study suggest that the family literacy environment can play a positive role in children’s acquisition of emergent literacy skills prior to entry into formal schooling. The results also suggest that exclusion by peers may play a negative role in children’s ease of academic skill attainment. Conversely, the results suggest that the academic skill level that children enter into formal schooling with may play a role in their social behavioural competency in a classroom setting.
Lineage Specification of Pluripotent Populations in Murine Development
Keywords: Lineage specification
Pluripotency
Imprinting

“The scientist, by the very nature of his commitment, creates more and more questions, never fewer. Indeed the measure of our intellectual maturity, one philosopher suggests, is our capacity to feel less and less satisfied with our answers to better problems.” ~G.W. Allport, Becoming, 1955 It will be interesting to look back at this thesis in a few decades and reflect on how the questions and interpretation of data in the field of developmental biology have changed. Indeed, a biologist currently in their twilight years might reflect on their youth, before the discovery of hereditary material, and compare that bookend with the range of genome sequences and related knowledge currently available. How long will it take before this thesis reads like a debate about whether the male or female contributed the ‘homunculus,’ a miniature preformed human to the embryo that grows into an adult? In this thesis I asked three related questions: whether the role of Oct4 during embryogenesis provides insight into its contribution to pluripotency; how surfaceome changes contribute to functional maturation of neural stem cells and to what extent the murine genome is imprinted. Our data indicate that Oct4 is required for posterior expansion. We propose that the function of the protein is conserved, but that its expression has been coopted to yield different cell types based on its combination with different factors. We show that fundamental aspects of cell biology are altered during the maturation from pluripotent populations to neural stem cells, and identify mediators of proliferation, survival and adhesion that distinguish neural stem cell regulation from their precursors. Finally, we validated discovery of a dozen novel imprinted transcripts using a genomic approach. These discoveries will contribute to a holistic view of the causes and consequences of imprinting, but do not support a paradigm shift in the scale and consequences of imprinting.
The Role of the X-chromosomal Porcupine Homolog Gene in Mouse Development
Keywords: Porcn
Wnt
Mouse
Embryogenesis
Gastrulation
Pre-implantation

WNT ligands are secreted proteins that act as signals between cells. WNTs activate several interconnected signaling pathways that are required for embryonic development as well as tissue homeostasis in adults. The X-chromosomal Porcn gene encodes a membrane-bound O-acyl transferase that is required for the acylation of all 19 WNT ligands encoded in the mammalian genome. Non-acylated WNTs fail to be secreted from the producing cell and thus do not activate downstream signaling targets. In my thesis research, I have investigated the function of Porcn in mouse embryonic development. In vitro, I have shown that Porcn is required for canonical WNT signaling in ES cells and further, for their differentiation into endodermal and mesodermal derivatives. Taking advantage of a mouse line carrying a conditional (floxed) Porcn allele that I have generated, I have focused my studies on the early embryonic roles of Porcn using Cre recombinase-mediated and X chromosome inactivation-based ablation of Porcn function in vivo. I have found that the earliest requirement for Porcn in mouse development is the induction of gastrulation. In contrast to findings from in vitro studies, I have provided evidence that Porcn is not required for pre-implantation development in vivo. Dissecting embryonic and extra- embryonic roles of Porcn, I have been able to show that Porcn is required in the extra-embryonic chorion in order to mediate chorio-allantoic fusion, whereas ablation in the extra-embryonic visceral endoderm had no apparent effects. The extra-embryonic requirement for Porcn results in a parent-of-origin effect in Porcn heterozygous females due to X chromosome inactivation. In contrast to the placentation defect causing embryonic lethality of maternal allele mutants, deletion of the paternal allele caused variable fetal defects resulting in perinatal lethality with only rare survivors to adulthood. Both fetuses and adults represent a mouse model for Focal Dermal Hypoplasia (FDH), the syndrome caused by mutations in the human PORCN gene. My studies highlight the importance of PORCN-mediated WNT signaling for gastrulation, placentation, and fetal development, but suggest that endogenous WNT secretion does not play an essential role in either implantation or blastocyst lineage specification.
Diamonds as Development: Suffering for Opportunity in the Canadian North
Keywords: Diamonds
Northern Development
Vocational Education
Northwest Territories

Despite the repeated collapse of mining towns and sites in the Great Slave Lake region, most residents embrace new resource projects as possibilities for creating viable futures. Situated at the intersection of socio-cultural and linguistic anthropology, this ethnographic investigation of the Canadian diamond boom of the 2000s illustrates how imagining stable livelihoods despite a record of impermanence and crises depends on integrating and reframing past failures with present aspirations for “the good life”. At the height of the diamond boom in 2007, future imaginaries were largely associated with high wage job creation in the rapidly expanding industrial sector. Based on 18 months of fieldwork among those said to benefit most from new industrial development: the Aboriginal under/unemployed, this dissertation’s ethnographic attention is on job training programs and employment interventions that promised local residents new futures. The fieldwork coincided with the global financial crisis and almost none of the 90 students followed through the research secured work in the industry at the conclusion of their training. Nevertheless, people continue to maintain faith in a future linked to resource development. Capturing people’s everyday re-makings of tomorrow in uncertain times, this dissertation reveals that while employment in global extractive industries is unable to provide economic security to those who seek it, its promises are productive for four reasons. First, they (re)define the natural world as ‘opportunities for work’. Second, the specific techniques of industry and statecraft that surround mining (impact and benefit agreements, and socio-economic monitoring) transform everyday events of difference and inequality into catastrophes which render industrial development sensible even urgent. Third, they orient public sentiment towards a “future anterior,” a form of temporal longing that I argue impedes a deep reading of the historical present and participates in a politics of deferral. Fourth, they rely on and reproduce a chronotopically constrained public debate on natural resource development.
The Roles of the E3 Ubiquitin Ligases RNF126 and Rabring7 in Membrane Traffic
Keywords: ubiquitin
trafficking
RNF126
Rabring7

Integral membrane proteins are targeted to discrete compartments through the action of a number of transport pathways. The post-translational modification of cargo with ubiquitin is a key regulator of protein sorting. Ubiquitinated cargo are bound by specific cargo sorting machinery and directed towards the appropriate destination. Therefore, the identification and characterization of the proteins involved in cargo ubiquitination is critical to understanding the regulation of protein sorting. In the work presented here, we characterize the role of the E3 ubiquitin ligases, RNF126 and Rabring7, in two distinct membrane trafficking pathways. First, we show that RNF126 and Rabring7 are involved in the ligand induced downregulation of cell surface receptors. RNF126 and Rabring7 associate with the EGFR, amongst other RTKs, and promotes its ubiquitination. RNF126 and Rabring7 are required for the efficient sorting of the EGFR through the late endocytic compartment. We also show that the depletion of Rabring7 attenuates the degradation of MET and that both RNF126 and Rabring7 regulate the sorting of CXCR4 from an early endocytic compartment. In addition, the depletion of RNF126 or Rabring7 destabilizes ESCRT-II and reduces the number of multivesicular bodies formed after EGF stimulation. Second, we found that RNF126 regulates the sorting of the CI-MPR. In cells transiently depleted of RNF126, the CI-MPR is dispersed into a transferrin receptor positive endocytic compartment. This effect is specific to the CI-MPR as other cargos that are sorted between the endosome at the Golgi remain unaffected. We found that RNF126 physically associates with the clathrin adaptor GGA3 and promotes its ubiquitination, suggesting that RNF126 regulates GGA3 mediated CI-MPR sorting. Together, this work furthers our understanding regarding the role of ubiquitin in membrane traffic.
Characterization of Signal Transduction Abnormalities Revealed Spleen Tyrosine Kinase as a Therapeutic Target in High-risk Precursor B Cell Acute Lymphoblastic Leukemia
Keywords: spleen tyrosine kinase
acute lymphoblastic leukemia
signal transduction

Currently, the intensive chemotherapy remains the first line treatment for B cell acute lymphoblastic leukemia (B-ALL). Although these regimens have significantly improved patient outcomes, their use is associated with debilitating morbidities and fatal relapses, highlighting the great need in new agents that target essential survival signals in leukemia. Thus, the overall goal of my project was to gain insights into the signaling abnormalities that regulate aberrant proliferation and survival of B-ALL cells in an effort to identify novel targets in this malignancy. This study demonstrated that pre-B cell receptor (pre-BCR)-independent spleen tyrosine kinase (SYK) activity was required for the survival and proliferation of a p53-/-PrkdcSCID/SCID mouse model of B-ALL. I extended this discovery to human disease, demonstrating that SYK was activated in primary B-ALL, independent of the pre-BCR expression. The small molecule SYK inhibitor fostamatinib (fosta) significantly attenuated proliferation of 79 primary diagnostic B-ALL samples at clinically achievable concentrations. Importantly, fosta treatment reduced dissemination of engrafting B-ALL cells into the spleen, liver, kidney and central nervous system (CNS) in a NOD.Prkdcscid/scidIl2rgtm1Wjl/SzJ xenotransplant model of B-ALL. Analysis of signaling abnormalities using a high-throughput phospho-flow cytometry platform demonstrated that pediatric and adult B-ALL samples exhibit variable basal activation of BCR, iii PI3K/AKT/mTOR, MAPK and JAK/STAT pathways. Importantly, we identified that fosta-mediated inhibition of SYK, PLC2, CRKL and EIF4E phosphorylation in B-ALL was predictive of its anti-leukemic activity, and was distinct from the cellular actions of other small molecule inhibitors of key nodal signaling pathways. Examination of molecular mechanism of fosta action by gene expression profiling revealed transcriptional effects of fosta treatment that included, most notably, potent inhibition of pathways involved in lymphocyte activation and inflammation. In conclusion, this study demonstrates that SYK signaling is crucial for B-ALL survival and provides detailed characterization of cellular and molecular mechanisms of fosta action in B-ALL. These data argue in favor of testing small molecule SYK inhibitors in pediatric and adult B-ALL.