VictorJuiz/thesis huggingface dataset:
Insights on the Xenobiotic Barrier Function of the Caenorhabditis elegans Pharynx Cuticle

Cuticles are specialized extracellular matrices that prevent water loss and provide a barrier to external, harmful chemical and infectious agents. In the model nematode C. elegans, I report the serendipitous discovery that certain lethal molecules accumulate in the heads of worms as crystalline and spherical objects. Using DIC microscopy, transmission electron microscopy, pulse-chase, RNA interference and fluorescent dyes, I confirmed that the crystalizing molecules preferentially accumulate in the non-luminal side of the anterior pharynx cuticle. A chemical screen of 2560 molecules revealed that crystal formation can be suppressed by 85 small molecules as well as the amyloid dyes Congo Red and Thioflavin S, but not by chitin dyes. Many crystallizers and suppressors have highly similar core scaffolds and are enriched for aromatic rings, with the ability to form hydrogen, halogen and π-π bonds. Interestingly, 43% of the substructures that are ≥1.5X enriched in crystallizing molecules compared to molecules that do not form objects and/or a random set of molecules, are known to bind amyloids. 33% of the core scaffolds of the crystal suppressors are found in known amyloid-interactors. Bioinformatic analysis of the C. elegans pharynx proteome revealed that during molting, the pharynx secretes in the cuticle large amounts of proteins/peptides with phase-separation ability, a property shared by amyloidogenic proteins. Through a genetic screen, I found that loss of components of a novel sphingomyelin synthase, SMS-5, or an ABC transporter, PGP-14, which are both expressed in the pharynx, abolishes crystal formation in the pharyngeal cuticle but also drastically alters the worm's overall sensitivity to xenobiotics/ This indicates that the proteins play a critical barrier function. Taken together, my Ph.D. work identifies the pharynx cuticle as a critical determinant of drug sensitivity, and implicates sphingomyelin, an ABC transporter and potentially phase-separated proteins/peptides as novel determinants of xenobiotics accumulation in C. elegans.

Keywords: C elegans
crystal
cuticle
drug resistance
model organism
pharynx
Advisor: Roy, Peter Dr
Antenatal Synthetic Glucocorticoid Programming of Post-natal Blood-brain Barrier Drug Transporters

The blood-brain barrier (BBB) is essential in protecting the developing brain from neurotoxins, through high expression of drug transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). However, external stimuli such as xenobiotic exposure may disrupt BBB protection. Antenatal synthetic glucocorticoid (sGC) therapy is a life-saving treatment administered to pregnant women at risk for delivering preterm, to aid in the maturation of several fetal organ systems, notably the lungs. The purpose of the studies described in this thesis were to investigate the long-term effects of antenatal sGC on the levels and function of BBB drug transporters. We investigated drug transporter levels and function in brain endothelial cells (BECs) of postnatal day (PND) 14 male and female offspring prenatally exposed to multiple or single course betamethasone (BETA) treatment. We have identified a reduction in the levels and function of P-gp in male offspring only, as well as sex differences in baseline P-gp function in BECs derived from juvenile guinea pigs. Importantly, we found a single course did not impact P-gp function. Recently miRNAs have had increased attention as a regulatory mechanism for drug transporter expression. As we observed decreased P-gp protein and function independent of changes in mRNA levels, we investigated miRNAs as a potential mechanism underlying these effects. Our targeted analysis of 7 miRNAs did not reveal differences after antenatal sGC. However, these initial studies have laid the groundwork for future investigations into miRNA regulation of BBB transporters. Finally, while levels of P-gp mRNA/protein, and function were unaltered in BECs derived from female offspring, once exposed to a challenge (infection mimic ssRNA, P-gp activity was markedly reduced. This is in-line with previous research that the long-term effects of antenatal sGCs do not always manifest themselves at baseline. The findings from this thesis have implications as decreased P-gp function can result in increased transfer of neurotoxic P-gp substrates into the brain of infant (e.g., bilirubin in hyperbilirubinemia) and pediatric (e.g., colchicine poisoning) populations. Additionally, long-term changes in drug transporter activity can have consequences for central nervous system (CNS) exposure to harmful agents across the entire life-course.

Keywords: blood-brain barrier
breast cancer resistance protein
glucocorticoid
p-glycoprotein
preterm birth
Advisor: Matthews, Stephen G
Genetically Engineering Microbes for Detecting and Treating Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) is a chronic medical condition characterized by inflammation in the intestine. Treating the patients starts by appropriately diagnosing the disease, followed by administering therapeutic drugs to reduce the symptoms and induce tissue repair that can help the patient achieve remission. However, as there are no definitive cures, treatment of IBD requires long-term management that involves periodically monitoring the extent of inflammation within the intestine and several treatment courses. Traditional methods for detecting the disease are highly intrusive, and the therapies are not entirely effective, often leading to relapse and sometimes even requiring surgical interventions. Therefore, there is a need for better diagnostic tools and treatment options to improve the outcomes and quality of life of IBD patients. Small, single-celled organisms, such as bacteria or yeast, have the unique ability to travel through the gastrointestinal tract and into the intestine- a natural habitat for microbes. Once inside this area, they come in close contact to the sites of inflammation, priming them for disease intervention. As these microbes are living organisms, they can also be genetically re-programmed to accomplish desired functions such as sensing molecular or environmental cues, or to deliver molecules with therapeutic properties. With the advances in genetic engineering tools and strategies, microbes can be engineered into diagnostic tools to monitor the inflammation within the patient’s intestine and as therapeutic delivery systems to provide targeted therapies at the source of the disease. The goal of this project was to contribute to the development of engineered microbes for detecting and treating IBD. These contributions include developing and validating genome editing tools, therapeutic protein expression systems, biosensors to detect reactive oxygen species, and a colorimetric yeast-based assay to detect biomarkers of IBD using a probiotic yeast strain Saccharomyces boulardii. I also discovered novel transcriptional markers of colitis within a probiotic bacteria strain, E. coli Nissle, that could be used to monitor intestinal inflammation. Overall, the results of this project should prove valuable for engineering microbial devices for medical applications.

Keywords: Escherichia coli Nissle
Genetic engineering
Genetically engineered microbes
Inflammatory bowel disease
Saccharomyces boulardii
Synthetic Biology
Advisor: McMillen, David
Methacrylic Acid-Based Biomaterials Promote Terminal Axon Growth

Peripheral innervation is essential for all tissue engineering strategies as all tissues are innervated. In absence of innervation, tissue regeneration is impaired. Here we show a new role for MAA-based biomaterials in promoting terminal axon nerve growth. Our results demonstrate that MAA-based biomaterials promote peripheral nerve growth in an Igf-1 and Shh dependent manner. The resulting nerves increased the sensitivity of treated mouse paws to nociception. iDISCO clearing showed that MAA increased the presence of peripheral nerve structures in whole explants. MAA was also able to increase the expression of key neuronal markers and growth factors in a peripheral neuropathy model, the diabetic db/db mouse, suggesting that MAA-based biomaterials may be relevant to treatment of peripheral neuropathy. Moreover, in a peripheral neuropathy model, MAA was able to up-regulate the expression of growth factors for an extended duration suggesting MAA may prevent degeneration through an effect on factors that promote survival. Through the use of single-nucleus RNA-sequencing we profile whole-explants of subcutaneous tissue following 21-day treatment with MAA and show that MAA explants contain a unique cell population that is a key source of factors that promotes axon growth. We also identify a new role for Nrg1-ErbB signalling in promoting MAA-induced peripheral nerve growth.Moreover, we also use an unbiased proteomics approach to further characterize PEG and MAA hydrogel explants. Our results demonstrate that MAA treatment results in the differential expression of proteins and show that MAA may have a temporal effect on protein expression. Proteomic profiling also identifies new signalling pathways that may be involved in the mechanism of MAA-induced axon outgrowth including GCPR, integrin, and mTOR signalling. As all tissues are innervated, MAA-based biomaterials could have broad applications in the promoting regeneration and preventing degeneration of peripheral nerves.

Advisor: Sefton, Michael V
Allosteric Regulation of the Helicobacter pylori NikR Transcription Factor in Response to Nickel and pH

Survival of the Helicobacter pylori pathogen within the acidic environment of the human stomach requires the nickel enzyme urease, which makes nickel an essential nutrient for this bacterium. Due to its inherent toxicity and limited availability, nickel homeostasis is maintained through a network of pathways that are coordinated by the nickel-responsive transcription factor, NikR. Nickel binding to H. pylori NikR (HpNikR) allosterically activates binding to several target promoters encoding genes involved in nickel homeostasis and acid acclimation to either activate or repress their transcription. HpNikR can also respond to a drop in pH and bind target promoters independently of nickel to regulate their transcription. In this work, the allosteric mechanisms by which nickel, and acidity activate DNA binding by HpNikR were explored using biochemical, spectroscopic, and computational methods. The results demonstrate that HpNikR adopts an equilibrium between an open conformation and more DNA-binding competent cis and trans states, regardless of nickel binding or increased acidity. The response to nickel involves an increase in protein rigidity that is propagated from the nickel-binding sites to the DNA-binding domains through a network of residues linked to the inter-domain interface. This response allows nickel binding to slow down conformational exchange and shift the equilibrium toward the DNA binding-competent states. The protein then becomes stabilized in a cis conformation upon binding a target promoter, in a manner consistent with conformational selection. In addition, the DNA sequence was found to play an important role in tuning the allosteric response. Experiments at lower pH levels revealed that the histidine residues of the nickel-binding sites are involved in the allosteric response to acidity. However, the mechanism of pH-responsive DNA binding is quite distinct compared to the response to nickel, and results in a unique DNA-bound cis conformation. Overall, this work provides novel insights into how HpNikR is able to respond to two important environmental cues for H. pylori – nickel and pH. This knowledge advances our understanding of the allosteric mechanisms employed by metalloregulators, and contributes to our understanding of how HpNikR helps this bacterial pathogen thrive in the harsh environment of the human stomach.

Keywords: Acid Adaptation
Allostery
Helicobacter pylori
Metal Homeostasis
Nickel
Transcription Factor
Advisor: Zamble, Deborah B
Shaman as Saviour? The Trope of the Amazonian Shaman in Recent Latin American Literature and Film

This dissertation analyzes the trope of the Amazonian shaman in recent Latin American literature and film. References to shamanism have become increasingly popular in recent years. Shamans have gained importance in environmentalism and struggles for Indigenous rights, as well as academic writing interested in radical social change. Meanwhile, shamanic tourism attracts thousands to the Amazon region each year. The books and films of this dissertation participate in this trend by mobilizing the shaman as a figure that holds privileged solutions to contemporary problems like ecological decline, climate change, and persistent coloniality. I argue that these shamanic solutions are often superficial and aimed at satisfying Western desires for progress and limitless growth. Furthermore, they reproduce problematic colonialist tropes, including Western fascination with the ‘wild man.’ Nevertheless, my readings also show how these works may suggest possibilities for change. I draw on critical environmental scholarship, including the work of Elizabeth Povinelli, Stacy Alaimo, Déborah Danowski and Eduardo Viveiros de Castro to show how the shaman characters’ risk-laden and grounded negotiations with materiality may sometimes challenge anthropocentrism and narratives of progress. Moreover, through their frequent references to the Amazonian archive of colonial texts, anthropology, literature and film, the works themselves model a similar process of negotiation that transforms the notion of shamanism. My own contribution via this dissertation is also an inevitable part of this ongoing engagement. Chapter 1 analyzes the influence of shamanic ayahuasca tourism and desires for reconciliation with Indigenous peoples over the abuses of colonialism in the film El abrazo de la serpiente (2015). Chapter 2 addresses how the film Ícaros: A Vision (2016) undermines the drama of shamanic miracle healing and tourism through a focus on the characters’ imbrication with materiality. Chapter 3 compares the ambivalence of shamanic pharmakon remedies in the novels Todas as coisas são pequenas (2008) and El último guerrero de’Aruwa (2006), and the film Chamán: El último guerrero (2016). Chapter 4 analyzes shaman Davi Kopenawa’s techniques for maneuvering restrictions on Indigenous discourse in The Falling Sky: Words of a Yanomami Shaman (2010).

Keywords: Amazon
anthropology
cinema
environment
literature
shamanism
Advisor: Antebi, Susan
Global Post-Transcriptional Regulation during Human Neuronal Development and Disease

Globally coordinated post-transcriptional mechanisms that regulate the transcriptome through mRNA stability and translation are relatively understudied during human neurodevelopment and disease. Our lab uses human induced pluripotent stem cells (iPSC) to model neurodevelopment in vitro by directed differentiation into neural progenitor cells (NPC) and cortical neurons. The iPSC model illuminates the mRNA regulatory programs that act during neurodevelopment and disease. Deficiency in the global transcription and chromatin regulator methyl-CpG-binding protein 2 (MECP2) leads to a neurodevelopmental disorder, Rett Syndrome (RTT). Recent molecular studies in RTT focused on transcriptionally altered genes. However, changes in the overall protein synthesis and microRNA biogenesis in RTT neurons point to the contribution of post-transcriptional mechanisms. I dissect the post-transcriptional regulatory layer by measuring mRNA synthesis, degradation, translation rates, and microRNA abundance in neurodevelopment and RTT. First, I described global changes in translation during neurodevelopment and identified genes bound by RNA-binding protein TIA1 that substantially influence MECP2 translation during differentiation to neurons. Second, I studied whether MECP2-deficient neurons have altered mRNA translation. I detected widespread and global changes in ribosomal engagement, agreeing with decreased protein synthesis in RTT neurons. I also captured translational misregulation of NEDD4-family E3-ubiquitin ligases. It highlights altered ubiquitination as a novel molecular signature in RTT. Third, I explored the contribution of mRNA stability to shaping the transcriptome during normal development and RTT. I observed significant shifts in median RNA stability during neuronal differentiation. This global change in RNA stability differentially affects gene isoforms, shaping the transcriptome. A concurrent shift in microRNA copy number per cell suggests a common genome-wide mechanism that drives the process. Next, I probed the degree of coupling between transcription and mRNA stability. I observed a widespread boosting and buffering of transcription rate shifts during neurodevelopment. This crosstalk between nuclear and cytoplasmic machinery is striking upon loss of MECP2. Reanalysis of mouse Mecp2-deficient neurons supported the existence of global buffering, suggesting that mRNA stability response is a conserved feature of RTT models. In summary, I identified a unique role of mRNA stability and translation in the regulation of transcriptome and in mounting a genome-wide balancing response to altered transcription.

Keywords: Neurodevelopment
Post-transcriptional
Rett
RNA
Advisor: Ellis, James
Improving Allele Calling in Highly Polymorphic and Repetitive Genomic Regions

Many regions of the human genome are highly polymorphic, containing numerous variants not found in the current reference genome and making it difficult for variant calling software to accurately identify variants. The effects are particularly pronounced when analyzing structural variants and repetitive regions with short-read sequencing owing to unmapped reads. Long-read sequencing greatly improves structural variant identification, but there is an abundance of data already available generated from short-read technology. Adapting current variant calling approaches for problematic regions of the genome can provide novel insights about variation from existing datasets. This thesis focuses on developing and assessing methods to improve variant calling for polymorphic repetitive genomic regions using the highly variable minisatellite-like zinc finger repeat array of the gene PRDM9 as a proof-of-concept region. I developed short-read models that utilize k-mer information to distinguish between the numerous PRDM9 alleles that differ by single nucleotide polymorphisms and copy number variants of zinc finger repeats. These models were able to call haploid and diploid PRDM9 genotypes with average F1 scores of up to 0.99 and 0.73, respectively, using simulated sequencing reads under typical conditions. Using real Illumina data, the models were able to genotype 43% of 101 samples correctly and ranked the correct genotype in the top 10 for 69% of the samples. I also developed long-read models that use realignment or consensus sequences to validate the short-read models and identify novel alleles. The models correctly genotyped 98% of 101 samples using PacBio reads and found five novel alleles. Finally, I assessed the use of graph-based reference genomes, which are flexible data structures that incorporate known variants to improve polymorphic representation. I constructed different PRDM9 reference graphs and observed modest improvements in read alignment over mapping to GRCh38, with larger improvements for samples with more divergent PRDM9 genotypes or with novel alleles. Overall, my thesis provides insight into how genotyping PRDM9 can be improved by avoiding reliance on mapping reads to the GRCh38 reference. These approaches—utilizing k-mers, long-read sequencing, and graph-based reference genomes—can be further refined and adapted to improve variant identification for additional polymorphic and repetitive genomic regions.

Keywords: genotyping
k-mers
pangenome
PRDM9
repetitive
Advisor: Awadalla, Philip
Simpson, Jared
Design, Fabrication, and Characterization of Filament Based Fiber Bragg Grating Sensors

In this thesis, femtosecond laser writing is harnessed towards advancing capabilities in the fabrication of novel fiber Bragg grating based sensors. Femtosecond laser pulses inscribed low- and high-contrast filament grating structures in single-mode fibers. The transmitted, reflected and radiated diffraction from these gratings have been tailored for various applications by optimizing the focal beam stretching, laser exposure, and grating period.In this work, the low-contrast laser filaments have been harnessed to exploit their properties for fabrication of all-fiber spectrometers and displacement sensors. The unique geometry of the filament shape as an optical scattering element is an enhancement of the first-order radiative emission of fiber-guided light into cladding modes. The opportunity exploited in this thesis is drawn from filament based FBGs which permits strong and directional radiation modes to escape from the fiber. The above advantages have been resulted in the designing and fabricating of all-fiber spectrometers. Furthermore, the thin geometry of laser filaments facilitates a precise off-centered positioning of filament arrays in the fiber core. This flexibility permits strain-optic responses for azimuthally resolved displacement sensing in single-mode fiber. Therefore, the special shape of the laser written filaments resulted in the designing and fabricating of opto-mechanical displacement sensors based on off-axis filament FBGs. For the case of high-contrast filaments, the laser-processing was further advanced to high energies sufficient to open arrays of hollow filament tracks inside the core waveguiding. The ordered Bragg gratings presented an usually large refractive index contrast of 0.4 into the fiber core, opening a new means for creating strong and compact photonic-stop band devices. The filament geometry underpinned a strong capillarity effect for wetting with various liquids which demonstrated high-resolution refractive index sensing through an intact fiber cladding. In another application, Nematic liquid crystal was introduced into the nano-hole grating arrays. Capillary alignment has resulted in a strong polarization extinction ratio within a broad Bragg resonance which enabled design of all-fiber, dynamically tunable, polarization filters. The present work is promising to transform how fibers shape the flow of light and sense the local environment from applications in biomedical probes through to large area communication networks.

Advisor: Herman, Peter