VictorJuiz/thesis huggingface dataset:
Insights into the Iimmune Mechanisms Leading to Lupus-like Autoimmunity in New Zealand Black Mice

Systemic lupus erythematosus (SLE) is a chronic, multi-organ autoimmune disease characterized by the production of antibodies against self nuclear antigens. Genetics play a dominant role in disease pathogenesis and functional examination of spontaneously-arising lupus-prone animal models has provided key insights into understanding the genetic complexity of the disease. The overarching goal of the work presented here is to identify the underlying immunologic abnormalities, together with lupus susceptibility loci that produce them, that promote the development of autoimmunity in the lupus-prone New Zealand Black (NZB) background. Chapter 2 identifies the critical role of CD40-CD40L interactions in the pathogenesis of disease in NZB mice. We showed that this interaction is required for the production of class-switched IgG autoantibodies and development of hemolytic anemia and kidney disease. Polyclonal B cell activation, expansion of plasmacytoid dendritic cells (pDC), and elevated gene expression of baff were maintained in CD40L-deficient NZB mice, despite the lack of IgG immune complexes. Chapter 3 utilizes bicongenic mice carrying both NZB chromosomes 1 and 13 to investigate the genetic complexity in disease pathogenesis. In addition to identifying new phenotypes, examination of bicongenic mice showed that chronic stimulation of pDC due to the persistence of nuclear antigens leads to a refractory state with a failure to produce more IFN-α upon subsequent stimulation. Chapter 4 identifies a novel lupus susceptibility locus on NZB chromosome 13 associated with impaired clearance of apoptotic debris, a key initiating step in the development of autoimmunity. Using subcongenic mice, this locus was localized and examined its impact on immune function. Work from this thesis will contribute to understanding the complex immunogenetic mechanisms that lead to development of SLE.

Keywords: Lupus
Autoimmunity
Advisor: Wither, Joan
Neuro-immune Elements of Inflammatory Disease

Interactions between the immune system and the nervous system are currently underappreciated, assumed to play minor mechanistic roles in disease pathogenesis. In contrast, our laboratory has demonstrated the importance of this relationship with significant impact, initially in Type 1 Diabetes (T1D). The experiments presented here build on our previous work to provide insights into the etiology of Multiple Sclerosis (MS) and Type 2 Diabetes (T2D). Transient Receptor Potential Vanilloid-1 (TRPV1) is an ion channel expressed on peripheral sensory afferent neurons that fundamentally control T1D pathogenesis. Here we show that mice with genetic ablation of TRPV1 are protected from EAE progression, attributable to reduced central nervous system (CNS) leukocyte passage. The pathogenic role of TRPV1 in permeabilizing the blood-CNS barriers may also translate to MS, as patients with progressive disease show a significant mutation bias within the TRPV1 gene. We were simultaneously intrigued by the growing worldwide obesity epidemic, and we observed that obese mice develop more severe EAE compared to lean mice. This was mechanistically linked to an expansion of TH17 cells, driven by sustained rises of IL-6 in obese mice. This research implies new therapeutic opportunities for the many obese patients with diverse autoimmune diseases. Finally, the immune system, obesity, and T2D are functionally linked, and we contributed to research that uncovered a large presence of immune cells in adipose tissue that drive insulin resistance. Manipulation of T cells and B cells affects local inflammation as well as whole-body insulin resistance and glucose homeostasis. Intriguingly, auto-antibodies in insulin resistant individuals are specific for a number of unique proteins, including glial fibrillary acidic protein (GFAP), initially shown by our laboratory to play a key role in T1D progression. We further characterized the autoimmune and neuronal progression elements that steer disease pathogenesis, and observed that administration of a vaccine containing GFAP is able to dramatically reduce weight gain and insulin resistance in mice. The data presented in this thesis provide a number of novel, mechanistic observations linking the immune and nervous systems in disease, and implies several potential avenues for treatment.

Keywords: Immunology
Autoimmunity
Neuroimmunology
Multiple Sclerosis
Diabetes
Advisor: Dosch, Hans-Michael
Organizing the Ubiquitin-dependent Response to DNA Double-Strand Breaks

DNA double-strand breaks (DSBs) are highly cytolethal DNA lesions. To protect genomic integrity and ensure cellular homeostasis, cells initiate a complex signaling-based response that activates cell cycle checkpoints, coordinates DNA repair, regulates gene expression and, if necessary, induces apoptosis. The spatio-temporal control of this signaling pathway relies on a large number of post-translational modifications, including phosphorylation and regulatory ubiquitylation. In this thesis, I describe the discovery and characterization of the E3 ubiquitin ligase RNF168, which cooperates with the upstream E3 ubiquitin ligase RNF8 to form a cascade of regulatory ubiquitylation at damaged chromatin. One of the main functions of RNF8/RNF168-dependent chromatin ubiquitylation is to generate a molecular landing platform for the ubiquitin-dependent accumulation of checkpoint and DNA repair proteins such as 53BP1, the breast-cancer associated protein BRCA1 and the RNF168-paralog RNF169. I present evidence that the hierarchical recruitment of these proteins to DSB sites is, in large part, organized through the use of tandem protein interaction modules. These modules are composed of a ubiquitin-binding domain and an adjacent targeting motif called LRM, which specifies the recognition of RNF8- and RNF168-ubiquitylation substrates at damaged chromatin. I conclude that the LRM-based selection of ligands is a parsimonious means to build a highly discrete ubiquitin-based signaling pathway such as the chromatin-based response to DSBs. Collectively, my results indicate that RNF168-mediated chromatin ubiquitylation is critical for the physiological response to DSBs in human cells. The importance of the ubiquitin-based response to DSBs is underscored by the finding that RIDDLE syndrome, an immunodeficiency and radiosensitivity disorder, is caused by mutations in the RNF168 gene.

Keywords: DNA double-strand breaks
ubiquitylation
DNA damage response
chromatin
Advisor: Durocher, Daniel
Novel Analytical Approaches for the Characterization of Natural Organic Matter in the Cryosphere and its Potential Impacts on Climate Change

Climate change is predicted to be the most pronounced in high latitude ecosystems, however very little is known about their vulnerability to the projected warmer temperatures. In particular, natural organic matter (NOM) in the high latitude cryosphere which includes dissolved organic matter (DOM) and cryoconite organic matter (COM) from glaciers and soil organic matter (SOM) in permafrost, is highly susceptible to climate change which may lead to severe consequences on both local and global carbon biogeochemical cycles. Examination of DOM in glacier ice by a novel 1H nuclear magnetic resonance (NMR) water suppression pulse sequence at its natural abundance revealed and quantified the composition and the organic constituents in ice samples from Antarctica. 1H NMR spectra of samples from several glaciers were acquired and compared to the dominant fluorescent DOM fraction. This comprehensive approach showed that glacier ice DOM was mainly composed of small, labile biomolecules associated with microbes. Examination of the organic debris found on glacier surfaces (COM) from both Arctic and Antarctic glaciers were determined to be derived from microbes. Samples from Arctic glaciers were more chemically heterogeneous with small inputs of plant-derived material detected after targeted extractions. Therefore the COM carbon composition was determined to be dependent on the local glacier environment, suggesting a site specific contribution to the carbon cycle. Finally, the distribution of extracted branched glycerol dialkyl glycerol tetraether (GDGT)microbial membrane lipids and the deuterium incorporation of plant-wax n-alkane biomarkers extracted from dated permafrost SOM (paleosols) were independently applied for Canadian Arctic climate reconstruction during the last glacial maximum. Overall, the branched GDGT based temperature reconstructions from the Arctic paleosols reconstruct higher temperatures, likely when bacterial activity was optimal. The deuterium composition of the C29 n-alkane plant lipids appears to integrate an average annual signal. Further analysis by both non-selective NMR spectroscopic and targeted biomarker techniques on these paleosol samples revealed that the major vegetative sources from this paleoecosystem originated from woody and non-woody angiosperms. This thesis demonstrates several novel analytical characterization techniques, along with the major sources and composition of NOM in the cryosphere while demonstrating its use in paleoclimate applications.

Keywords: Organic Geochemistry
NMR Spectroscopy
Natural Organic Matter
Cryosphere
Paleoclimate
Chromatography
Isotope Geochemistry
Biomarkers
Advisor: Simpson, Myrna J.
Examination of the Transcriptional Regulation and Downstream Targets of the Transcription Factor AtMYB61

The mechanisms behind how a transcription factor elicits a given phenotype can be complex. The aim of the research presented herein was to provide experimental evidence to characterise the upstream and downstream regulation of the Arabidopsis thaliana R2R3-MYB transcription factor, AtMYB61. To address these aims, three separate experiments were undertaken. First, three direct downstream target genes of AtMYB61 were predicted based on a two-stage complete transcriptome analysis, using publicly available microarray datasets in combination with a custom microarray dataset comparing the transcriptomes of WT, atmyb61 and 35S::MYB61 plants. These candidate target genes encode the following proteins: a KNOTTED1-like transcription factor, a caffeoyl-CoA 3-O-methyltransferase and a pectin-methylesterase. AtMYB61 bound the 5’ non-coding regulatory regions of these target genes, as determined by electrophoretic mobility shift assay. Second, the preferred DNA-binding sites of recombinant AtMYB61 protein were assessed with a cyclic amplification and selection of targets (CASTing) assay. Key interactions between amino acids in the AtMYB61 DNA-binding site and nucleotides in the preferred DNA targets were predicted by molecular modeling. While recombinant AtMYB61 was sufficient to drive gene expression from CASTing-identified target DNA sequences in yeast, it did so in a manner that was not entirely consistent with predicted DNA-binding affinities determined by a nitrocellulose filter binding assay. Finally, the molecular components that function upstream to modulate AtMYB61 expression were determined. AtMYB61 was determined to be de-repressed by sucrose in a mechanism involving its second intron. An over-represented motif was conserved within the second intron of Brassicaceae AtMYB61 homologues and this motif functioned as a binding target for a putative sugar-mediated repressor, as determined by EMSA. Putative AtMYB61 repressor proteins that bound this motif in the absence of sucrose were affinity purified and characterised using LC-MS/MS, and the proteins identified based on their MS fingerprints.

Keywords: AtMYB61
DNA-binding
Arabidopsis
Biotechnology
Advisor: Campbell, Malcolm M.
Microbial Community Composition and Activities Across Northern Peatlands

Northern peatlands are large repositories of carbon and little is known about the effect the microbial community has on carbon mineralization rates, and there is concern that a loss of microbial diversity due to environmental change may lead to reduced ecosystem functioning. Microbial communities vary among peatland types and abiotic variables such as temperature and pH have a large influence on carbon dioxide production, but distinguishing between abiotic controls and the role of microbial community structure has proved challenging. Microbial activity and community composition was characterized in three peatlands within the James Bay Lowlands, Ontario. Similar dominant microbial taxa were observed at all three peatlands despite differences in nutrient content and substrate quality and geographic location. In contrast, microbial activity differed among the sites, indicating that it is influenced by the quality of the peat substrate and the presence of microbial inhibitors. A series of reciprocal field and laboratory transplant experiments were conducted at a rich and poor fen near White River, Ontario to more explicitly distinguish between the abiotic and microbial controls on carbon mineralization. The effect of transplantation differed between the laboratory and field studies and when viewed individually could lead to different interpretations of the effect of substrate change. Surprisingly, intensive sampling within both fens was unable to reveal a difference between the rich and poor fen microbial community due to high within site temporal and spatial variation. Thus studies with small sampling effort will have a very incomplete understanding of microbial community structure and thus microbial ecology. A reciprocal sterilization transplant experiment was also conducted to examine how different microbial communities adapted to various peat substrates influenced C-mineralization patterns. Post-inoculation/incubation bacterial communities across peatlands converged towards a similar community structure, suggesting that abiotic variables are the dominant control on peatland microbial activity and community composition. The studies presented in this thesis collectively show that across a broad range of temperate and sub-arctic peatland types dominant members of the microbial community are generally similar, and decomposition rates can be predicted by broader controlling environmental factors rather than temporal niche or distributional constraints of the microbial community.

Keywords: carbon dioxide
microbial community
microbial activity
peatlands
methane
TRFLP
Advisor: Basiliko, Nathan
Jurisdiction and Settler Colonialism: The Algonquins of Barriere Lake Against the Federal Land Claims Policy

This dissertation analyzes tensions between Indigenous and Canadian authority over land and governance through a critical inquiry into jurisdiction. I examine jurisdiction in the context of the Algonquins of Barriere Lake’s territory, located about three hours north of Ottawa in the northernmost boreal region of Quebec. To undertake this study of overlapping jurisdiction, I analyze the struggle over resource management across the past thirty years on the territory and their struggle against the federal land claims policy. I map the ways in which space is differentiated under competing legal orders, where on the one hand, jurisdiction is produced by the sovereign territorial state through operations of economic and political calculation, and on the other, by the Algonquin nation through a kinship nexus of allocated hunting and trapping grounds, and by the daily caretaking practices associated with Anishnabe life on the territory. I raise questions as to how simultaneous operations of law may take place in a single area, across distinctive epistemological and ontological frameworks, and how jurisdictions are produced in this context. My dissertation examines how the Algonquins of Barriere Lake have contested the socio-spatial production of state sovereignty claims through the exercise of jurisdiction over their lands. My focus on jurisdiction in this dissertation turns our attention to the practices of settler colonial sovereignty in Canada, and especially to the role Indigenous law plays in resisting intervention on their lands. I examine the role Indigenous law plays in shaping the political economy of this country, seeking to identify whether a “distinct form of accumulation” emerges in the dialectic of settler colonialism and Canada’s staple state economy. The interpretive framework of jurisdiction allows us to examine the overlapping authority claims between Indigenous, state, regional, and private interests, and to parse out the ways in which these jurisdictional claims produce different kinds of political space.

Keywords: colonialism
land claims
jurisdiction
Canada
Advisor: Prudham, W. Scott
Cowen, Deborah
Dust and Gas in Different Galactic Environments

This thesis encompasses the study of the mid-infrared (IR) dust properties in diffuse high latitude cirrus and in the dense environments of supernova remnants (SNRs) in the plane of our Galaxy. Unlike the well known emission properties of dust grains in the diffuse ISM in the far-IR and submillimeter, the mid-IR spectrum is still relatively unconstrained. We extend the correlation of dust emission with H I column densities to mid-IR wavelengths and look for evidence of variations in the emissivity of dust associated with local and halo gas. This is accomplished by spatially correlating the IR maps from the IRIS/IRAS survey at 12, 25, 60 and 100 μm with H I column density maps inferred from 21-cm line emission observations obtained with the GBT (at a 9′ resolution). We find that IVCs (halo clouds thought to be part of the Galactic fountain) show color ratios consistent with a dust evolution scenario in which large dust grains are shattered into smaller ones (VSGs). The low 12 μm emission found suggests a reduced abundance of PAHs in IVCs. We also address the IR extragalactic emission seen in our residual maps and quantify its power spectrum behaviour. Continuing with the mid-IR theme, we conducted a comprehensive study of the morphology and energetics of SNRs in the plane of our Galaxy. We make use of the Spitzer MIPSGAL (at 24 and 70 μm) and GLIMPSE (at 8 μm) surveys to detected infrared counterparts to SNR candidates in Green’s catalog. We find that a third of the sample shows IR emission and calculate the corresponding fluxes. We explore the relation between IR colors to place constraints on the different IR SNRs emission mechanisms. Aided by archival radio data, we find that most candidates detected show IR-to-radio ratios consistent with SNRs with a few exceptions displaying ratios seen in H II regions. Finally, we explore the connection between the IR and the high-energy X-ray emission of SNRs and find a good morphological association between the 24 μm emission and the X-ray features in younger remnants. The IR power is often greater.

Keywords: Dust
ISM
Advisor: Martin, Peter
State Power for Low-Carbon Development: A Comparative Investigation into the Effectiveness of Carbon Finance Projects in Tanzania, Uganda and Moldova

Empirical investigation into afforestation and bioenergy carbon finance projects in Tanzania, Uganda and Moldova demonstrates that effective projects—both in terms of sustainable development and the generation of genuine carbon credits—are more likely to result when the state is able to bring carbon finance initiatives into alignment with national development objectives. Amongst the countries investigated, the most important factor in such alignment was, paradoxically, commitment liberal economic reforms. Contrary to the expectation that the performance of projects under the Kyoto Protocol’s Clean Development Mechanism (CDM) would be the same in states with similar administrative capacities, carbon finance projects were more effective in Uganda and Moldova than Tanzania. Commitment to liberal economic reforms in Uganda functions as an animating set of ideas that allows the state apparatus to work in a more purposeful manner and establish institutions and organizations which allow it to generate state power for low-carbon development. For CDM forest and bioenergy projects, the risk of unsustainability is mitigated by a land tenure system and investment regime that (i) offer opportunities for individual smallholders to engage directly with the carbon market and create incentives for domestic investors while (ii) also accommodating historical land governance practices. Genuine carbon credits were associated with project developers who possessed a latent organizational capacity for implementation and were motivated to pursue market opportunities—state forest agencies in Uganda and Moldova. However, the ability of the state to retain latent organizational capacity was restricted to sectors such as forestry that are less sophisticated technically; in the energy sector, such capacity was ceded to the private sector in Uganda and Moldova during structural adjustment. More skeptical of liberal economic policy, Tanzania has retained capacity in the energy sector; however, for the same reasons, it has not treated the CDM as a genuine opportunity. At current carbon prices, CDM projects investigated were effective when the state was able to play a developmental role in the economy. Whether commitment to liberal economic reforms can have similar developmental effects in other parts of the developing world is questionable—a different animating set of ideas may be important.

Keywords: Carbon market
Development studies
Climate policy
Forestry
Bioenergy
Comparative public policy
Political economy
Advisor: Bernstein, Steven Franklin
Handley, Antoinette
Skogstad, Grace
Understanding the Fungicidal Activity of Lipopeptides on the Basis of their Biosurfactant Properties

Many biosurfactants show antimicrobial activity and some are found to be superior for isolating membrane proteins. This study was aimed towards a general understanding of the interactions of biosurfactants with lipid membranes on a molecular level. To this end, a new, fluorescence lifetime-based membrane leakage assay has been established that does not only quantify membrane permeabilization more precisely but reveals also the leakage mechanism. This mechanism, referred to as graded or all-or-none leakage, is crucial for interpreting potential biological activities and modes of action. Lipopeptides of the surfactin, fengycin, and iturin families as produced by Bacillus subtilis were studied along with synthetic surfactants. Their membrane permeabilizing activity and selectivity mirrored, to some extent, the active concentrations and fungicidal selectivity of the compounds in vivo. Furthermore, the effects of co-surfactants and co-solvents (glycerol, urea, DMSO) have been investigated to better understand and predict means of improving the performance of fungicidal products as well as conditions for membrane protein solubilization.

Keywords: biosurfactant
lipopeptides
antimicrobial
lipid bilayer
detergents
vesicle leakage
calcein
fluorescence lifetime
membrane permeabilization
biophysical
Advisor: Heerklotz, Heiko