VictorJuiz/thesis huggingface dataset:
Molecular Mechanisms Involved in Insulin and Palmitate Actions on Clonal, Hypothalamic Cell Lines Expressing Neuropeptide Y and Agouti-related Peptide

Type 2 diabetes mellitus (T2DM) ensues from diminished insulin sensitivity and abated compensatory insulin secretion. While diminished insulin secretion has a strong genetic origin, environmental factors are central in the development of insulin resistance; these include hyperinsulinemia and lipotoxicity. Insulin resistance results in the dysregulation of hypothalamic neurons that mediate its central actions: a key intermediary neuron is the neuropeptide Y/agouti-related peptide (NPY/AgRP) neuron. The hypothesis therefore was generated that insulin directly regulates NPY/AgRP neurons, and that prolonged insulin or palmitate, a prevalent free fatty acid (FFA), exposure inhibits neuronal insulin signaling. Using well characterized hypothalamic cell lines, mHypoE-46 or mHypoE-44, which express NPY, AgRP and insulin receptor signaling machinery, this hypothesis was examined in three studies. Correspondingly, insulin decreased NPY and AgRP mRNA expression in the mHypoE-46 cells, through an extracellular signal-regulated kinase (ERK) dependent mechanism; whereas prolonged exposure of NPY/AgRP cells to insulin or palmitate attenuated insulin signaling, determined by analysis of phosphorylated Akt. Insulin induced insulin receptor substrate-1 (IRS-1) serine 1101 phosphorylation in mHypoE-46 cells, utilizing the mTOR-S6K1 pathway, as the mTOR inhibitor rapamycin prevented IRS-1 serine phosphorylation. Insulin also decreased insulin receptor and IRS-1 protein levels; this was prevented by lysosomal and proteasomal pathway inhibitors, 3-methyladenine and epoxomicin, respectively. Importantly, rapamycin, epoxomicin or 3-methyladenine pre-treatment decreased the attenuation of insulin signaling during long-term insulin exposure. On the other hand, palmitate activated c-Jun N-terminal kinase (JNK), the apoptosis effector caspase 3, and induced endoplasmic reticulum (ER) stress in mHypoE-44 cells: JNK inhibition prevented ER stress. In an attempt to avert the deleterious effects of palmitate, the neuronal cells were treated with the 5`AMP-activated protein kinase (AMPK) activator AICAR, a possible insulin sensitizer. Interestingly, AICAR attenuated JNK and caspase 3 activation, and restored insulin signaling. These findings demonstrate that insulin directly regulates NPY/AgRP neuronal cells, and that insulin and palmitate provoke neuronal insulin resistance through different mechanisms. These findings substantiate the idea that environmental factors known to trigger peripheral insulin resistance may have consequences at the level of the individual hypothalamic neuron, which may ultimately contribute to the resulting pathophysiological states of obesity and T2DM.

Keywords: Hypothalamus
Insulin resistance
Hyperinsulinemia
Type 2 diabetes mellitus
Free fatty acids
Palmitate
Neuropeptide Y
Agouti-related peptide
Cell biology
Molecular biology
Advisor: Belsham, Denise
The Ecological Consequences and Adaptive Function of Nectar Secondary Metabolites

Plants are under selection to simultaneously attract pollinators while deterring herbivores. This dilemma can lead to tradeoffs in floral traits, which are traditionally thought to be optimized for pollinators. My dissertation addresses the ecological costs and putative functional significance of nectar secondary metabolites, a paradoxical but widespread phenomenon in the angiosperms. I investigate this issue from the pollinator’s perspective using a series of controlled laboratory investigations focused primarily on the bumble bee Bombus impatiens and the nectar alkaloid gelsemine, from Gelsemium sempervirens. I begin by demonstrating that nectar enriched with the alkaloid gelsemine significantly deters visits from bumble bees at a range of natural alkaloid concentrations. However, this aversion can be mitigated by increasing the sucrose concentration such that the alkaloid-rich nectar is more rewarding than its alkaloid-free counterpart. I then demonstrate that the consumption of gelsemine-rich nectar can inhibit oocyte development and protein utilization in bees, but that this effect is limited to bees of suboptimal condition. Continuous consumption of the nectar alkaloid gelsemine also leads to a reduction in the pathogen load of bumble bees infected with Crithidia bombi, but direct interactions between the pathogen and the alkaloid have no impact on infection intensity. Gelsemine also fails to inhibit floral yeast growth, suggesting that nectar alkaloids may not be universally antimicrobial. Finally, I demonstrate that gross nectar cardenolides from the genus Asclepias are strongly correlated with gross leaf cardenolides and that the majority of individual cardenolides found in nectar are a subset of those identified in leaves. This pattern suggests that nectar cardenolides are a consequence of defense for Asclepias; however, they may not be a costly corollary because bumble bees show an overall preference for nectar cardenolides at mean concentrations. Altogether, my dissertation provides a new perspective on the role of chemical defenses against herbivores in plant-pollinator interactions.

Keywords: plant-animal interactions
chemical ecology
pollination biology
toxic nectar
Bombus impatiens
Gelsemium sempervirens
Asclepias
Crithidia bombi
behavioural ecology
Advisor: Thomson, James
Involvement of Reactive Metabolites in Idiosyncratic Drug Reactions

Idiosyncratic drug reactions (IDRs) represent a significant medical problem and pose a great challenge to drug development. Circumstantial evidence suggests that, in most cases, reactive metabolites of the drug are responsible. The major focus of this thesis is the identification of reactive metabolites and the synthesis of analogs required to test several hypotheses related to involvement of metabolism and covalent binding in the mechanisms of IDRs. Minocycline is unique among tetracyclines in causing a significant incidence of a lupus-like syndrome and autoimmune hepatitis. In this study, we demonstrated that minocycline is oxidized to reactive intermediates by myeloperoxidase/H2O2/Cl-, HOCl, horseradish peroxidase/H2O2, or hepatic microsomes. When trapped with N-acetylcysteine (NAC), two adducts with protonated molecular ions at m/z 619 were isolated and analyzed by NMR. One represents attack of the aromatic D ring by NAC meta to the N, N-dimethylamino group, implying that the reactive intermediate was a quinone iminium ion. The other adduct, which was not observed when minocycline was oxidized by hepatic microsomes, indicates that the NAC is attached at the junction of the B and C rings, suggesting that the HOCl added across the double bond of the B ring leading to a reactive molecule, and then NAC displaced the chloride ion. Nevirapine, an anti-HIV drug, is associated with idiosyncratic skin rashes in humans. The goal of this project was to investigate whether the 12-hydroxylation pathway is responsible for the skin rash. To test a part of this hypothesis, 12-trideuteronevirapine, 12-OH-NVP sulfate, and several other analogs of nevirapine were synthesized. D-penicillamine is known to cause idiosyncratic autoimmune reactions in humans. The goal of this project was to test whether D-penicillamine covalently binds to macrophages and triggers downstream events leading to autoimmunity. To test a part of this hypothesis, D-penicillamine conjugated to biotin was synthesized. In summary, reactive metabolites of minocycline were found that likely explain why minocycline has an IDR profile unique among the tetracyclines. In addition, analogs of nevirapine and D-penicillamine required for mechanistic studies of nevirapine and D-penicillamine-induced IDRs were synthesized. These studies provide additional support for the involvement of reactive metabolites in the mechanisms of IDRs.

Keywords: drug metabolism
mass spectrometry
adverse drug reactions
drug safety
LC/MS
Advisor: Uetrecht, Jack
Investigation of Unilamellar Phospholipid Vesicle Interactions with PNIPAM Based Hydrogel Beads

Phospholipid liposome binding to hydrogel beads based on poly(N-isopropylacrylamide) (PNIPAM) is accomplished employing either avidin/biotin conjugation or hydrophobic modification of the microgels, and the ability to form single supported lipid bilayers is explored. The co-monomer acrylic acid (AA), evenly distributed or localized to the shell of the microgel, is included to facilitate post-polymerization chemical modification of the hydrogel beads. The degree of chemical modification of the microgels as well as the thermal behavior is monitored via 1H and 13C nuclear magnetic resonance (NMR). Liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phoshocholine (POPC) and a small amount of commercially available biotinylated-lipid are shown to bind as intact entities while sequestering internal contents to biotinylated hydrogel beads, utilizing avidin as the coupling agent. Under fusogenic conditions, these bound liposomes remain as individual vesicles. Alternatively, POPC liposomes are shown to bind to microgels modified to display single chain alkyl groups or cholesteryl moieties, and remain as intact vesicles. It is demonstrated that these liposomes become permeable at high hydrophobe content. Bound liposomes will fuse into larger structures under high hydrophobe content conditions, but remain permeable. The volume phase transition (VPT) characteristic of PNIPAM microgels is shown to influence the permeability of hydrophobically bound (low hydrophobe content), but not avidin/biotin conjugated, liposomes. The degree of liposome binding, as well as their resulting structures and permeability are investigated utilizing 31P NMR, fluorescence spectroscopy and microscopy. The microgel-bound liposome and microgel-supported lipid bilayer hybrid systems would be ideally suited to drug delivery and tissue engineering applications. The microgel-supported single lipid bilayer system would, in addition, potentially act as a cell model system for membrane dynamics and embedded amphiphile NMR studies.

Keywords: Vesicle
PNIPAM
Microgel
NMR
Advisor: Macdonald, Peter Moore
The Challenge of Guinean Independence, 1958-1971

Since the end of French colonial rule in Guinea, “independence” has held a central place in its political culture. Implying both dignity and self-determination for the sovereign people which possesses it, independence is a concept that has meaning only in relation to other nation-states and cultures. Yet the political elite that dominated Guinea’s First Republic constructed a new national culture around this concept. The Challenge of Guinean Independence, 1958-1971 examines Guinea’s assertion of its right to independence and the response of powerful Western players, especially the United States and France, as Guinea challenged their assumptions about the nature of African sovereignty. Considering the history of the international relations of a single African state that enjoyed limited international power and prestige challenges conventions in the historiography of both Africa and international relations. It illuminates and contextualizes expectations concerning the meaning of modernity, African sovereignty as a matter of international law, and the end of formal colonial rule coinciding with the tensions and competitions of the Cold War. The study demonstrates that the international context played a crucial role, both in conditioning the timing and form of decolonization and in shaping the international community’s adaptation of colonial patterns of economic and political interaction to the new reality of African nation-states. Focusing on the invention, development and reception of one country’s insistence on independence in turn illuminates significant issues and events: the end of French colonial rule; limitations on the sovereignty of non-European postcolonial states; the advent of neocolonialism and the failure of the nominally anti-colonial United States to oppose it; the ideological appeal of African unity as a means of safeguarding sovereignty and the compromises that its institutional form entailed; foreign aid and the notion that development for modernization could be stimulated from outside; and the implications of unlimited internal autonomy for a state’s people. Guinea’s independence ultimately challenged developing norms of Western economic and political interaction with new African states by complicating assumptions about the universality of Western notions of economic development, justice and morality.

Keywords: Guinea
International relations
Africa
United States
Advisor: Pruessen, Ronald W.
The Effect of Insulin and Insulin Resistance on Glucagon-like Peptide-1 Secretion from the Intestinal L Cell

Glucagon-like peptide-1 (GLP-1) is secreted from the enteroendocrine L cell following nutrient ingestion. Although GLP-1 regulates several aspects of nutrient homeostasis, one important function is to enhance glucose-dependent insulin secretion. In type 2 diabetes, post-prandial GLP-1 secretion is impaired. Insulin resistance, which is required for the pathogenesis of type 2 diabetes, is also associated with impaired GLP-1 secretion. I, therefore, hypothesized that insulin modulates GLP-1 secretion from the intestinal L cell and, furthermore, insulin resistance directly impairs the function of the endocrine L cell. In well-characterized L cell models, I established that insulin stimulates GLP-1 secretion through the MEK1/2-ERK1/2 pathway, and induction of insulin resistance in vitro attenuated insulin- and heterologous secretagogue-induced GLP-1 release. Furthermore, glucose-stimulated GLP-1 secretion was decreased in hyperinsulinemic-insulin resistant MKR mice, demonstrating that insulin resistance is associated with impaired L cell function. I next examined the role of the actin cytoskeleton in insulin-stimulated GLP-1 secretion. Insulin treatment transiently induced actin depolymerization, and depolymerization of the actin cytoskeleton potentiated insulin-stimulated GLP-1 release from the L cell, demonstrating that the cytoskeleton functions as a permissive barrier. Central to insulin’s effects on actin dynamics is the Rho GTPase, Cdc42, as siRNA-mediated knockdown and over-expression of a dominant-negative mutant, prevented insulin-stimulated actin remodeling and GLP-1 release. Insulin also promoted activation of PAK1, the downstream kinase of Cdc42, and over-expression of a kinase-dead PAK1 mutant attenuated insulin-stimulated GLP-1 release. In cells that expressed dominant-negative Cdc42 or kinase-dead PAK1, activation of ERK1/2 following insulin treatment was attenuated, demonstrating that the Cdc42-PAK1 axis regulates the activity of the canonical ERK1/2 pathway. In summary, this thesis demonstrates, for the first time, that insulin is a GLP-1 secretagogue, and this effect of insulin is mediated through the canonical ERK1/2 pathway and the Cdc42-PAK1 axis. Insulin resistance in the L cell impairs the responsiveness of the L cell to heterologous secretagogues. Collectively, these findings suggest that an alternative approach to treat type 2 diabetes and/or insulin resistance may be to directly improve the function of the L cell, thereby enhancing endogenous GLP-1 release.

Keywords: insulin
diabetes
insulin resistance
glucagon-like peptide-1
actin cytoskeleton
intestine
Advisor: Brubaker, Patricia
Mechanisms of Cell Nucleation, Growth, and Coarsening in Plastic Foaming: Theory, Simulation, and Experiment

This thesis highlights a comprehensive research for the cell nucleation, growth and coarsening mechanisms during plastic foaming processes. Enforced environmental regulations have forced the plastic foam industry to adopt alternative blowing agents (e.g., carbon dioxide, nitrogen, argon and helium). Nevertheless, the low solubilities and high diffusivities of these viable alternatives have made the production of foamed plastics to be non-trivial. Since the controls of the cell nucleation, growth and coarsening phenomena, and ultimately the cellular morphology, involve delicate thermodynamic, kinetic, and rheological mechanisms, the production of plastics foams with customized cell morphology have been challenging. In light of this, the aforementioned phenomena were investigated through a series of theoretical studies, computer simulations, and experimental investigations. Firstly, the effects of processing conditions on the cell nucleation phenomena were studied through the in-situ visualization of various batch foaming experiments. Most importantly, these investigations have led to the identification of a new heterogeneous nucleation mechanism to explain the inorganic fillers-enhanced nucleation dynamics. Secondly, a simulation scheme to precisely simulate the bubble growth behaviors, a modified heterogeneous nucleation theory to estimate the cell nucleation rate, and an integrated model to simultaneously simulate cell nucleation and growth processes were developed. Consequently, through the simulations of the cell nucleation, growth, and coarsening dynamics, this research has advanced the understanding of the underlying sciences that govern these different physical phenomena during plastic foaming. Furthermore, the impacts of various commonly adopted approximations or assumptions were studied. The end results have provided useful guidelines to conduct computer simulation on plastic foaming processes. Finally, an experimental research on foaming with blowing agent blends served as a case example to demonstrate how the elucidation of the mechanisms of various foaming phenomena would aid in the development of novel processing strategies to enhance the control of cellular structures in plastic foams.

Keywords: Plastic Foaming
Cell Nucleation
Cell Growth
Cell Coarsening
Computer Simulation
Foaming Mechanisms
Cellular Structure
Advisor: Park, Chul B.
Viral Mineralization and Geochemical Interactions

Viruses are ubiquitous biological entities whose importance and role in aquatic habits is beginning to take form. However, several habitats have undergone limited to no examination with viral-geochemical parameters minimally examined and viral-mineral relationships in the natural environment and the role of mineralization on viral-host dynamic completely lacking. To further develop knowledge on the presence and abundances of viruses, how viruses impact aquatic systems, and how viral-host interactions can be impacted under mineralizing conditions, viruses were examined under a variety of habitats and experimental conditions. Water samples were collected from the deep subsurface (up to 450 m underground) and acid mine drainage (AMD) systems in order to determine the presence, abundance, and viral-geochemical relationships within the systems. Samples were also collected from a variety of freshwater habitats, which have undergone limited examination, to determine viral-geochemical and viral-mineral relationships. Lastly, bacteriophage-host dynamics were examined under authigenic mineral precipitation to determine how mineralization impacts this relationship. Results reveal that not only are viruses present in the deep subsurface and AMD systems, but they are abundant (up to 107 virus-like particles/mL) and morphogically diverse. Viruses are also the strongest predictor of prokaryotic abundance in southern Ontario freshwater systems where potential nutrients are rich. Geochemical variables, such as pH and Eh, were shown to have negative impacts of viral abundance indicting that AMD environments are detrimental for free viruses (i.e. not particle associated). Direct evidence of viral-mineral interactions was found using transmission electron microscopy as viral particles were shown attached to iron-bearing mineral phases (determined through elemental analysis). In addition, evidence of viral participation in mineralization events was found in both AMD and freshwater environments where inverse correlations were noted between viral abundance and jarosite saturation indices (r = -0.71 and r = -0.33, respectively), and goethite saturation indices were also noted to be the strongest predictor of VLP abundance in freshwater habitats explaining 78% of the variability in the data. Lastly, iron precipitation and/or metal ion binding to bacterial surfaces greatly reduced phage replication (~98%) revealing bacterial mineralization has a protective benefit strongly hindering viral replication.

Keywords: microbial geochemistry
viral ecology
bacteriophage
biomineralization
extreme environments
geomicrobiology
Advisor: Ferris, F. Grant
Sequential Agroforestry systems for Improving Fuelwood Ssupply and Crop Yield in Semi-arid Tanzania

Promotion of agroforestry practices in sub-Sahara Africa may help sustain subsistent food and wood production by integrating trees and crops on farmlands to replenish soil fertility and improve crop yield. Using rotational woodlot and pigeonpea intercropping systems in semi-arid Tanzania as case studies, my research screened suitable tree species to increase fuelwood supply and examined mechanisms for reducing tree-crop competition. By adopting nutrient use efficiency (the ratio of biomass yield to nutrient uptake) as a criterion, I found that selecting tree species of low wood nutrient concentrations would minimize nutrient exports by 42 – 60 %, thus reducing soil nutrient depletion while concurrently sustaining local fuelwood supply harvested from rotational woodlots. Currently smallholder farmers cannot afford to replenish soil fertility because of high fertilizer costs. However, 5-year tree fallowing raised soil N and P levels for maize culture as high as those from recommended fertilizer applications. Post-fallow maize yield was also increased significantly over natural fallow practices. Apparently there is a trade-off between yields of maize and fuelwood under rotational woodlot culture providing farmers the choice to proportion tree and crop composition based on priority demands. An alternative practice of intercropping pigeonpea with maize may also rapidly replenish soil fertility as well as enhance maize yield when competitive interactions between trees and crops are controlled. Vector analysis revealed that such interactions suppressed biomass yields of maize and pigeonpea by 30 % and 60 %, respectively, due to limited soil nutrients and/or moisture. Optimizing yields of both crops would require prescribed fertilizer addition when intercropped, but dose rates can be lowered by half under the improved fallow system due to alleviating interspecific competition. My findings form the basis of a plea for greater use of rotational woodlot and pigeonpea intercropping systems in semi-arid areas. I conclude that smallholder farm management of rotational agroforestry systems can be significantly improved by refining tree selection criteria and mitigating nutrient competition between trees and crops to maintain food and fuelwood production.

Keywords: Acacia
Improved fallows
Litter quality
Manure
Nutrient use efficiency
Soil fertility
Sustainability
Vector analysis
Advisor: Timmer, Vic