This thesis concerns the role of nutrients in cognitive and behaviour problems associated with child attention deficit hyperactivity disorder (ADHD). Study 1 investigated relationships between Conners' ADHD Index ratings, fatty acid deficiency symptoms (FADS), and cognitive performance in a normal population of children. Studies 2 and 3 comprised a 30 week intervention trial investigating effects of n-3 PUFA supplementation on ADHD symptoms in 7-12 year old children with high ADHD scores.

Trace amounts of pharmaceuticals have been detected in water, from nanograms per liter to micrograms per liter, and have a negatively effect in the aquatic environment and an increased potential risk of drug poisoning for human and animals. In order to address the problem, drug degradation catalyzed by chloroperoxidase (CPO) has been investigated. CPO is a heme-containing glycoprotein secreted by the fungus, Caldariomyces fumago, it catalyzes two major types of oxidations, two one-electron oxidations as catalyzed by most peroxidases and two-electron oxidations which are rare for conventional peroxidases. Five common drugs from a variety of classes which were persistent in the environment have been studied. The metabolites of each drug were identified and the pathways of degradation were proposed. All of them were found to be 100% degradation efficiency in the CPO-H2O2-Cl- system which the catalyzation only required low concentration of CPO (normally nanomolar level) as well as relatively low concentration of H2O2 as cofactor. This degradation method is economic and highly efficient, the results of my experiment extensively support the hypothesis that CPO has a great potential in the environmental application. A new mutant of CPO has been constructed to investigate the role of histidine 105 in the active site of distal pocket. Histidine 105 was suggested to play an essential role in modulating the chlorination activity by forming hydrogen bond with glutamic acid 183, histidine has been replaced by arginine to generate CPO H105R mutant. The construction and transformation were a success but the protein was expressed as apoenzyme, suggesting the mutagenesis to a larger arginine residue at position105 disturbed the heme incorporation.

<p>Cardiovascular diseases (CVD) and their risk factors remain the leading cause of morbidity and mortality in North America. Food-derived bioactive peptides (BAP) have been shown to play a role in regulating physiological pathways of CVD risk factors including hypertension, diabetes, and chronic inflammation. Common sources of BAP include dairy and plant proteins. In addition to being an alternative protein source, it is now accepted that edible insect proteins can also confer health benefits beyond nutrition. However, as with any novel protein source, allergenicity remains a major concern surrounding edible insect consumption. </p> <p>This dissertation aimed to: 1) Evaluate the bioactive potential of peptides from an edible cricket species and; 2) determine the effects of BAP production methods on immunoreactivity. First, peptide-rich extracts were generated from farmed food-grade crickets via enzymatic hydrolysis techniques with the commercial protease Alcalase™. To measure the <i>in vitro</i> bioavailability, cricket peptides were also subject to simulated gastrointestinal digestion (SGD). Peptides and their digests were tested for relevant bioactivities and active groups were further fractionated by chromatographic methods to identify the major peptides responsible for the bioactivity. When tested for <i>in vitro</i> antihypertensive and anti-glycemic properties, cricket peptides were found to inhibit the activities of angiotensin converting enzyme, dipeptidyl peptidase-4, α-amylase, and α-glucosidase. The anti-inflammatory potential was expounded using RAW-264.7 macrophages and human umbilical vein endothelial cells (HUVEC). Cricket peptides (after SGD) effectively lowered NF-κB, MCP-1, and IL-6 production in cells without affecting their viability. Proteomic analyses identified 18 sequences from the enriched cationic peptide fraction that showed the highest activity. Three novel peptides were identified via molecular docking, as potent ACE-inhibitors and binding was similar to that of the commercial drug captopril. Key binding characteristics included interaction with hydrophobic amino acids (Phe, Pro, Leu) near the C-terminal position and coordination with Zn (II) ions near the ACE active site.</p> <p>Immunological reactivity was measured by IgE-binding from shrimp-allergenic patient sera to antigens present within cricket peptides. Our studies demonstrate that immunoreactivity was impacted by enzymatic hydrolysis, depending on the conditions and heating source used. Tropomyosin (a major shrimp allergen) was extracted from both untreated crickets and protein hydrolysates, and verified as the major reactive protein. Tropomyosin reactivity decreased (under both partial and extensive hydrolysis) or retained (under conditions which prevented epitope cleavage). However, using microwave-assisted enzymatic hydrolysis was effective at decreasing tropomyosin reactivity in all immunoassays tested (IgG and IgE). Proteomic and immunoinformatic analyses revealed prominent allergen binding regions of cricket tropomyosin available for cleavage during enzymatic hydrolysis. Conserved antigen regions showed greater homology with other crustacean species, but not with other well studied allergenic insect proteins (i.e., cockroach). Lastly, LC-MS/MS and FT-Raman spectrometry suggests that reactivity was affected due to distinct epitope cleavage within the protein instead of decreased antigen extractability/solubility. </p> <p>The findings of this dissertation support that edible cricket proteins are a potential source of bioactive peptides for functional food or nutraceutical development. Additionally, using protein extraction methods such as microwave-assisted hydrolysis seems a promising tool for minimizing the immunoreactivity of the allergen present in this edible cricket species.</p>

Fabry disease (FD) is an X-linked recessive lysosomal storage disorder (LSD) known to affect approximately 1 in every 40,000 males, and a smaller number of females. FD results from a deficiency of functional α-galactosidase (α-GAL), which leads to the accumulation of terminally α-galactosylated substrates in the lysosome. The predominant treatment is Enzyme Replacement Therapy (ERT), requiring the regular infusion of recombinant human α-GAL. More than half of individuals receiving ERT experience a range of adverse infusion reactions, and it has been reported that as many as 88% of patients receiving ERT develop neutralizing IgG antibodies against the drug. In aim of designing a non-immunogenic treatment candidate for Fabry disease ERT, we have engineered the active sites of α-GAL and another homologous family 27 exoglycosylase named α-N-acetylgalactosaminidase (α-NAGAL) to have interconverted substrate specificities. 11 of 13 active site residues are conserved between these two enzymes, and we have shown that their substrate specificities can be interconverted by mutating the two non-conserved active-site residues. We report the kinetic properties of these two mutants along with wild type controls, and use western blotting to show that both mutant enzymes retain their respective wild type enzyme antigenicity. Structural data obtained by X-ray crystallography on the α-GAL mutant (called α-GALSA ) reveals the mechanism by which substrate specificity is dictated between these two proteins, and provides explanations for the mutant’s reduced catalytic efficiency.

<p>The human multidrug resistance transporter P-glycoprotein (P-gp) is highly expressed at blood- tissue barriers, including the blood-brain barrier (BBB), and poses a serious challenge for the delivery of therapeutics to the brain. Additionally, expression of P-gp is also detected in some blood cancers, and is thought to limit the uptake of therapeutics, leading to a multidrug resistant phenotype (MDR). P-gp has multiple substrate binding sites and uses the energy of ATP hydrolysis to actively transport a variety of hydrophobic compounds out of the cell from the plasma membrane. Our goal is to take advantage of the polyvalency of the substrate binding site to create P-gp inhibitors from substrates through dimerization. Herein, we demonstrate the synthesizes and characterization two libraries of dimeric prodrug inhibitors of P-gp based on the substrates temozolomide and dasatinib, a glioblastoma chemotherapeutic and chronic myelogenous leukemia chemotherapeutic, respectively. In addition to inhibiting P-gp, by containing reversible tethers, these dimers are designed to act as prodrugs and release the therapeutics inside the cell. To improve the efficacy and solubility of our dimers, we synthesized heterodimers with the known substrate quinine to generate libraries of temozolomide-quinine and dasatinib-quinine molecules with varying tether lengths. Both libraries were shown to be potent inhibitors of P- gp efflux at low micromolar concentrations.</p>

Preterm birth is the number one cause of death in neonates, accounting for 35% of neonatal mortality. The preterm birth rate in the U.S. in 2021 was 10.5%, disproportionally affected by race and ethnicity. Obese women have an increased risk of preterm pregnancy, and if delivered before 37 weeks of gestation, the offspring have higher rates of complications that extend from the neonatal period into life-long metabolic and immune adverse outcomes. In the early months after delivery, preterm infants have higher rates of adverse health outcomes than term infants, including infections, extrauterine growth restrictions, respiratory, metabolic, and neurological complications, necrotizing enterocolitis (NEC), and bronchopulmonary dysplasia (BPD). Diet for the preterm infant is crucial for infection prevention, and maternal breast milk is most beneficial when given in the first few days after birth. Expectant and breastfeeding mothers should consume appropriate food and supplements to optimize their nutrition. In addition to nutrients, bioactive components, vitamins, and minerals found in breast milk (BM), there is evidence that microbes (microbiome) are a significant factor in infant development, contributing to protection against pathogens and playing a role in the development of the immune and nervous systems. Maternal BM composition and microbiome are affected by many factors, including maternal body mass index (BMI), maternal health, antibiotics use, mode of delivery, maternal parity, gestational age, and time and duration of lactation. Maternal body composition, however, and not maternal nutritional status, is associated with breast milk nutritional composition. Altogether, these maternal factors may modify the premature infant gut microbiome. We examined the role of maternal adiposity and how it impacts the composition of human breast milk, specifically hormones, nutrient composition, short and long chain fatty acids, and microbiome. We also examined the role of maternal adiposity and how it impacts the short-chain fatty acids and microbiome in infant fecal samples. We found that maternal adiposity affects breast milk hormones, potentially modulating infant metabolism. Additionally, we found that maternal adiposity does not alter the nutrient composition of breast milk; however, differences in both short and long chain fatty acids and maternal adiposity were detected. In our small cross-sectional cohort of preterm infants, we did not observe differences in short-chain fatty acids in the preterm infant stool samples compared to maternal adiposity. Concerning maternal adiposity and its impact on the microbiomes of breast milk and infants, we observed differences in phyla and genera between the maternal BMI groups on the outcome of breast milk and preterm infant microbiomes but no statistical significance in alpha and beta diversities between the groups. Thus, our results indicate that maternal adiposity impacts hormonal, microbial composition, and short-chain fatty acid profiles in breast milk, which tremendously influences infant growth and development.

<p>In atherosclerotic plaques, macrophages ingest modified LDL and turn to foam cells. Others have shown that SR-BI expression levels inversely correlated with cellular cholesterol levels, and is independent of well characterized cholesterol sensing pathways; SREBP and LXR. Thus the mechanism of regulation of SR-BI is unclear. In this study, we showed that treating macrophage with agents known to increase cellular cholesterol levels, namely acLDL, LDL, MβCD:Cholesterol, resulted in reduction in HMGCoAR mRNA and SR-BI expression levels. In contrast, acLDL did not reduce SR-BI mRNA levels in macrophages from acLDL SR-A KO mice, demonstrating that acLDL mediate suppression of SR-BI was dependent on SR-A. Fucoidan, a known competitive inhibitor of acLDL binding to SR-A, and subsequent degradation, also suppressed SR-BI expression levels. Unlike acLDL, however, fucoidan induced mRNA levels corresponding to the pro-inflammatory genes iNOS and IL-6 mRNA levels, and its effects were not altered by the lack of SR-A. Instead, fucoidan mediated stimulation of iNOS and IL-6 and suppression of SR-BI mRNA levels was prevented by an anti-CD14 blocking antibody, demonstrating that the fucoidan mediated effects were dependent on CD14. Interleukin-15, a pro-inflammatory cytokine that binds to a distinct receptor, also induced iNOS and IL-6 mRNA levels and reduced SR-BI expression, suggesting that inflammatory signaling in general can reduce SR-BI expression levels. Treatment of macrophages with the lipoproteins acLDL, LDL or HDL suppressed the induction of iNOS and IL-6 mRNA by fucoidan or IL-15. Macrophages foam cells in an atherosclerotic plaque may have reduced SR-BI due to exposure with modified LDL or inflammatory cytokines or both in an atherosclerotic plaque. SR-BI expression in macrophages protects against atherosclerosis development. Our data suggests that modified lipoproteins as well as inflammatory stimuli suppress SR-BI expression in macrophages and this may contribute to their pro-apoptotic properties.</p> / Master of Science (MSc)

<p>Parkinson’s disease (PD) is a complex neurodegenerative movement disorder involving protein misfolding, mitochondrial dysfunction, and oxidative stress. The current dissertation, motivated by a lack of valid biomarkers and sustainable therapies, examined the potential application of a novel target for therapeutics and diagnostics of PD — the multifunctional, heat-shock like protein Catecholamine-Regulated Protein 40 (CRP40). The goal of this program of research was to elucidate further the implications of CRP40 in PD using a variety of molecular biology, bioinformatics, and clinical approaches through integrative collaborations with academia, government, and industry partners to translate scientific findings into real world solutions. Chapters 2 and 3 explored the potential therapeutic use and structure-function relationships of CRP40 through elucidating the smallest functional piece of this protein that was six times smaller, and validating a negative control for these experiments (Heat-Shock Protein 47). These initiatives could eventually lead to a small drug that could cross the blood-brain barrier and be targeted to the specific brain regions affected in PD. Chapter 4 examined the potential mechanisms of CRP40, and suggested that this protein may protect neurons from oxidative stress, maintain energy levels, and mitochondrial homeostasis, with important future implications for a variety of disorders. Finally, Chapter 5 presented compelling evidence for the potential use of CRP40 as a valid biomarker for early detection of PD and monitoring of disease progression. Overall, findings suggest that CRP40 may be a critical target for future breakthroughs in the diagnosis and treatment of PD.</p> / Doctor of Science (PhD)

<p>lmmunocompromised, burned, and cystic fibrosis patients are highly susceptible to severe and chronic <em>Pseudomonas</em> infections. Extracellular virulence factors, such as type IV pili (T4P), contribute to the establishment and maintenance of infection in these hosts. T4P are hairlike appendages involved in attachment to and colonization of biotic and abiotic surfaces, DNA uptake, biofilm formation, virulence and twitching motility. In<em> Pseudomonas aeruginosa</em>, the pilus fibre-primarily composed of PilA-is directed by the inner membrane subcomplex PilM/N/O/P to PilQ, the secretin pore. FimV is an inner membrane protein that contains a periplasmic region that binds peptidoglycan and a cytoplasmic region containing tetratricopeptide repeat (TPR) protein-protein interaction domains. FimV is essential for twitching motility in <em>P. aeruginosa</em>, but its exact function is not well understood. Here we investigate the role of the cytoplasmic region of FimV in the T4P system. Co-purification studies revealed that PilM and PilG, a protein proposed to be involved in T4P chemotaxis, interact with the cytoplasmic region of FimV. Fluoresence microscopy was used to test the role of FimV in the localization of a functional PilG-YFP fusion. In the wild type, PilG is polarly localized, while in a <em>fimV</em> mutant, PilG becomes diffuse. The interactions between FimV with PilG and PilM may play a pivotal role in twitching motility as <em>fimV</em> mutants lacking the cytoplasmic region are incapable of twitching. In this study, we have shown that FimV is interacting with components of the T4P chemotaxis system, which may be important for cAMP regulation.</p> / Master of Science (MSc)

<p dir="ltr">Cyclic peptides are a growing drug class with over 40 FDA-approved drugs, with natural product cyclic peptides being the inspiration for many of these medicines (e.g., the antibiotic daptomycin, the immunosuppressant cyclosporine A, and the antifungal caspofungin). Two major types of cyclic peptide natural products exist: (1) the ribosomally synthesized and post-translationally modified peptides (RiPPs) and (2) those made by nonribosomal peptide synthetases (NRPSs). Even though many uncharacterized cyclic peptide biosynthetic gene clusters (BGCs) exist, the discovery of new cyclic peptide natural products remains a challenge because many BGCs are cryptic. Herein, we describe the development of a new approach to access the products of these cryptic NRPS BGCs and the discovery of many novel bioactive compounds from this approach. Specifically, we utilized bioinformatics programs to predict the amino acid sequences associated with the multimodular NRPS and the presence of a nearby penicillin-binding protein (PBP)-like thioesterase (TE, PBP-TE) to determine that the predicted peptides are head-to-tail cyclized. Following the bioinformatics-guided predictions, solid phase peptide synthesis (SPPS) followed by solution phase cyclization enabled access to a library of predicted cyclic peptides that we then screened for interesting bioactivities. From this screening, we have identified molecules that stimulate the proteasome, inhibit the growth of free-living amoebas, selectively lyse Gram-negative bacteria, and suppress the immune system. Structure activity relationships for these molecules have been determined, with more potent analogs being discovered. Future directions include mechanism of action studies, as well as expanding the peptide library to include more off-loading methods and tailoring enzymes that might modify the peptide after release from the NRPS.</p>