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Introduction: Several factors such as thermal processing and cooking practices may affect food allergen structure and digestibility, as well as their ability to stimulate an immunemediated adverse reaction, such as immunoglobulin E (IgE)-mediated peanut and egg allergies. Baked matrices, including muffin and biscuit, may help to promote tolerance to these foods. However, the impact of the gluten matrix in such baked products on the bioaccessibility of allergens in the gastrointestinal tract is not well understood. The transport of food proteins and peptides across the gut epithelial cells may also be affected by baked food matrices, studies require modifying their presentation to the immune system. Methods: Muffin containing egg and peanut was prepared using a recipe previously used for oral immunotherapy. Commercially available weaning cookies were purchased. Food samples were subjected to in vitro oral-gastro-duodenal batch digestion model and the progress of protein digestion monitored by 1D SDS-PAGE, immunoblotting, and inhibition ELISA using allergen-specific antibodies. The IgE-reactivity of digests was assessed by immunoblotting using human sera and plasma samples from peanut and egg-allergic patients, respectively. Second, the transport of ovalbumin in muffin and biscuit digests, as well as the purified ovalbumin, was investigated using gut epithelial models: Caco2 cell alone and the Caco2/HT29-MTX co-culture model. In order to characterize the co-culture system, DESI-MS imaging was used to map the distinct lipid species of the intestinal epithelial cell lines Caco2 and HT29-MTX cells in both the positive and negative ionization modes. The transported ovalbumin in different matrices across Caco2 cell monolayer and the co-culture monolayer was then evaluated by inhibition ELISA using an ovalbumin-specific antibody. Results: The bio-accessibility of ovalbumin and the peanut allergens Ara h 1 and 3 was reduced in the muffin compared to the biscuit in the gastric digestion. However, during duodenal digestion, whilst Ara h 1 and Ara h 3 were rapidly digested from the muffin, ovalbumin formed a stable high molecular weight intermediate digestion product, whilst, the peanut allergen was Ara h 2 was highly resistant to gastric digestion. The IgE reactivity of peanut proteins and their digested products remained unchanged after proteolysis by pepsin, whilst that of egg proteins was reduced. Caco2 cells were characterised using MSI in positive and negative mode showed several lipid species, such as cholesterol ester, phosphatidylcholine, triglyceride, and fatty acids. However, HT29-MTX cell characterization showed a different lipid profile, such as phosphatidylethanolamine, phosphatidylinositol in addition to phosphatidylcholine. The transport of ovalbumin to the basal side was low in the co-culture model compared to the Caco2 cell model, indicating the accumulation of ovalbumin in the co-culture was higher than in the Caco2 cell culture. The barrier integrity of Caco2 monolayer was affected after the transportation of muffin digests compared to a biscuit or purified ovalbumin, but it was not changed in the co-culture model. Conclusion: The differences in the structure of the matrices used in this study may explain why allergen bio-accessibility was greater from the biscuit than the muffin. The biscuit matrices are formed from a sugar glass in which starch, gluten, and fat are embedded whilst muffins are formed from a high-water batter and have a sponge-type structure. It is likely that the biscuit sugar-glass will disintegrate readily on hydration, releasing incurred allergens. In contrast, the muffin matrix requires the action of proteases and amylase before it will disintegrate and release incurred allergens, as has previously been observed for bread. DESI-MSI confirms the Caco2 cells were differentiated in the culture as well as functioning as mature cells in regard to their cellular metabolic activity (in particular lipids). The transport of muffin digests has an impact on the barrier integrity of the Caco2 cell monolayer while not in the co-culture monolayer. Sequestered ovalbumin from either matrix, this may result either from ovalbumin becoming trapped in the mucus layer or becoming taken up into the cells. The level of uptake was lower from the muffin, perhaps reflecting the lower levels of the bio-accessible allergen from this matrix. These data indicate that the slower release of allergens from the muffin matrix may make this a more suitable matrix for oral tolerance induction.

Poultry are vital to food security, with 60 billion chickens reared worldwide per annum and demand fast accelerating. For many years antibiotic growth promoters have been used to promote energy retention from the diet and control intestinal bacterial growth. Antibiotic use for prophylaxis or growth-promotion in farmed animals is prohibited under EU Directives due to human health concerns, but a pressing need exists to maintain the efficiency of animal production by finding alternatives. Antimicrobial peptides (AMPs), part of the innate immune system exist naturally in most species and could provide a vast array of potential therapeutics. Microbial resistance to AMPs is unlikely due to their relatively unspecific mode of action, their ability to target multiple sites within a cell and diverse immune-modulatory activities. The avian egg provides antimicrobial protection through many mechanisms including AMPs which are incorporated into the egg white by the hen. The ovodefensin family and ‘transiently expressed in neural precursors’ (TENP) have been identified as potential novel antimicrobials in egg white and therefore formed the basis of the peptide portfolio of this study. TENP was first identified as having a role in neurological development but has since been shown to be an important egg component constituting ~0.1-0.5% of the total protein. TENP is conserved across avian species being found in chicken, turkey, duck and zebra finch. Its homology with the bacterial permeability-increasing family of innate immune genes suggests it may contribute to antimicrobial function in the egg. This study confirmed that expression of TENP is confined to the albumen forming region of the oviduct in adult hens and is under gonadal steroid control, typical of an oviduct and egg specific gene. The ovodefensin family are β defensin related antimicrobial peptides thought to be restricted to the albumen producing region of the avian oviduct. This study identified twenty five novel ovodefensin members through genome analysis, expanding the ovodefensin family to include reptiles for the first time. Phylogenetic analysis showed a unique example of the evolution of a cysteine spacing motif alongside traditional sequence evolution. The expression of eight ovodefensins was shown to be oviduct specific supporting the hypothesis that ovodefensins evolved to protect the egg. Antimicrobial activity for three ovodefensins from chicken and duck was investigated against gram negative organisms E. coli and Salmonella including pathogenic strains as well as a gram positive organism, S. aureus, for the first time. The spectrum of activity varied greatly between peptides suggesting a link between structure and function. Inclusion of recombinant ovodefensin peptides in the feed of chickens showed beneficial effects on the gut microbiome, metabolite profile and most crucially an increase in mean body weight. This demonstrates the potential of antimicrobial peptides as alternatives to antibiotic growth promoters in poultry.

The aim of this research was to identify the molecular mechanisms that promote spontaneous activation in rat eggs after their recovery from the oviduct. Typically, mammalian eggs await fertilisation arrested at the second metaphase II of meiosis. However, ovulated rat eggs spontaneously enter anaphase II when exposed to in vitro culture. After extrusion of the second polar body, these spontaneously activated eggs do not proceed to interphase but become arrested at metaphase III stage with chromatids scattered in the egg cytoplasm. This instability may be one factor that has made it more difficult to establish reliable protocols for somatic cell nuclear transfer in rats. The triggers of spontaneous activation and signalling pathways leading to the metaphase III progression are largely unknown. Analyses of signalling pathways that are involved in the regulation of final stages of meiosis during fertilisation revealed several anomalies that were associated with spontaneous activation and the transition from metaphase II to metaphase III. Metaphase II arrested eggs usually exhibit an increased level of maturation promoting factor (MPF) activity. Spontaneous activation in rat eggs was associated with a drop in MPF activity at the time of the second polar body extrusion. MPF is composed of a catalytic subunit, CDK1, and a regulatory subunit, cyclin B1. Interestingly, the level of cyclin B1 was stable throughout spontaneous activation. Post-translational modifications of CDK1 can influence MPF activity: whereas no inhibitory phosphorylation on Tyr15 of CDK1 was found; a decrease in activating Thr161 phosphorylation of CDK1 was associated with the time of the second polar body extrusion, and hence could contribute to the transient MPF inactivation. MAPK (p42/p44) activity has been shown to decrease during egg activation in fertilisation. By contrast, during spontaneous activation, MAPK (p42/p44) remained active and thus resembled the profile usually found between two meiotic divisions (metaphase I to metaphase II). Securin, a protein which prevents premature chromatid separation, was degraded in eggs going through spontaneous activation. Cytostatic factor (CSF) is a biochemical activity, which enables stable metaphase II arrest in ovulated eggs of vertebrates. Recently, the endogenous meiotic inhibitor 2, EMI2, was confirmed as the major component of CSF. For egg activation to occur, the CSF must be destroyed. At the beginning of egg activation, Ca2+/calmodulin kinase (CaMKII) promotes posttranslational modifications of EMI2, leading to its degradation. In the rat, inhibition of CaMKII activity stably prevented the onset of spontaneous activation in a subset of metaphase II eggs. However, no degradation of EMI2 protein was found at the start of abortive metaphase II exit. This finding revealed that one of the central elements of the CSF pathway, EMI2, could be preserved in the rat eggs going through spontaneous activation. In order to study the mechanisms regulating EMI2 stability in rat oocyte maturation and spontaneous activation, functional analysis of ectopically expressed synthetic mRNA was performed. The mechanism enabling EMI2 degradation became active 12 hours after the start of oocyte maturation. The C-terminal fragment of EMI2, known to be non-degradable in Xenopus oocyte maturation, was significantly more stable than the full-length counterpart in matured rat eggs but not during oocyte maturation. Interestingly, C-terminal EMI2 became degraded in parthenogenetic rat embryos. This indicated that additional not previously reported mechanisms responsible for EMI2 degradation might exist in the rat. The microinjection of metaphase II rat eggs with the C-terminal fragment of EMI2 or IVT full-length EMI2 protein had little effect on the progression of spontaneous activation. Taken together, these observations suggest that abortive spontaneous activation in rat eggs was a result of incomplete engagement of signalling pathways normally triggered in fertilisation or parthenogenetic activation. Activation of CaMKII initiated pathways that allowed anaphase entry and chromatid segregation. At the same time, not all pathways normally triggered during fertilisation or parthenogenetic activation were fully engaged, possibly due to the presence of non-degraded component of CSF. Abortive incomplete activation results in the re-establishment of high level of MPF activity in metaphase III eggs. Early prevention of CaMKII activation, perhaps by blocking [Ca2+ i] signalling, may provide a means of holding ovulated eggs at metaphase II prior to enucleation and somatic cell nuclear transfer.

This dissertation investigated aerodynamic, glottographic, and acoustic differences between habitual and clear speech. Nine normal-speaking individuals (five cis female, 4 cis male) were asked to read six short sentences in four reading conditions: habitual reading, habitual reading while holding a mask to the face to capture airflow and oral air pressure, clear reading, and clear reading while holding the mask to the face. Mask-off conditions in both habitual and clear reading manners were used for acoustic analyses, and mask-on conditions were used for aerodynamic and glottographic analyses. The instruction for eliciting habitual speech was “Read each sentence as if you are talking with a friend across the table.” The instruction for eliciting clear speech was “Read the sentences as clearly as possible by enunciating well, as if someone is having trouble understanding you.” Acoustic and time-related results indicated that from habitual to clear speech: (1) sentence duration increased, (2) speaking rate decreased, (3) duration of stressed vowels and unvoiced fricatives increased, (4) voice onset time increased for some unvoiced plosives, (5) stop gap duration increased, (6) fundamental frequency did not change except for two stressed vowels in female speakers for which fo increased, and (7) intensity of stressed vowels and stop consonants increased, but not for unvoiced fricatives (except for /ʃ/). Aerodynamic results indicated that from habitual to clear speech, there was greater (1) oral air pressure, (2) average airflow, (3) total air volume, and (4) peak flow during the release of the voiceless bilabial stop, suggesting the influence of greater subglottal pressure. In contrast, there was little to no change in glottal dynamics such as EGG width, EGG height, EGG contact and open quotients, and glottal airflow timing measures. In this study, it might be inferred that clear speech was a phenomenon that is more related to subglottal pressure and oral cavity kinematics than to vocal fold adduction and vibratory dynamics.

Diet is known to be a strong determinant of gut microbiome composition and function, and it is also known that gut microbiome dysbiosis is strongly linked to the risk of chronic disease. However, it is not clear how to intervene through diet or supplementation to beneficially alter the gut microbiome in individuals who have dysbiosis or who are at risk for chronic diseases such as cardiovascular disease and Alzheimer’s disease (AD). Hypothesis driven studies to determine how dietary changes affect the diet-gut microbiome-host interrelationship are critical for the discovery of solutions to improve health and mitigate disease risk. In this dissertation, three chapters are included to discuss important aspects of study design and prebiotic selection, and the outcomes of a study investigating the effects of a prebiotic supplement.In the first chapter, critical factors in successful study design for diet-microbiome research are discussed. For example, the importance of detailed dietary data collection and analysis, tight control of background diet, and stabilizing diet in the days prior to sample collection is critical for reducing noise and amplifying the signal that can be detected of the effects of the intervention diet. Specific recommendations such as including multiple consecutive sampling per study timepoint and collection of multiple consecutive days of diet records, particularly the days prior to sample collection, are discussed. High interindividual variability in gut microbiome composition is a well-established phenomenon that has been known since the early results from the Human Microbiome Project. Because of this high interindividual variability, some study designs are not well suited to discovering the effects of a dietary intervention. Instead, we discuss the double-blinded, randomized order, placebocontrolled, cross-over study design is the optimal study design for gut microbiome research, enabling the detection of the effects of even a subtle diet intervention. We also discuss the importance of precision nutrition approaches for nutritional recommendations. As an example, we discuss nutritional recommendations for egg intake, taking into account the link between egg-derived components (i.e., choline and cholesterol) and their interaction with the gut microbiome and subsequent health outcome measures important in cardiovascular disease.The second chapter emphasizes why we care about changes in gut microbiome in human health and how we can better utilize certain dietary interventions such as prebiotics to modulate human health and diseases focusing particularly on AD. Prebiotics are oligosaccharide and polysaccharide structures isolated from plants or synthesized from sugars that are shown to modulate the microbiome and are associated with health benefits. We summarized the importance of dietary fiber intake for maintaining a healthy gut microbiota, with a focus on short chain fatty acid production, mucus secretion, and protection from pathogens. The specific links between healthy gut physiology, gut immune function and the brain are also discussed. The importance of anti-inflammatory metabolites produced by gut microbes consuming specific substrates in modulating critical signaling pathways in the brain is discussed in the context of AD prevention. We conclude by discussing which specific bacterial genera are increased and decreased in AD patients and highlight the potential of increasing bifidobacteria abundance through the intake of specific prebiotic substrates as a promising approach for beneficially modulating the gut microbiome and improving health outcomes in AD patients.The final chapter summarizes the findings from our human clinical trial in healthy subjects consuming a low fiber diet (< 15 g/day) to determine whether a low-dose, easy to use bifidogenic prebiotic supplement formulation increases bifidobacteria and produces measurable metabolomic and metagenomic changes over the course of 4 weeks. We used the double-blinded, placebo-controlled, randomized order crossover study design highlighted as the optimal study design for diet-microbiome studies in chapter 1, to study the effects of the prebiotic supplements in 20 otherwise healthy young individuals consuming diets low in fiber, typical of the average American diet. As hypothesized, the relative abundance of bifidobacteria significantly increased after the prebiotic supplement, as did the gene counts of genes associated with prebiotic utilization, acetate production, and choline oxidation. Importantly, these changes in gut microbiome composition and function were reflected by changes in the concentrations of microbially-derived metabolites in the plasma, and point to novel pathways by which increasing the abundance of bifidobacteria may confer beneficial health effects on the host.

Microbial studies have led to various ecological and medicinal discoveries. Amphibian eggs, like those of Ambystoma maculatum, exhibit natural resistance to bacterial invasion that may be microbiome related. The purpose of this research was to sequence the external and internal microbiome of A. maculatum egg masses and compare with the environmental microbiome. It was hypothesized that the external and internal microbiome would bear more similarity to each other than the water. A significant difference was observed between the microbiome of the inside surface of the egg mass and the water but not the outside surface of the egg and the water, suggesting that the internal environment consists of unique microbes possibly acquired through vertical transmission or interactions with other organisms.

Food Insecurity (FI) is defined as a condition in which individuals lack access to adequate food due to limited financial resources. FI is estimated to impact 12% of households in United States. Adults and children who experience FI are an increased risk for developing metabolic disease such as type 2 diabetes, obesity, hypertension, and cardiovascular disease. The negative health outcomes associated with FI are multi-factorial, however, many of them may be caused by limited nutritional intake and poor diet quality. Efforts to better understand the obstacles food insecure populations face is critical in order to improve nutrient intake and minimize the negative health outcomes involved. Dietary intake of eggs may serve as applicable solution for FI families who are challenged by limited nutritional intake. Eggs contain a variety of nutrients that support metabolic health. For instance, eggs are a complete source of high quality protein. Diets abundant in high quality protein are shown to improve body composition, nutrient intake, and markers of cardiometabolic health. In addition, eggs contain sixteen vitamins and minerals, and are one of the few sources of naturally occurring vitamin D. Furthermore, eggs are cost-efficient. When comparing the relationship between foods based on calories and unit cost, the energy cost of eggs is significantly less when compared to other animal protein foods such as meat, poultry and fish. However, dietary intake of eggs is controversial in-regards to cardiovascular health and it is unclear whether or not regular and long-term consumption is appropriate. Therefore, the objective of this thesis was to determine if habitual egg intake improves body composition, cardiometabolic markers, and nutrient status is adults and children from food insecure households

Objective:Art song is a unique genre in the realm of European classical music, which embraces the combined beauty of vocal melody, instrumental accompaniment, and text. In a performance context, the same composition can be performed with a variety of emotional interpretations. The purpose of this study was to determine sound production differences relative to two emotional interpretations in performing an excerpt from a classical art song. Methods/Design:The first author, a soprano with a master’s degree in vocal performance, recorded an excerpt from “La Mort d’Ophelie” composed by Hector Berlioz (1803-69). The excerpt was sung in two contrasting musical interpretations: an “empathetic legato” approach, and a “sarcastic” approach with emphatic attacks. Microphone, airflow (Glottal Enterprises MSIF-2), and electroglottography (EGG; Kay Model 6103) signals were digitized. These recordings were analyzed for acoustic, airflow, and glottographic measures. The vowels in the musical excerpt were analyzed in terms of intensity, long term average spectra (LTAS), fundamental frequency vibrato rate and extent, vowel onset, intensity comparison of harmonic frequencies, and glottal measures based on electroglottograph waveforms. Results & Conclusions:Data analyses revealed that stressed vowels, when performed with the emphatic approach compared to the legato approach, had faster vowel onset, increased glottal adduction (relative to the EGGW25 measure), increased intensity of harmonics in the 1500 to 3000 Hz range, inferred increase in subglottal pressure, increased airflow for the /f/ consonant, and greater aspiration airflow for the plosives /t/ and /p/. The vibrato extent for both fo and airflow were both greater for the emphatic approach. Findings also revealed larger amplitude values of the EGG waveform, but this finding was not statistically significant. Long-term average spectrum (LTAS) analyses of the entire production displayed minor increases across all formant frequencies for the emphatic approach. While this is a single-case objective study, it emphasizes the reality and informative nature of physiological, aerodynamic, and acoustic production differences in the pedagogical and interpretive aspects of art song performance. Vocal performers, teachers, and other music educators may benefit from applying findings like those of this study to developing performance strategies while maintaining healthy vocal production.

Abstract: Osteoporosis is a common disease afflicting a quarter of the population of 50 years old and above worldwide. The osteoporotic fracture brings great pain/inconvenience to the patients, and sometimes causes immobility or even mortality. Drugs are available for treating osteoporosis but are associated with serious side-effects. There is an interest in searching food components for improving bone health. Egg yolk phosvitin is one of the most phosphorylated proteins in nature with potential for bone health. The overall objectives of this thesis were to develop a scalable method of phosvitin extraction and to study the effects of phosvitin and phosvitin hydrolysate on bone cells as well as osteoporosis rat model. Since inflammation stimulates bone cells and causes bone loss, the effects of phosvitin and phosvitin hydrolysate on bone cells under inflammatory stimulation were investigated. The first experiment studied the effects of pH, low centrifugal force, use of industrial separators and different water dilution factors on the extraction. The results suggested that decreasing pH from neutral to acidic range and increasing water dilution ratio from 1/1 to 1/4 (yolk/water, weight/weight) increased phosvitin purity and recovery. Low centrifugal force resulted in low phosvitin recovery, although the purity remained at high level. None of the industrial separators (decanter and disc stack separators) were sufficient to separate pellet from egg yolk and thus resulted in significantly lower yield (~20-30%), while the purity was comparable (~70%). Phosvitin and its hydrolysate promoted osteoblast differentiation by upregulating expression of runt-related transcription factor 2 (RUNX2), alkaline phosphatase, osteocalcin production and collagen synthesis. Tumor necrosis factor alpha (TNF-ɑ) induced production of inflammatory proteins including regulated on activation, normal T cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1) and cyclooxygenase-2 (COX-2). Phosvitin and phosvitin hydrolysate inhibited the production of these proteins in osteoblast cells. In another experiment, both phosvitin and phosvitin hydrolysate inhibited receptor activator of nuclear factor kappa-Β ligand (RANKL) induced osteoclastogenesis in RAW264.7 cells. Inflammatory biomarkers, such as TNF-ɑ, MCP-1, RANTES, and inducible nitric oxide synthases (iNOS), were also significantly suppressed by phosvitin and phosvitin hydrolysate. In the animal study with ovariectomized (OVX) rats, 2% phosvitin hydrolysate in the diet improved bone micro structure and prevent bone loss measured by Micro computed tomography. Higher concentrations of serum osteocalcin (OCN) and N-terminal propeptide of type 1 collagen (P1NP) and lower serum Tartrate-resistant acid phosphatase isoform 5b (TRACP 5b) were also detected in the same animal group, indicating the activities of phosvitin hydrolysate might be a combination of both anabolic and anti-resorptive effects. The findings of this thesis suggest that phosvitin and phosvitin hydrolysate have good potential to be incorporated into functional foods to reduce bone loss and/or promote bone growth.

The mosquito Aedes aegypti is the primary vector of dengue, yellow fever, chikungunya, and Zika viruses. Despite decades of effort to curb transmission of these diseases, this mosquito’s continued status as the most proficient arbovirus vector highlights the need for new and improved vector control methods. Manipulating entire mosquito populations in the wild is a promising avenue by which mosquitoes may be controlled. Typically, such strategies aim either to suppress a wild population by limiting reproduction or to replace a population with mosquitoes with a desirable trait (such as refractoriness to disease). Regardless of the type of manipulation, these strategies require that altered individuals (usually reared in laboratories or factories) are deployed into the wild to compete with and mate with wild individuals. While mosquito reproduction has been studied for decades, many questions still remain to be answered that could help to improve mosquito release strategies. The purpose of this dissertation was to answer fundamental questions surrounding Ae. aegypti reproduction that will contribute to the development of new or enhanced mosquito control strategies. Specifically, it addresses the timeline with which several reproductive events occur and lays a foundation for future investigations to describe the cellular and molecular underpinnings of important reproductive events. First, I addressed the question of when Ae. aegypti females mate more than once. While consensus in the literature suggested that they do occasionally mate more than once, some studies suggest that re-mating occurs soon after a female’s first mating, and others claim that females only mate after they have undergone several gonotrophic cycles. Using males with fluorescently labeled sperm, I demonstrate that females are most likely to mate a second time within 2 h of their first mating, that almost no re-insemination occurs after 24 h post-mating (hpm), and that no females re-mate after they have begun laying eggs. Given this 24 hpm period in which behavior drastically shifts, I wondered if other aspects of reproductive physiology also follow similar timelines. Studies of other mosquitoes’ sperm suggested that sperm are initially equipped with a thick glycocalyx, but that this outermost layer is removed some time after mating. To understand whether this occurs in Ae. aegypti, when it occurs, and if it is typical of all sperm, I used cryo-electron microscopy to image sperm from the male prior to insemination and at different times post-insemination from the female. Within 24 hpm, sperm indeed shed a thick outer glycocalyx. Motility assays over the same 24 h period indicated that within 8 h, sperm had achieved their maximum motility. Finally, I tested whether oviposition is initiated and fertility is achieved during this period. Both reached their peak at ~16 hpm, with fertility gradually setting in beginning at 4 hpm and oviposition experiencing a sharp increase after 12 hpm. After investigating Ae. aegypti reproduction in the laboratory, I aimed to describe aspects of its reproductive biology in an epidemiologically relevant field setting. I intensively sampled Ae. aegypti in Medellín, Colombia, by aspirating resting adults from homes. By recording data on each female’s mating status, parity status, and whether she had taken a blood meal and developed eggs, I compared virgin and mated females’ behavior and physiology. Females frequently took blood meals prior to mating. However, blood fed virgins’ abdominal distension was significantly lower than that of mated females, suggesting that virgins may take smaller blood meals. Some virgins also produced eggs, and those that did produced a number of eggs similar to their mated counterparts. These observations provide the first comprehensive comparison of mated and virgin Ae. aegypti feeding behavior and reproductive physiology in a field setting. Knowing when critical reproductive events occur is a critical first step to understanding how they occur. To enable future investigations of the cellular and molecular mechanisms that govern reproductive events, I developed a comprehensive proteome for both Ae. aegypti sperm and seminal fluid. Building on previous work by Sirot et al. (2011) and using state-of-the-art mass spectrometry technology, I was able to identify over 870 putative sperm proteins and 280 putative seminal fluid proteins. These findings were largely corroborated and supported by transcriptomes that I generated from the testes and male accessory glands. The findings in this dissertation will support future investigations of crucial reproductive processes that may be manipulated for the purposes of vector control.

Finite element models were developed with the purpose of finding an optimal radio frequency (RF) heating setup for pasteurizing shell eggs. Material properties of the yolk, albumen, and shell were measured and fitted into equations that were used as inputs for the model. When the egg was heated by itself, heating tend to be focused at the air cell to result in a “coagulation ring.” The focused heating near the air cell of the egg prevented satisfactory pasteurization of the egg, but deeper analysis of the simulation results offered a new perspective on how non-uniform RF heating could occur in heterogeneous food products, especially those that contain air bubbles. By immersing the egg in deionized water, the “coagulation ring” disappeared and heating was concentrated in the yolk after RF heating. Extrapolation of the model suggested that 3 log reductions of Salmonella can be achieved within 37 min of RF heating followed by hot water immersion. In addition, the contamination of the water surrounding the egg was shown to have a positive impact on the heating rate up to a threshold, at which point further contamination decreased the effectiveness of the process. The knowledge gained in this study could be used to design pasteurization equipment for shell eggs that are faster than conventional equipment. Advisor: Jeyamkondan Subbiah

Abstract: Hypertension afflicts ~22% of Canadian adults and 80% of the Canadian population over the age of 65. Due to the inevitable side effects associated with drug use, there is increasing interest in developing functional foods or natural health products as an alternative for the prevention and management of hypertension. Bioactive peptides with inhibitory activity against angiotensin converting enzyme (ACE), a key enzyme for regulating blood pressure, may reduce blood pressure. Our previous study has identified three potent ACE inhibitory peptides, IRW (Ile-Arg-Trp), IQW (Ile-Gln-Trp) and LKP (Leu-Lys-Pro) from egg white protein ovotransferrin. The specific objectives of this thesis were to 1) evaluate the anti-inflammatory and anti-oxidant activities of these peptides on endothelial cells and to understand their structural requirements for these activities, 2) test their in vivo anti-hypertensive activities using spontaneously hypertensive rats (SHRs), and 3) explore the possible molecular mechanism of action of egg peptide through gene expression analysis. The anti-oxidant and anti-inflammatory activities were studied using human umbilical vein endothelial cells (HUVECs). IRW significantly inhibited tumor necrosis factor (TNF) stimulated expression of intercellular cell adhesion molecule-I (ICAM-1) and vascular cell adhesion molecule-I (VCAM-1). IQW and LKP, however, only inhibited the expression of ICAM-1 or VCAM-1, respectively. The anti-inflammatory activities of IRW and IQW were mediated differentially through transcription factor nuclear-kappa B (NF-κB). Both IRW and IQW exhibited anti-oxidant activity by reducing superoxide generation, whereas LKP did not show any effect. The structural integrity of these peptides (IRW and IQW) was essential for their activities, since dipeptides or the combination of their constituent amino acids did not exhibit the same effect. In vivo antihypertensive activity was studied in SHRs using the telemetric method. Oral administration of IRW, IQW, and LKP (15 mg/kg BW) significantly lowered mean arterial pressure (MAP) by 40, 19, and 30 mmHg, respectively, over a period of 18 days. IRW treatment led to decreased angiotensin II (Ang II) levels, restored nitric oxide (NO) dependent vasorelaxation, and decreased expression of inflammatory markers such as ICAM-1 and VCAM-1 as well as tissue fibrosis. Therefore, the antihypertensive effect of IRW was likely mediated through ACE inhibition, endothelial nitric oxide synthase, and anti-inflammatory properties. IQW and LKP treatments also reduced plasma Ang II level and restored NO-dependent vasorelaxation; however, only IQW treatment reduced ICAM-1 expression and nitrotyrosine levels in the arteries. The antihypertensive activities of IQW and LKP were likely mediated through similar pathways involving increased NO-mediated vasorelaxation and ACE inhibition, but only IQW reduced vascular inflammation and oxidative stress. Since IRW (at 15 mg/kg BW) exhibited the most pronounced anti-hypertensive, anti-inflammatory, and anti-oxidant activities compared to other two peptides, further transcriptomics study was used to delineate the molecular mechanism. The study revealed that IRW treatment could significantly modulate the gene expression in mesenteric artery. Interestingly, IRW treatment could significantly up-regulated the expression of angiotensin converting enzyme-2 (ACE-2), a vasodilatory enzyme of the renin-angiotensin system (RAS). Upregulation of ACE-2 reduces the hyperactivity of the RAS and reduces blood pressure. In addition, IRW could reduce the gene expression of pro-inflammatory adhesion molecules, ICAM-1 and VCAM-1 that further supported the anti-inflammatory activity of egg peptide. Thus the present study confirmed the anti-hypertensive activity of these three egg protein derived peptides. ACE inhibitory peptides were thought to function through the inhibition of ACE. Our study added new insights that the antihypertensive activity of these peptides could be due to anti-inflammation and antioxidant activities, restored NO-dependent vasodilation, and up-regulation of ACE-2. Findings from this study support the potential of egg protein derived peptides for uses in the prevention and management of hypertension.

The 14-3-3 (YWHA) is a highly conserved, ubiquitously expressed protein family regulating important cellular processes including cell cycle. This work, for the first time, explored the differential expression and roles of 14-3-3 isoforms during mouse oocyte maturation. All seven mammalian 14-3-3 isoforms were identified in mouse eggs and ovarian follicular cells including oocytes, by Western blotting. Immunocytochemical and immunohistochemical staining confirmed the presence of all 14-3-3 isoforms in oocytes, eggs and ovarian follicles with characteristic similarities and differences in their distributions. Mammalian oocytes are arrested at meiosis prophase I by an inhibitory phosphorylation on Cyclin-Dependent Kinase I (CDK1), released by CDC25B phosphatase which is bound and inactivated in phosphorylated form by 14-3-3 in oocyte cytoplasm. Here, in situ Proximity Ligation Assays (PLA) revealed that all 14-3-3 isoforms interact with CDC25B in oocytes, with reduced interactions in eggs. Phosphorylation of CDC25B at Ser-149 was found to be reduced in eggs compared to oocytes. Microinjection of a translation-blocking morpholino oligonucleotide against 14-3-3eta mRNA caused germinal vesicle breakdown in significantly higher percentage of oocytes compared to oocytes injected with morpholinos targeting other 14-3-3 isoforms. Thus, interaction of 14-3-3eta with CDC25B is required for maintaining prophase I arrest in oocytes. Protein 14-3-3eta was observed to accumulate and co-localize with alpha-tubulin at both meiosis I and II spindles during mouse oocyte maturation in vivo as well as in vitro. It interacts directly with alpha-tubulin with an accumulation of the interactions at meiotic spindles, detected by in situ PLA. In a significant 76% of mouse oocytes microinjected with the morpholino against 14-3-3eta mRNA, meiotic spindles were deformed or absent with clumped chromosomes, no accumulation of 14-3-3eta and no polar body formation. All control eggs showed normal, bipolar spindles with accumulation of 14-3-3eta. Therefore, 14-3-3eta is essential for normal meiotic spindle formation during in vitro maturation of mouse oocytes, in part by interacting with alpha-tubulin, to regulate the assembly of microtubules. These studies reveal 14-3-3 isoform-specific interactions with key proteins involved in mouse oocyte maturation, such as CDC25B and alpha-tubulin. The results help to elucidate the roles of 14-3-3 proteins in mammalian oogenesis and reproductive development.

Abstract: Eggs are an economical source of nutrient rich food containing essential nutrients but are also one of the eight most common foods causing allergy today. Eggs are present in a wide variety of foods including sauces, bread and pasta. A novel method for the production of hypoallergenic egg ingredients should be developed. Fermentation of egg white proteins in sourdough was studied and revealed that ovotransferrin was degraded. No significant change was noted in ELISA while immunoblot showed elimination of IgE binding to the single protein ovotransferrin. Fermentation of egg white solutions and extracted ovomucoid was also studied and revealed that A. oryzae fermented samples showed a decrease in IgE binding with a minor shift in ovalbumin-related protein Y, characterized by LC ESI-MS and MS/MS. The use of fermentation as a pretreatment in ovomucoids’ susceptibility to enzymatic hydrolysis showed that there was no significant difference before and after fermentation.

Abstract: Phosphopeptides derived from egg yolk phosvitin have been reported to exhibit various physiological functions. However, commercial use of phosvitin phosphopeptides (PPPs) has been hampered due to the lack of a simple and economic method to prepare PPPs. The purposes of this study were to develop a protocol to extract phosvitin from egg yolk, and to prepare and characterize phosphopeptides derived from enzymatic hydrolysis of phosvitin. The phosvitin prepared in this study had comparable or higher purity and recovery than previous methods. The atomic ratio of nitrogen to phosphorus (N/P) describes the density of phosphorus in peptide sequences. A lower value relates to higher density of phosphorus and high phosphorus content is preferred for bioactivities. The phosvitin hydrolysate showed comparable or lower N/P ratio than purified casein phosphopeptides. Novel phosphopeptides with high phosphorylation degree were identified. The protocols developed in this study could be used for laboratory uses and industrial scale-up. The phosphopeptides identified in this study are expected to exert multi-physiological functions and to be developed as functional food ingredients.

A dynamic model for the growth of Salmonella spp. in liquid whole egg under continuously varying temperature was developed and validated. A primary model (Baranyi model) was fitted to each temperature growth data and corresponding maximum growth rates were estimated. Pseudo-R2 values were greater than 0.97 for primary models. The maximum growth rates obtained from each primary model were then plotted against temperature and modeled using the modified Ratkowsky model. The pseudo-R2 and root mean square error were 0.99 and 0.06 h-1, respectively for the secondary model. A dynamic model for the prediction of Salmonella spp. growth under varying temperature conditions was developed by solving a combination of primary and secondary models using fourth-order Runga-Kutta method. The developed dynamic model was validated for two sinusoidal temperature profiles, 5-15°C and 10-40°C with corresponding RMSE values of 0.28 and 0.23 log CFU/ml, respectively between predicted and observed values. The developed dynamic model can be used to predict the growth of Salmonella spp. in liquid whole egg under varying temperature conditions. The germination and outgrowth of B. cereus and C. perfringens spores during the egg white hydrolysate (EWH) manufacturing process was evaluated. EWH was prepared by enzymatic hydrolysis of the inoculated egg white solution and maintained at optimum pH for enzymes (trypsin and chymotrypsin) for 24 h at 37°C. B. cereus population was increased by > 3.5 log CFU/ml during the EWH preparation using chymotrypsin, while minimal increase was observed during trypsin hydrolysis. C. perfringens populations were reduced during EWH preparation irrespective of the enzyme used. Hydrolysis of major egg proteins by chymotrypsin was completed within 4 h of incubation, while minimal hydrolysis of egg white proteins was observed with trypsin subsequent to 24 h incubation. New enzymes other than trypsin and chymotrypsin should be exploited for manufacturing egg white hydrolysates. Advisor: Harshavardhan Thippareddi

In-shell thermal pasteurization of eggs has adverse effects on the appearance and functionality of eggs. Ozone-based processing is an alternative technology with potentially fewer adverse effects as it is less thermally intrusive. There are no consumer acceptance studies published on either the appearance or the taste of ozone-treated eggs. This study examines whether consumers can detect differences when comparing ozone-treated eggs to commercially available pasteurized eggs and to fresh unpasteurized eggs. A visual difference test evaluating the appearance of freshly cracked uncooked eggs and a consumer affective test evaluating microwave scrambled eggs made from freshly cracked whole shell eggs was conducted. Visual panelists evaluated eggs on cloudiness of the albumen and yolk, spread of the albumen and yolk, height and color of yolk and overall visual appeal using 10-point linear scales. The yolks and albumens of the thermal treated and ozone-treated eggs were perceived as significantly cloudier than the unpasteurized control while the ozone-treated were perceived as more similar to the control (p < 0.05). The yolks of the ozone-treated eggs had a significantly lower heights and greater spreads than the other treatments (p < 0.05). Despite these differences, overall visual appeal of ozone-treated eggs was not significantly different from control. In the consumer affective test, panelists used 9-point hedonic scales to evaluate overall liking, appearance, aroma, flavor, and texture. Just-About-Right scales were used to rate the appeal of color, moistness and texture. There was no treatment effects on degree of liking on any of the attributes. The major difference in egg appeal was in moistness, where thermal treated and ozone-treated eggs were perceived as drier relative to the control but not compared to each other. This did not affect overall liking scores. This shows no adverse effects on consumer acceptance of eggs treated by the ozone process, with acceptance not different from unprocessed control on overall appearance or taste. These findings are useful when considering ozone-pasteurization to enhance the safety of fresh whole shell eggs to meet the goals of the U.S. Egg Safety Action Plan.

Proteins in the tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (14-3-3) family are involved in the regulation of many intracellular processes. We have examined the interaction of 14-3-3 with peptidylarginine deiminase, type VI (PADI6), an abundant protein found in mammalian oocytes, eggs and early embryos. Peptidylarginine deiminases catalyze the posttranslational modification of peptidylarginine to citrulline. PADI6 is associated with oocyte cytoplasmic sheets containing intermediate filaments and it has been shown that PADI6-deficient mice are infertile due to disruption of development beyond the two-cell stage. We found that PADI6 undergoes a dramatic developmental change in phosphorylation during oocyte maturation. This change in phosphorylation is linked to an interaction of PADI6 with14-3-3 in the mature egg. Recombinant glutathione-s-transferase (GST) 14-3-3 pull-down experiments and transgenic tandem affinity purification with liquid chromatography/mass spectrometry demonstrates a binding interaction between 14-3-3 and PADI6 in mature eggs, but not in the immature oocytes. 14-3-3 proteins modulate or complement intracellular events involving phosphorylation dependent switching or protein modification. These results indicate that phosphorylation and/or 14-3-3 binding may serve as a means of intracellular PADI6 regulation.

The transparent, extracellular layers surrounding the eggs of the sea urchin Arbacia punctulata account for approximately 3--11% of maternal energy in egg production, and thus likely play an important role in successful fertilization of this free-spawning organism. In this research, we have proposed an indirect approach to investigate the protection role of the jelly coat. We utilized shear flow experiment to determine critical flow rates and critical shear stresses that resulted in jelly coat detachment, and to determine deformations induced by the gradient of the velocity of the fluid flow. Our experimental results showed that jelly coats became detached at a shear stress of 22Pa; egg without jelly coat was destructed with shear stress of 24Pa. The egg and jelly layer exhibited nonlinear behavior under external loads. Shear flow produced stresses on the surface of the egg or jelly layer, which satisfied the universal deformation condition due to traction, under assumptions of an isotropic, homogeneous material. With the data from the shear flow experiment, material properties of the egg and jelly coat were found using an inverse finite element method (IFEM) approach, involving a fluid-solid interaction algorithm. The hyperelastic coefficient of the egg was found to be in the range of 110--160 Pa. Some jelly coats had a shear modulus close to that of the egg, while some had 48--64 Pa. With these material properties, simulations were done to mimic the situation that larger egg with or without jelly coat passed through the smaller oviduct during spawning. The results showed that existence of the jelly coat dramatically reduced the maximum Von Mises stress, for compressive loads. Future work will focus on determining the relative importance of the jelly coat's protective role, and its other possible functions in sperm attraction. In this work, experiments using a microfluidic device were initiated to measure the motility coefficient, and the effect of the chemoattractant on the sperm movement can be investigated based on the analysis of convection-diffusion system. By coupling these with more detailed experiments with various dilutions of jelly coats and water, we aim to compare the possible mechanical role of jelly layers, with some of its other, putative functions in a complete way.

The traditional roles of stable isotopes in paleontology are as indicators of diet or climate. An emerging field of paleontology and geochemistry is to use isotopes in order to make inferences about extinct and fossil animal physiology and behavior. Using bones from animals that minimally remodel their hard tissues, it may be possible to use isotopes to infer processes that occurred during early skeletal development. To examine potential isotope signatures of early development, I constructed a calcium isotope growth series for extant domestic chicken (Gallus gallus) embryos. I obtained Fertilized domestic chicken eggs and determined Ca isotope composition of the albumen, yolk, eggshell, and embryonic bone. The eggs ranged in ontogenetic age from 10-20 days after oviposition. Yolk and shell components have δ⁴⁴Ca values roughly consistent with those generated by other researchers on unfertilized eggs. The δ⁴⁴Ca values of all egg components showed significant heterogeneity among individuals. Prior studies of the Ca isotope composition of adult vertebrates (including fish, mammals, and some reptiles) showed that the δ⁴⁴Ca values of food and soft tissue were approximately equal, but mineralized bone lower by about 1.3‰. In contrast, the embryonic bone measured in my study was characterized by a δ⁴⁴Ca value that was higher than the primary source of Ca (shell) by about 0.4‰ and about the same as the secondary source of Ca (yolk). This result indicates yolk is a much more important source of calcium for the developing bone than was previously thought, or the egg environment is causing a fractionation of calcium isotopes opposite of that which has been seen in prior studies. This unique isotope composition of embryonic bone may, pending further study, provide a chemical marker of oviparity. Additional research is needed in order to reach a definitive conclusion about why calcium isotopes are behaving differently in eggs of Gallus than in previous studies

The aim of this study was to document the degree and causes of life history variation within and among populations of a small percid fish, the Johnny Darter. There is a significant variation in egg size, growth rate, size of individuals at a given age, size and age at maturity, total mass of eggs produced, and the mass of eggs produced relative to female body mass. To determine what accounts for this variation, and to test predictions of life history theory, I surveyed 30 populations of this species and attempted to find natural indices for factors hypothesized to be causes life history variation. The life history of Johnny Darters is influenced by the length of time during which growth occurs and the relative favorability of the habitat during the growing period. The former has a significant effect on size at a given age, relative fitness of offspring, and the mass of eggs produced relative to body size. Habitat favorability was estimated from the width of otolith growth increments during larval growth. Habitat favorability is negatively correlated with egg size and positively correlated with the size of individuals at a given age. The assumption that otoliths may be used as an index of growing conditions was tested by examining (1) the relationship between otolith growth rate and the size of individuals at one year of age and (2) the relationship between the size and growth rate of paired larval and otolith samples from laboratory-reared larvae. The results of these tests support the proposition that otolith growth rate can be used to study the growing conditions of larval Johnny Darters. The most important findings of this study are, (1) egg size has a significant effect on the size and growth of Johnny Darters, (2) otolith growth may be used as an indication of growing conditions for larvae, (3) growing conditions are good predictors of egg size, and (4) offspring fitness is highly influenced by the length of the growing season and this has a significant effect on the schedule and quantity of energy allocated to reproduction.