In animals, the accepted model of carbohydrate digestion and absorption involves reduction of disaccharides into the simple sugars glucose, fructose and galactose. Previous studies have shown the presence of disaccharides maltose and trehalose in the blood of several crab species, the crayfish and the American lobster. In 2011, a gene for a distinct disaccharide sucrose transporter (SCRT) was first found in Drosophila melanogaster and characterized using a yeast expression system. The purpose of the current study was to identify and characterize a putative disaccharide transporter analog in crustaceans using the American lobster, Homarus americanus. Brush border membrane vesicles purified from the hepatopancreas were utilized. After identification of a sucrose transporter in the brush border membrane of the hepatopancreas, transport kinetics experiments were used to characterize it using 14C radio-labeled sucrose and a Millipore filter isolation technique. Lack of glycyl-sarcosine inhibition of sucrose uptake into vesicles indicated that the highly non-specific dipeptide transporter PEPT1 was not the functional transporter of sucrose. A more acidic pH of 4 was shown to drive sucrose transport in the absence of sodium. Sodium was then shown to also significantly stimulate sucrose uptake, which resulted in an overshoot at 1 minute over a hyperbolic potassium uptake curve, suggesting that both sodium and acidic pH were capable of driving disaccharide transport. Experiments that used a variety of monosaccharides and polysaccharides indicated that the disaccharides maltose and trehalose were the only sugars to significantly inhibit carrier-mediated sucrose transport (maltose P = 0.017, trehalose P = 0.023 using a one-way ANOVA) (Km = 0.1951 ± 0.0630 mM sucrose, Jmax = 0.5884 ± 0.0823 nmol/mg protein x 1 minute), suggesting specificity of the transporter. Sucrose in the presence of 20 mM maltose had a Km of 0.5847 ± 0.1782 mM sucrose (P = 0.030) and a Jmax of 0.6536 ± 0.1238 nmol/mg protein x 1 minute (P = 0.006). ANOVA P-values indicate the difference between the sucrose control curve and the maltose curve. The highly significant reduction between the Km values of the control sucrose curve and the maltose curve suggests competitive inhibition between the two sugars. These two disaccharides could utilize the same transporter, and are appropriate for the physiology of the animal in this case, as lobsters commonly digest glycogen and chitin, polymers of maltose and trehalose, respectively. These findings suggest there is a brush-border proton-, or sodium-dependent, hepatopancreatic carrier process, shared by sucrose, maltose, and trehalose, that may function to absorb disaccharides that occur from digestion of naturally-occurring dietary constituents.

Prior work using a contractility bioassay determined that the serotonin (5-HT) receptor subtype 5-HT2A is present in bovine lateral saphenous veins and plays a role in ergot alkaloid-induced vascular contraction in steers grazing endophyte-infected (Epichloë coenophiala) tall fescue (Lolium arundinaceum). A study was conducted to determine what 5-HT receptors are involved in vasoconstriction of bovine gut vasculature. The findings of this study indicate that 5-HT2A is present and may play a role in ergot alkaloid induced vasoconstriction. A second study was conducted to determine if ergot alkaloids were transported in the small intestine. The active transporter, peptide transporter 1 (PepT1), was evaluated for its role in the transport of various concentrations of ergot alkaloids across Caco-2 cell monolayers. Results indicate that CEPH, ERT, EXT, and LSA do move across Caco-2 cell monolayers, but appear to utilize PepT1 at larger concentrations. Overall, the demonstrated presence of 5-HT2A receptors in the bovine gut vasculature established a potential for vascular interference by ergot alkaloids entering the bloodstream through transepithelial absorption.

Hyperpolarization-activated, cyclic-nucleotide gated ion channels (HCN channels) are activated by membrane hyperpolarization and modulated by cyclic nucleotides. HCN channels are important to maintain the resting membrane potential and input resistance in neurons and have important physiological functions in the brain and heart. Four mammalian HCN isoforms, HCN1-4, and the isoform cloned from sea urchin, spHCN, have been extensively studied. Among these, only spHCN channel shows a voltage dependent inactivation. Previous studies have shown that the ligand binding in mHCN2 channel is activity dependent: cAMP binding increases along with channel opening or channels in the open state have higher binding affinity for cAMP. But to date, information pertaining to the ligand binding to an inactivated ion channel or desensitized receptor is lacking. To address this gap, we used fluorescently labelled cAMP analogues in conjunction with patch clamp fluorometry (PCF) to study the ligand binding to the spHCN channel in various conformational states. We show that inactivated spHCN channel shows reduced binding affinity for cAMP, compared to that of the closed or open channel. Parallelly, we noticed significant changes to channel function when a combination of laser and photosensitizer was used to study ligand binding. A reactive oxygen species called singlet oxygen has been confirmed to be the major player in this process. Both photo-dynamically generated and chemically generated singlet oxygen modifies spHCN channel by removing the inactivation. The effect of singlet oxygen on channel can be abolished by the mutation of a key histidine (H462) residue in the ion conducting pore. Taken together, these two projects expanded our understanding about the physicochemical nature of fluorophores from two aspects: (i) the release of photon as a valuable tool to study the conformational dynamics in proteins; (ii) the generation of singlet oxygen as an effective modulator of protein function.

Atherosclerotic cardiovascular disease (CVD) is hallmarked by inflammatory immune activation, particularly by the induction of a response by monocytes. Classical (CD14++CD16-) are anti-inflammatory mediators under homeostatic conditions, while intermediate (CD14++CD16+) and non-classical (CD14LowCD16++) monocytes promote inflammation following activation. Monocyte activation and functionality is dependent upon receptor expression and ligand production by a variety of cells, including monocytes. Alterations in the expression of surface receptors often have a direct impact upon monocyte function, such as the increased pro-inflammatory cytokine production in response to activation that accompanies elevated CD14 expression or increased chemotaxis that is elicited by increased CCR2 expression. Ligand-receptor interactions also play a significant role in cell fate, including survival, proliferation, and differentiation. Monocytes are capable of differentiating into phagocytic cells known as macrophages in response to specific ligand-receptor interactions. Macrophages play a significant role in the pathogenesis and progression of CVD. Imbalance between pro-inflammatory M1 and anti-inflammatory M2 macrophages can to lead disease development and progression, such as the skewing toward the M1 phenotype that occurs in CVD. Elucidation of these mechanisms will allow for the development of targeted interventions, including pharmacological and non-pharmacological physical interventions, such as physical exercise. Therefore, this dissertation investigates the role of CD14 and CCR2 monocyte subset receptors that impact immune-mediated inflammation following ST segment elevation myocardial infarction (STEMI) as well as physical activity and cardiorespiratory endurance related differences in the acute exercise response of monocyte signaling, recruitment, and macrophage polarization and their potential role in CVD prevention.

Obesity is a major health problem in the United States and worldwide. It increases the risk for type-2 diabetes and cardiovascular diseases. A chronic low-grade inflammation occurring in white adipose tissue (WAT) is causally linked to the development of insulin resistance (IR), metabolic syndrome and obesity-associated chronic diseases. The aim of this dissertation research was to elucidate the WAT function in metabolic syndrome using genetic (overexpression of an adipose pro-inflammatory hormone, angiotensinogen) and nutritional manipulations/approaches (caloric restriction and omega-3 fatty acids), with specific emphasis on the role of inflammation. Previous research indicates that WAT renin-angiotensin system (RAS) is overactivated in obesity. However, its role in the pathogenesis of IR is hitherto unknown. Using mice overexpressing angiotensinogen (Agt), the only precursor for the hypertensive hormone angiotensin (Ang) II, in WAT, we showed that adipose-specific RAS overactivation leads to systemic IR. This is at least in part due to Ang II, NADPH oxidase and NF-kB-dependent increases in WAT inflammation. Caloric restriction is the main dietary intervention to treat obesity-associated metabolic disorders. While most health agencies recommend a low-fat diet, energy-restricted high-fat diets (HFR) are also claimed to be effective in this regard. Here, we show that weight loss due to HFR is accompanied by improvements of IR but only partial resolution of WAT inflammation. Further, this diet negatively impacted the adipokine profile supporting the current recommendations for low-fat diets. Dietary interventions targeted at reducing WAT inflammation have not been explored in detail. Eicosapentaenoic acid (EPA) is an omega-3 polyunsaturated fatty acid of marine origin with anti-inflammatory properties. We show that EPA is able to both prevent and reverse high-fat diet-induced IR and hepatic steatosis via modulation of WAT inflammation. In conclusion, primary changes occurring in WAT, such as overexpression of Agt, can lead to WAT inflammation and systemic IR. Moreover, nutritional interventions targeting at reducing adiposity (caloric restriction) and inflammation (EPA) can both lead to improvements in systemic IR. Our findings support the current recommendation of low-fat diets for improvement in metabolic profile and show that dietary modulation of WAT function can be used to improve metabolic derangements in obesity.

Mis-folded proteins and their associated aggregates are a contributing factor in some human diseases. In this study we used the protein lysozyme as a model to define aggregation structures under denaturing conditions. Sasahara et al. (2007), Frare et al. (2009, 2006), and Rubin et al. (2008) observed conditions where heat denatured lysozyme formed fibril structures that were observed to be 8-17 nanometers in diameter under the electron microscope. Even though the crystal structure of lysozyme is known, the denatured form of this protein is still unknown. Therefore, we used Rosetta++ protein folding and blind docking software to create in silico models of the protein at denaturing temperatures and subsequently docked them into aggregates. Here we compare those structures and select forms consistent with the fibril structure from the previous papers. The next step is to be able to use the predicted models of the fibrilar forms of denatured lysozyme to help us understand the exact conformation of fibril structures. This will let us confirm the docking interactions during the fibril aggregation process. The ultimate goal is to use the validated denatured structures to model interactions with heat shock proteins during the dis-aggregation process.

Human essential hypertension affects over 75 million people in the United States, and can lead to death due to its several serious health complications such as hypertension-related cardiovascular disease. The purpose of this research was to understand how hypertension could cause physiological changes to the microcirculation, specifically the PO2 dependence of oxygen consumption (VO2) in skeletal muscle of normotensive and hypertensive rats. The Spontaneously Hypertensive Rat (SHR) strain was used as the diseased model, and Wistar-Kyoto (WKY) rats were used as controls to conduct this study. The SHR strain develops hypertension between 5-6 weeks after birth with an average systolic blood pressure of 150 mmHg. By arresting blood flow using an objective-mounted inflatable airbag, PO2 measurements were obtained along with an oxygen disappearance curve (ODC), which was used to calculate VO2 over various ranges of physiological PO2 values. PO2 and VO2 curves were analyzed based on Hill’s equation to fit the data and describe the PO2 dependence of VO2. When compared to the healthy Wistar-Kyoto rats, the SHRs exhibited a higher Vmax, or maximum rate of oxygen consumption. The average maximal rate of consumption by the hypertensive animal models could be a consequence of a “mitochondrial uncoupling” or some disconnect in the mitochondrial oxygen consumption and the normal corresponding ATP production. In conclusion, this project demonstrated that in situ muscle tissue from hypertensive and normotensive rats had a PO2 dependence of oxygen consumption over a wide range of physiological PO2 values and the hypertensive rats consumed oxygen at a higher maximal rate.

Ectothermic animals are susceptible to temperature changes such as cold shock with seasons. To survive through a cold shock, ectotherms have developed unique strategies. My interest is focusing on the physiological function of during cold shock and prolonged cold exposure in the fruit fly (Drosophila melanogaster) and crayfish (Procambarus clarkii). I used Drosophila melanogaster as a model system to investigate cardiac function in response to modulators (serotonin, acetylcholine, octopamine, dopamine and a cocktail of modulators) in acute cold shock and chronic cold shock conditions as possible mechanism to regulate heart rate in the cold. To examine if the dampened heart rate in the cold could still be enhanced by modulators or calcium loading, modulators and light-sensitive channelrhodopsin proteins were utilized to stimulate the heart. This light induced cardiac activation increased heart rate in all conditions, and potentially can be used for cardiac therapy in mammals. Also, the acute and chronic cold conditioned heart showed responsiveness to the above mention modulators. In examining how synaptic transmission is influenced by acute and chronic cold, the crayfish neuromuscular junction was used as a model. This is a good model as there are high and low output synapses to be investigated. The low output neuromuscular junction was enhanced in response to acute cold. The high output nmj increased in synaptic response to acute cold. In addressing chronic cold conditions, the nmj were physiologically assayed in their response to acute warm changes as well as influence of serotonin and octopamine. In chronic cold condition, the synaptic output was varied in enhanced and dampened responses to an acute warm environment. These junctions were enhanced in their synaptic output by serotonin and octopamine (100nM). In assessing, by HPLC assay, octopamine concentration increased in chronic cold crayfish. This suggests compensation in synaptic transmission in cold acclimation possibility via endocrine responses.

The demise of reef-building corals potentially lies on the horizon, given ongoing climate change amid other anthropogenic environmental stressors. If corals cannot acclimatize or adapt to changing conditions, dramatic declines in the extent and health of the living reefs are expected within the next half century. The primary and proximal global threat to corals is climate change. Reef-building corals are dependent upon a nutritional symbiosis with photosynthetic dinoflagellates belonging to the group Symbiodinium. The symbiosis between the cnidarian host and algal partner is a stress-sensitive relationship; temperatures just 1°C above normal thermal maxima can result in the breakdown of the symbiosis, resulting in coral bleaching (the loss of Symbiodinium and/or associated photopigments) and ultimately, colony death. As ocean temperatures continue to rise, corals will either acclimatize or adapt to changing conditions, or will perish. By experimentally preconditioning the coral Acropora millepora via sublethal heat treatment, the coral acquired thermal tolerance, resisting bleaching during subsequent hyperthermal stress. The complex nature of the coral holobiont translates to multiple possible explanations for acclimatization: acquired thermal tolerance could potentially originate from the host itself, the Symbiodinium, or from the bacterial community associated with the coral. By examining the type of in hospite Symbiodinium and the bacterial community prior acclimation and after thermal challenge, it is shown that short-term acclimatization is not due to a distinct change in the dinoflagellate or prokaryote community. Though the microbial partnerships remain without considerable flux in preconditioned corals, the host transcriptome is dynamic. One dominant pattern was the apparent tuning of gene expression observed between preconditioned and non-preconditioned treatments, showing a modulated transcriptomic response to stress. Additionally several genes were upregulated in association with thermal tolerance, including antiapoptotic genes, lectins, and oxidative stress response genes. Upstream of two of these thermal tolerance genes, inhibitor of NFκB and mannose-binding lectin, DNA polymorphisms were identified which vary significantly between the northern and southern Great Barrier Reef. The impact of these mutations in putative promoter regions remains to be seen, but variation across thermally-disparate geography serves to generate hypotheses regarding the role of regulatory element evolution in a coral adaptation context.

Purified brush border membrane vesicles (BBMV) were isolated to characterize primary cellular transport mechanisms for white shrimp. The ultimate goal is to determine the effective components of a shrimp’s diet, thereby enhancing growth, as well as nutrient content. Juvenile shrimp are dependent on plant material as a food source. Potassium is a key component of plants, thus it may play a role in nutrient transport. In addition, divalent metals have been shown to act as co-transporters in several other organisms, thus they may serve as a transport mechanism for shrimp. Fresh, live, white or brown shrimp were obtained, and from them 15-30 hepatopancreases were dissected to prepare the BBMV. Methods for preparing BBMV were based on the Mg2+ precipitation technique developed by Kessler et al., (1978) and Biber et al. (1981) for mammalian eipithelia and applied to crustaceans. The results suggest that there is a sodium/potassium-dependent glucose transport system that resembles the SGLT1 system of vertebrates, except the shrimp transporter can accept both sodium and potassium as cofactors, while the vertebrate system is restricted to sodium stimulation. Potassium showed strong stimulation of L-histidine uptake by shrimp BBMV, suggesting that a crustacean isoform of the insect potassium-dependent carrier protein (KAAT1) might be present in shrimp, and contribute to amino acid uptake. Amino acids also appear to form bis-complexes with divalent metals, that are transported by an analog of the dipeptide transporter (PEPT1). The metals appear to be accommodated, with varying affinities. PEPT1 has been described as a very non-specific carrier process because it transports such a wide range of di- and tripeptide combinations.