Thesis (M.S.)--University of Wisconsin--La Crosse, 2002. / Includes bibliographical references.

In coordination with the New York City Medical Examiner's Office, we received the sequence of a mutated SCN5A gene that was found in a five-week-old girl who died in her sleep. SCN5A codes for the voltage-gated cardiac sodium channel alpha subunit (Nav1.5) and is responsible for the fast depolarization in phase zero of the cardiac action potential. The mutations that were present in the girl's SCN5A gene were a missense mutation, Q1832E, and a truncation mutation, R1944X. In order to gain an understanding of the conditions that led to the patient's death, we carried out a functional analysis on the mutant channels and measured how their properties differed from wild type Nav1.5 properties. For our functional analysis we carried out mutagenesis reactions to produce three experimental constructs in order to examine independent effects of Q1832E or R1944X, and to examine their interaction (mutant Nav1.5 that contains both Q1832E or R1944X; as was found in the genetic screen). These constructs were transfected into HEK 293 cells and studied using the patch clamp analysis using the whole cell configuration. Experiments were carried out to test the Nav1.5 current voltage relationships, the recovery from inactivation properties, and steady state inactivation properties. The data demonstrated that each of the three constructs resulted in a significantly reduced current density when compared to wild type Nav1.5 currents. The gating properties of the mutant channels were similar to those of wild type Nav1.5, though Nav1.5-R1944X did show a statistically significant slower recovery from inactivation than the wild type channel. Though more experimentation is needed to determine the mechanism behind the reduced current in the mutant channels, our data shows that each of the mutations is sufficient to produce a severely dysfunctional channel and this is likely the cause of the patient's death.

Hypertension is both a domestic and international health issue – diagnosed in 1 in 3 U.S. adults and classified by the World Health Organization as the number one risk factor for mortality worldwide. It has been established that salt plays a role in the development of hypertension, and that a salt-sensitive phenotype indicates heightened sensitivity to salt consumption. Here, we studied the roles of the afferent renal nerves, which travel from the kidney to the central nervous system, and the sodium-chloride cotransporter in fluid and electrolyte homeostasis and blood pressure regulation. Our laboratory utilized a novel technique of afferent renal nerve ablation on Sprague-Dawley rats to examine the effects of afferent renal nerve mechanoreceptors and chemoreceptors in response to acute sympathoinhibitory challenges. Additionally, salt-sensitive and salt-resistant rats were randomly subjected to chronic normal salt (0.6% NaCl) or high salt (8% NaCl) diets, and examined for levels of norepinephrine and substance-P release. A different group of salt-resistant and salt-sensitive rats were subcutaneously infused with terazosin, a selective -1 adrenoreceptor antagonist, or propranolol, a selective -adrenoreceptor antagonist, and then randomly subjected to normal salt (0.6% NaCl) or high salt (4% NaCl) diets for 21 days. We subsequently examined these rats, and analyzed the effects of high salt intake on blood pressure, sodium-chloride cotransporter activity, and expression of the sodium-chloride cotransporter and its relevant kinases. In response to an acute mechanoreceptor-specific stimulus, Sprague-Dawley rats that underwent afferent renal nerve ablation were unable to modulate blood pressure or natriuresis after regaining consciousness. Chronic high salt (8% NaCl) consumption in salt-sensitive rats resulted in increased levels of plasma norepinephrine, renal norepinephrine, and norepinephrine-evoked Substance-P release. In addition, salt-sensitive rats subjected to a 21-day high salt (4% NaCl) diet exhibited increased blood pressure, elevated sodium-chloride cotransporter activity, and upregulated levels of the sodium-chloride cotransporter and the kinases that regulate it. However, these observed increases in blood pressure, protein activity, and protein expression were abolished in salt-sensitive rats experiencing -1 adrenoreceptor antagonism due to terazosin administration. In conclusion, our findings indicate that mechanoreceptor-driven afferent renal nerve activation is needed to maintain fluid and electrolyte homeostasis and regulate blood pressure in response to acute sympathoinhibitory challenges and chronic high salt intake. In addition, our data demonstrates that the sodium-chloride cotransporter is aberrantly upregulated in salt-sensitive rats through a norepinephrine-1-adrenoreceptor gated pathway, and this this upregulation results in excessive salt reabsorption. Thus, our experiments have generated new data that reveals selective 1-adrenoreceptor antagonism and renal denervation as potential treatment options for hypertensive individuals.

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Tomato (Lycopersicon esculentum Mill.) is an important commercial crop. High quality tomatoes for the local and export markets are grown hydroponically. In this study, the effect of electrically activated sodium bicarbonate and non-ionized sodium bicarbonate solutions on productivity and postharvest quality of hydroponically grown tomatoes was tested. Sodium bicarbonate is a ready source of bicarbonate. By using a novel technique obtained from Radical Waters (Pty) Ltd., ionized bicarbonate (anolyte) can be produced from sodium bicarbonate and applied to plants to stimulate photosynthesis, and improve yield and postharvest quality. The effect of ionized and non-ionized bicarbonate solutions on productivity and postharvest quality was studied by measuring the following: (1) Preharvest growth rate (leaf length, stem height and diameter); total yield; starch concentration in leaves; fruit count; leaf chlorophyll content; CO2-uptake; soluble solids and concentration of K+, Na+ and NO3¯ in leaves. (2) Postharvest rate of respiration and transpiration; ethylene production; total soluble solids content; ascorbic acid content; pH; fruit firmness; fruit mass; fruit diameter; longevity; concentration of K+, Na+ and NO3¯ at one-third towards maturity, two-thirds towards maturity, at maturity and during ripening, and rate of ripening at 12 and 23°C. During the preharvest period, plants treated with anolyte showed increased growth rate, starch concentration, chlorophyll content, soluble solids content and improved yield. During the growth period, the concentration of K+ and NO3¯ was higher in plants treated with anolyte than in plants treated with non-ionized sodium bicarbonate. The concentration of Na+ during the preharvest period was higher in plants treated with non-ionized sodium bicarbonate than in plants treated with anolyte. After harvest, fruits from plants treated with anolyte had a reduced rate of ethylene production, rate of respiration and transpiration, total increased soluble solids and ascorbic acid content than fruits from plants treated with non-ionized sodium bicarbonate. Fruits from plants treated with anolyte had reduced rate of ripening and were larger and heavier than fruits from plants treated with non-ionized sodium bicarbonate. Anatomical and ultrastructural studies revealed that treatment with anolyte stimulated cell growth and photosynthesis. These results were confirmed by comparing the CO2-uptake of treated and untreated plants. Application of anolyte stimulated photosynthesis, thus improving yield and fruit quality. Treatment with non-ionized sodium bicarbonate resulted in salt- stress and calcium deficiency. Some of the fruits on plants treated with non-ionized sodium bicarbonate suffered from blossom-end rot due to salt-stress. / Prof. C.S. Whitehead

No description available.

Degree Not Listed / Department of Diagnostic Medicine/Pathobiology / Roman Reddy R. Ganta / Anaplasmataceae family rickettsial bacteria are mostly vector-transmitted pathogens causing important diseases in several vertebrates, including humans, canines, and ruminants. Ehrlichia chaffeensis, a tick-transmitted intraphagosomal rickettsial bacterium, is the causative agent of human monocytic ehrlichiosis (HME). Little is known about how this and other related rickettsial organisms are able to reside and replicate within an acidified phagosome environment. Similarly, it is unclear how the infectious form of the bacterium maintains pH homeostasis in the extracellular milieu where the pH is about 7.35-7.45, before its infection to a naïve host cell. Sodium/cation: proton antiporters are integral membrane proteins reported from a wide range of species. They exchange sodium or other monovalent cations against protons across a plasma membrane in maintaining the cytoplasmic pH of a cell. We recently described a mutation within the Ech_0379 gene of E. chaffeensis that is predicted to encode for a Na+/H+ antiporter protein. The mutation caused the attenuated growth of the organism in vertebrate hosts, resulting in a reduced level of the bacterial presence in the circulation. In this study, we evaluated several antiporter protein genes of E. chaffeensis. Its genome contains 10 coding sequences encoding for polypeptides which may form at least six functional proteins. To define their function, a sodium sensitive Escherichia coli strain having a mutation in two of its three antiporter protein genes (EP432) is used to carry out the functional complementation assay with E. chaffeensis genes from their respective promoters. The EP432 strain has a growth defect during its replication in the presence of NaCl that can be restored with functional complementation. All six E. chaffeensis genes could complement the growth defect of EP432 under acidic pH, while Ech_0379 and Ech_0179 also complemented at basic pH. Ech_0179 complemented at neutral pH as well. The complementation of all genes at neutral and basic pHs, except Ech_0179 and Ech_0379, made EP432 E. coli strain be more sensitive to the presence of 200 mM NaCl. The channeling activity is verified independently by constructing a proteoliposome in vitro with the recombinant protein Ech_0379. The recombinant protein showed antiporter activity at all three pHs in the presence of 100 or 200 mM NaCl when assessed using the recombinant proteoliposome. This research is the first description of antiporter proteins of E. chaffeensis.