Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 63-69).

During conditions of osmotic stress, some plants accumulate compatible osmolytes such as glycine betaine or choline-0-sulphate. Choline is required as a precursor for synthesis of both osmolytes and choline is also required by all plants as a component of phospholipids. In the betaine accumulator spinach, choline synthesis requires three sequential N-methylations of phosphoethanolamine (PEA) to generate phosphocholine (PCho), with the first N-methylation being catalyzed by S-adenosyi-L-methionine: PEA Nmethyltransferase (PEAMeT). Choline synthesis and, more particularly the activity of PEAMeT, are up-regulated by salinity (Summers and Weretilnyk, 1993). This thesis reports on the partial purification and preliminary characterization of PEAMeT from spinach. A variety of column chromatography matrices including DEAE Sepharose, phenyl Sepharose, w-aminohexyl agarose, hydroxylapatite, phenyl Superose, Mono Q and adenosine agarose, have been used to purify PEAMeT. A 5403- fold purified preparation yielded a specific activity of 189 nmol· min-1 • mg-1 protein. SDS-PAGE analysis of this preparation revealed a number of polypeptide bands but only one which photoaffinity cross-linked to [3H]SAM. The estimated native molecular weight (MW) of PEAMeT was found to be 77 kDa by gel filtration chromatography and an estimated MW of 54 kDa was determined by SDS-PAGE. SDS-PAGE analysis of samples photoaffinity crosslinked to [3H]SAM gave a slightly higher estimated MW of 57 kDa. Effects of various factors on PEAMeT assay conditions were evaluated using partially purified PEAMeT preparations. PEAMeT activity as a function of pH gave a unimodal curve with an apparent pH optimum at 7.8 with 1 00 mM HEPES-KOH buffer. In vitro PEAMeT activity was inhibited by phosphate, PCho, S-adenosyi-L-homocysteine, ca+ 2, Mn+2 and co+2 but not by choline, betaine, ethanolamine, mono- and dimethylethanolamine or Mg+2 • Phosphobase N-methyltransferase activities present in preparations enriched for PEAMeT activity can catalyse the reaction sequence PEA- PMEA - PDEA - PCho. Under optimized assay conditions using PEA as the sole substrate, PMEA, PDEA and PCho were quantified and were detected in the order: PMEA (77%) > PDEA (17%) > PCho (6%). Thus a single enzyme, PEAMeT, is capable of converting PEA to PCho in leaves of spinach. The existence of a second enzyme which converts PMEA to PCho has also been reported for leaves and roots of spinach (Weretilnyk and Summers, 1992). The presence of two enzymes with overlapping activities raises questions regarding the roles of these two enzymes in choline metabolism. For example, do these enzymes also have overlapping functions in choline synthesis, particularly under conditions of osmotic stress? / Thesis / Master of Science (MSc)

Molecular dynamics simulations are an increasingly valuable tool to biochemical researchers: advances in computational power have expanded the range of biomolecules that can be simulated, and parameters describing these interactions are increasingly accurate. Despite substantial progress in force field parameterization, recent simulations of protein molecules using state-of-the-art, fixed-charge force fields revealed that the interactions among and within protein molecules can be too favorable, resulting in unrealistic aggregation or structural collapse of the proteins being simulated. To understand why these protein-protein interactions are so over-stabilized, I first assessed the ability of simulation force fields to represent accurately the interactions of individual amino acids, employing an osmotic pressure simulation apparatus that enabled direct comparison with experiment. Surprisingly, simulations of most of the amino acids resulted in behavior that was in strong agreement with experiment. A number of amino acids, however—notably those that contain hydroxyl groups and those that carry a formal charge—interacted in ways that were clearly inaccurate. Additionally, some commonly-used force fields failed to accurately represent the interactions of amino acids in a consistent manner. By further investigating the interactions of the functional groups of these amino acids, I was able not only to determine some of the root causes of individual amino acid inaccuracies, but also to implement simple modifications that brought the interactions of these small molecules and amino acids in stronger accord with experiment. These studies have highlighted some of the shortcomings in popular simulation force fields, and have proposed useful modifications to address them. Still, there is additional work that must be—and is being—conducted in order to correctly model the interaction behavior of proteins in simulation.

<p>Irrigation with saline waters is an agricultural practicethat is becoming increasingly common as competition for freshwater increases. In this thesis the mechanisms behind salinityand drought stress has been studied using data from fieldexperiments in combination with a modelling tool, theCoupModel. Measurements from field experiments on salinity,boron toxicity and drought stressed tomatoes grown during twoclimatically different seasons in the Arava desert, Israel,showed a linear relationship between relative growth andevapotranspiration, for all treatments and seasons. Data fromthe spring was used to concurrently simulate growth andtranspiration, hence accounting for feedback mechanisms betweenthe plant and the environment. Salinity stress was modelled asan osmotic effect (reduction of water uptake at high soilsalinities, W approach) or a toxicity effect (direct reductionof photosynthesis with soil salinity, G approach). Goodagreement between simulated growth and transpiration wasachieved with both salinity stress approaches, with twoexceptions. When growth and transpiration were simulated withthe W approach at different salinity levels, transpiration wasunderestimated at high stress. The G approach resulted in anunderestimation of growth at high water stress under moderatesalinity. A direct decrease of photosynthesis leads to adecreasing water-use efficiency with salinity while water-useefficiency remains constant with salinity when the salinitystress is modelled as a reduction in water uptake. Measurementsshowed decreasing water-use efficiency for the salinitygradient, explaining why the W approach was not applicable. Itwas not possible to detect any considerable differences betweenthree different approaches for water uptake tested in thestudy.</p><p><b>Keywords:</b>Water-use efficiency; osmotic effect; iontoxicity; semi-arid.</p>

A series of mutant strains of the yeast Saccharomyces cerevisiae that are sensitive to osmotic stress and also have a defect in vacuolar biogenesis have been isolated (M. Latterich, PhD Thesis 1992). The mutations that cause this pleiotropic phenotype are termed ssv, for salt sensitive vacuolar mutants. Complementation analysis has revealed that ssv mutations fall into one of 18 complementation groups. A MAP kinase related signal transduction pathway, termed the HOG pathway for High Osmolarity Glycerol, has been identified in yeast. This pathway senses osmotic stress and invokes the cellular response, one aspect of which is the accumulation of intracellular glycerol (Brewster et. al, 1993). Mutations in the HOG pathway often cause an osmosensitive phenotype similar to that shown by ssv mutations. This work sets out to characterise several ssv strains for defects in the HOG pathway. These strains were subjected to osmotic stress and the intracellular and extracellular glycerol determined and compared to control strains and conditions. Many of the strains showed reduced, or even elevated in one case, glycerol levels compared to wild-type strains. No correlation could be made between these glycerol levels and the activity of the rate-limiting enzyme, glycerol-3-phosphate dehydrogenase (GPDH) determined in an independent study. Transcription of the GPDH gene is under the control of the HOG pathway. In a separate study, the nucleotide sequence of a short region of yeast chromosome VII was determined. Approximately 11,000 bases of DNA from the right sub-telomeric region was sequenced. Analysis of the DNA sequence showed four potential open reading frames. One of these encoded the YORl gene and another a protein related to PAU1 The remaining two ORFs, termed ORFl and ORF2, encoded potential proteins of unknown function. Disruption cassettes containing the LEU2 selectable marker were constructed for both ORFl and ORF2. Successful disruption of ORFl was achieved, but no viable transformants were ever recovered after attempted disruption of 0RF2..ORFl gene knockouts are viable and show no observable phenotype under a range of growth conditions. Subsequent analysis of ORFl and 0RF2 after the completion of the Yeast Genome Project, shows that both ORFl and 0RF2 are members of different sub- telomeric associated gene families. 0RF2 encodes a putative Y' protein.

Com o objetivo de validar um protocolo de indução de diarréia osmótica e avaliar comparativamente a eficácia de três diferentes tratamentos, foram utilizados 18 bezerros holandeses hígidos, com idade entre oito e 30 dias, e peso variando entre 37 e 50 Kg. A diarréia e a desidratação foram induzidas por via oral, através da administração de leite integral (16,5 ml/Kg), de quatro gramas de sacarose por quilo e de dois tipos de diuréticos (espirolactona e hidroclorotiazida, em dose de 2mg/Kg), a cada oito horas, por um período de 48 horas. Os bezerros foram distribuídos aleatoriamente por três grupos experimentais, cada um com seis animais, que receberam as seguintes soluções: solução salina hipertônica à 7,5% (SSH-4ml/kg IV), solução eletrolítica oral (SEI-60ml/kg) e a associação dessas duas soluções (SSH+SEI), sendo a SEI administrada por mais duas vezes com um intervalo de oito horas. O exame físico e as colheitas de sangue para determinações do hemograma, de dosagens hemogasométricas e de componentes bioquímicos séricos e plasmáticos foram realizadas nos seguintes momentos: 0h (antes da indução), 24 e 48 horas pós-indução(PI) de diarréia, e com 1, 3, 6, 12, 24, 48 e 72 horas pós-tratamento (PT).As amostras de fezes e urina foram colhidas às 0h, 24 e 48 horas PI, e 24, 48 e 72 horas PT. O protocolo de indução da diarréia obteve 100% de resposta produzindo diarréia aquosa e desidratação severa (equivalente a 13% do peso corpóreo) acompanhada de azotemia pré-renal, aumentos no volume globular, no teor de hemoglobina e na proteína total; hipercalemia; hiperlactemia; hiperfosfatemia; acidose metabólica; elevado déficit de volume plasmático; aumentos da densidade urinária, da concentração de uréia e creatinina urinárias e do lactato urinário e fecal; diminuições da pressão venosa central, do pH urinário e fecal. Os animais tratados com SSH+SEI diminuíram de forma rápida e substancial os valores do volume globular e do déficit de volume plasmático; aumentaram o pH sangüíneo e a pressão venosa central entre 1hPT e 3hPT, além de manterem as concentrações de glicose próximas aos valores basais durante todo o período de tratamento. A SEI também promoveu o restabelecimento das variáveis anteriormente citadas, porém de forma mais lenta quando comparada com o uso da SSH+SEI. Os animais tratados apenas com SSH exibiram, no monitoramento através de exames físico e laboratoriais, sinais de desidratação, de depressão clínica e de acidose metabólica ainda às 24hPT, indicando que o uso isolado dessa solução não é eficaz no tratamento da diarréia em bezerros neonatos. / With the purpose of validating a protocol for osmotic diarrhea induction and to comparatively evaluate the efficacy of three different treatments, 18 healthy Holstein calves, with eight to 30 days old, and 37 to 50 Kg of body weight were used. The diarrhea and dehydration were orally induced, through the administration of whole milk (16.5 ml/Kg bwt), sucrose 4g/Kg bwt and two types of diuretics (spironolactone and hydrochlorothiazide, in a dosage of 2mg/Kg bwt), every eight hours, for a total period of 48 hours. The calves were randomly distributed into three experimental groups, each one with six animals, which received the following solutions: 7.5% hypertonic saline solution (HSS - 4ml/Kg bwt, IV), oral electrolytic solution (OES - 60ml/Kg bwt) and the association of both solutions (HSS+OES), being the OES administered two more times with an interval of eight hours. The physical examination and blood sampling for hemogram determinations, hemogasometric dosages and serum and plasmatic biochemical components were done in the following moments: 0h (before the induction), 24 and 48h post-induction (PI) of diarrhea, and within 1, 3, 6, 12, 24, 48 and 72 hours post-treatment (PT). The protocol for osmotic diarrhea induction had a 100% response with aqueous diarrhea and severe dehydration (corresponding to 13% of body weight), followed by pre-renal azotemia, increases in the globular volume, and hemoglobin and total protein levels; hyperkalemia; hyperlactemia; hyperphosphatemia; metabolic acidosis; high plasmatic volume deficit; increases of urinary density, urinary urea and creatinine concentrations and urinary and fecal lactate; diminished central venous pressure and urinary and fecal pH. The animals treated with HSS+OES rapidly and substantially diminished the values of globular volume and plasmatic volume deficit; had the blood pH and central venous pressure increased between 1hPT and 3hPT, besides they maintained glucose concentrations near basal values along the period of treatment. The OES also promoted the reestablishment of the cited variables, although in a slower manner when compared with the use of HSS+OES. The animals treated only with HSS showed, through the physical and laboratory exams, signs of dehydration, clinical depression and metabolic acidosis still at 24hPT, indicating that the isolated use of this solution is not efficient in the treatment of neonatal diarrhea in calves.

The goal of this thesis was to investigate the interplay between sodium, glycosaminoglycans, vascular stiffness, and hypertension. In contrast to the traditional view of salt-dependent hypertension, recent studies have found that sodium accumulation can occur without commensurate fluid retention. Researchers hypothesise that this sodium is stored non-osmotically via association with negatively charged glycosaminoglycans (GAGs) in the extracellular matrix. The interaction of sodium and GAGs, the influence of sodium on GAG production, and the ability of GAGs to affect vascular stiffness are of key interest. This thesis first investigates the link between hypertension, vascular stiffness, and GAGs in ex vivo human aortae. Aortae from hypertensive donors were found to be stiffer than normotensive controls even after controlling for both pressure and age, a novel finding in humans. In these aortae, hypertension was associated with GAG remodelling, but not with changes in total GAG content. Next, an interventional rat study is presented to examine the effects of dietary salt on vascular stiffness and GAGs, and to distinguish between salt-dependent and blood pressure-dependent effects. In vivo vascular stiffness was found to be salt-dependent but pressure-independent, with ex vivo stiffness unaffected by salt. Ex vivo stiffness was also independent of aortic GAG content, similar to the human aortae described previously. GAG content in the skin was both salt-dependent and pressure-dependent. Finally, this thesis closes with an interventional study in humans. This study was designed to examine the effects of diuretic-induced salt loss on sodium storage, GAGs, and haemodynamics. An eight-day diuretic course corresponded to a ~10% reduction in skin sodium content, without associated water loss or cardiovascular changes. GAG mRNA expression was decreased in the skin, suggesting reduced GAG content. Pilot work from this study supports the use of 23Na MRI as a non-invasive measurement of skin sodium, but only for pre- vs post-treatment comparisons rather than absolute quantification. In conclusion, this thesis demonstrates that both salt and blood pressure influence GAG accumulation and distribution, but that GAGs do not directly affect vascular stiffness. However, GAGs do play a direct role in osmotically inactive sodium storage, which may modulate development of hypertension.

"When cells are exposed to environmental stress, they respond by compartmentalizing mRNA and translation proteins in stress granulates to protect mRNA. However, the mechanism through which external stress is communicated into the cell to form stress granules is unknown. Phospholipase Cβ (PLCβ) is activated by Gq on the plasma membrane in response to sensory stimuli to initiate calcium signals resulting in a variety of cellular responses. Here, we show that PLCβ binds to major proteins that organize stress granules as well as the main component of the RNA-induced silencing machinery, Argonaute-2 (Ago2). Under stress, PLCβ moves from the plasma membrane to the cytosol to escort Ago2 into stress granules and potentially inhibit mRNA degradation by regulating microRNAs (miRs) expression. Using a model muscle cell line functionally adapted to handle stress, we find that upon osmotic stress, the movement of PLCβ into the cytosol to move Ago2 into stress granules changes the population and distribution of miRs, and in particular, members of the let family. The impact of changes in let is to acutely affect glucose metabolism allowing cells to adapt to stress conditions. Our studies present a model in which PLCβ relays information about external stress to promote stress granule formation and protect mRNAs."

abstract: Environmental changes are occurring at an unprecedented rate, and these changes will undoubtedly lead to alterations in resource availability for many organisms. To effectively predict the implications of such changes, it is critical to better understand how organisms have adapted to coping with seasonally limited resources. The vast majority of previous work has focused on energy balance as the driver of changes in organismal physiology. While energy is clearly a vital currency, other resources can also be limited and impact physiological functions. Water is essential for life as it is the main constituent of cells, tissues, and organs. Yet, water has received little consideration for its role as a currency that impacts physiological functions. Given the importance of water to most major physiological systems, I investigated how water limitations interact with immune function, metabolism, and reproductive investment, an almost entirely unexplored area. Using multiple species and life stages, I demonstrated that dehydrated animals typically have enhanced innate immunity, regardless of whether the dehydration is a result of seasonal water constraints, water deprivation in the lab, or high physiological demand for water. My work contributed greatly to the understanding of immune function dynamics and lays a foundation for the study of hydration immunology as a component of the burgeoning field of ecoimmunology. While a large portion of my dissertation focused on the interaction between water balance and immune function, there are many other physiological processes that may be impacted by water restrictions. Accordingly, I recently expanded the understanding of how reproductive females can alter metabolic substrates to reallocate internal water during times of water scarcity, an important development in our knowledge of reproductive investments. Overall, by thoroughly evaluating implications and responses to water limitations, my dissertation, when combined previous acquired knowledge on food limitation, will enable scientists to better predict the impacts of future climate change, where, in many regions, rainfall events are forecasted to be less reliable, resulting in more frequent drought. / Dissertation/Thesis / Doctoral Dissertation Biology 2019

Graduation date: 1995