Genetic recombination is an ubiquitious process in living organisms and is one of the most elaborate activities that DNA undergoes. As such, the recombination process potentially interacts with all of the major metabolic events involving DNA. This study examined one of these interactions, specifically that occuring between recombination and DNA replication in phage T4. This interaction was investigated from both the mechanisitic and evolutionary viewpoints. In phage and bacteria, the central reactions of homologous recombination are those of homologous pairing and strand insertion. These reactions are generally catalyzed by a single enzyme. The E. coli recA gene product serves as the paradigm for this class of enzyme. Four cloned wild type "recA" genes from the bacterial species E. coli, A. caviae, B. pertussis, and the uvsX gene from phage T4, were used to test for complementation of both E. coli recA⁻ and phage T4 uvsX⁻ mutants. The B. pertussis recA⁺ gene was able to complement an E. coli recA⁻ mutant with respect to the repair of UV induced DNA damage and general recombination. In a phage Mode 2 DNA synthesis assay, all three bacterial recA genes complemented a uvsX⁻ mutant to some extent, but the B. pertussis RecA protein allowed "runaway" DNA synthesis to occur. In contrast, none of the three bacterial genes were able to increase DNA synthesis of a uvsX⁺, uvsY⁻ phage T4. In a one step growth experiment, only the B. pertussis RecA protein complemented a phage uvsX⁻ mutant. However, none of the three bacterial recA genes complemented the same phage mutant in a UV survival assay. These results imply that normal wild type Mode 2 phage DNA synthesis is an example of a specific interaction between the DNA replication and homologous recombination "machines" of phage T4. Multiplicity reactivation (MR) is a type of recombinational repair. MR experiments were performed using a gene 42(ts) mutant phage to study the effect of this member of the phage's DNA replication machinery on recombinational DNA repair. The data show that MR is dependent on the gene 42 gene product suggesting that the phage DNA replication and recombination metabolic pathways are coupled through a protein-protein interaction involving gpuvsX and gp42. A hypothesis concerning the molecular coevolution of DNA replication and recombination pathways is developed. This hypothesis is used to explain the significantly different ways in which each of the enzymatically equivalent "recA" proteins substitutes for the E. coli RecA protein and gpuvsX.

The disease pertussis, more commonly known as whooping cough, is caused by the gram negative bacterium Bordetella pertussis. In order to cause disease, the pathogen produces a battery of potential virulence factors which assist the bacteria in colonizing and surviving in the human host. Among these factors is a soluble adenylate cyclase which is thought to interfere with host immune defenses. The adenylate cyclase toxin, along with several other B. pertussis virulence factors, undergo a phase shift between states of expression and repression. The gene for the adenylate cyclase was isolated by use of transposon tagging to localize the region of the structural gene. In addition, primer extension was done to localize the promoter region of this gene. Studies with this promoter fused to a promoterless galactokinase gene were done in Escherechia coli and demonstrated that the adenylate cyclase promoter was unable to be activated in the heterologous E. coli system.

A theoretical probability distribution function for neutron triplet correlation that is amenable to experimental determination of the reactivity of a neutron multiplying system has been obtained. A two-dimensional data collection method designed for the experiment was implemented with the aid of an interface counting board installed in a microcomputer system. An efficient non-linear least-squares code was developed to fit experimental data to the theoretical distribution function to obtain reactivity. The system developed for data collection and analysis is fully portable, requiring only a microcomputer to implement. Reactivities obtained from measurements carried out on the University of Arizona Subcritical Assembly and the TRIGA Reactor were compared to results determined by other methods. The range of prompt reactivities measured is -34.2 dollars to -6.1 dollars.

Aging has been associated with alterations in the physiological responses to physical stressors. This study investigated the regional and systemic hemodynamic responses and venous plasma catecholamine responses to three commonly encountered physical stressors (dynamic exercise, isometric exercise, and orthostasis) in groups of young (25.9 ± 0.7 yr, mean ± SE, range 21 to 29) and older (65.0 ± 0.8 yr, mean ± SE, range 60 to 72) healthy males. In separate experimental sessions, subjects performed supine leg cycling at three submaximal levels (∼45%, ∼65%, and ∼85% of peak oxygen uptake), isometric handgrip to exhaustion (30% of maximal force), and two types of orthostatic stress (lower body negative pressure (LBNP) and standing). In general, there were no differences between the young and older subjects in any baseline hemodynamic variable or baseline plasma catecholamine concentrations. In response to all levels of leg cycling, the young and older subjects had similar arterial blood pressure responses. However, the older subjects did demonstrate an augmented forearm vasoconstriction, mediated at least in part, by greater sympathetic vasoconstriction in the skeletal muscle circulation, perhaps necessary to help offset a lower cardiac output response. In response to isometric handgrip, there was a lesser heart rate response in the older subjects, but there were no age-related differences in the selected regional hemodynamic responses, arterial blood pressure response or the plasma norepinephrine responses. Likewise, in response to both types of orthostatic stress, the older subjects demonstrated a lesser tachycardic response, but there was no decline in their ability to maintain arterial pressure. Therefore, the main conclusion of these studies is that, although aging may alter the regulatory scheme, the arterial pressure response to these forms of acute physical stress is not impaired in older humans.

This study was designed to characterize two DNA replication proteins from herpes simplex virus type 1 (HSV-1): the major DNA binding protein (ICP8) and the DNA polymerase. The two proteins are among the seven proteins required for viral DNA synthesis. Four areas were investigated. First, the ICP8 protein was purified from an overproducing cell and showed to behave the same as the viral protein in interacting with DNA. To define the DNA-binding domain of the protein, a proteolytic fragment with the same DNA-binding specificity as the intact protein was identified by a protein blotting assay. N-terminal protein sequencing located the fragment between residues 300 and about 849 in the intact protein. This fragment contains some features important for DNA binding. Second, the DNA polymerase was found to replicate non-processively in vitro. The ICP8 protein slightly stimulated the polymerase activity at lower concentrations and inhibited its activity as the ICP8 concentration increased. Third, to define the functional domain of the DNA polymerase, a fragment of the polymerase predicted to contain both the polymerase and 3$\sp\prime$-5$\sp\prime$ exonuclease activities was overexpressed in Escherichia coli. The fragment was partially purified and its enzymatic activity was examined. Finally, to identify polymerase residues involved in substrate binding, three amino acid changes in polymerase mutants with altered drug sensitivities have been identified by DNA sequencing. Two mutations, affecting nucleotide binding, occur near two highly conserved regions which constitute part of a putative nucleotide binding site. This result indicates that two more residues are important for substrate binding. The third mutation, affecting pyrophosphate binding, occurs upstream of any previously known mutations. This result may indicate a new region involved in substrate binding.

A cell-free system has been developed from cells of an Escherichia coli strain, carrying cloned genes 1 (DNA polymerase) and 8 (terminal protein) of bacteriophage PRD1, that catalyzes protein-primed DNA synthesis. DNA synthesis in vitro is entirely dependent upon the addition of PRD1 DNA-terminal protein complex as template, Mg²⁺, and four deoxyribonucleoside triphosphates. The origin and direction of PRD1 DNA replication in vitro were determined by restriction enzyme analysis of ³²P-labeled PRD1 DNA synthesized in this system. Replication starts at either end of the linear PRD1 DNA template. Analysis by alkaline sucrose gradient centrifugation and alkaline agarose gel electrophoresis of DNA synthesized in vitro showed that full-length PRD1 DNA is synthesized. DNA elongation in this system is inhibited by the drug aphidicolin. On the other hand, DNA initiation is inhibited by phenylglyoxal, an arginine-specific α-dicarbonyl reagent. In vitro studies have also demonstrated that linear duplex, protein-free DNA molecules containing an inverted terminal repeat (ITR) sequence of the PRD1 genome at one end can undergo replication by a protein-primed mechanism. No DNA replication was observed when the ITR sequence was deleted or was not exposed at the terminus of the template DNA. The minimal origin of replication was determined by analyzing the template activity of various deletion derivatives. It was shown that the terminal 20 bp of ITR are required for efficient in vitro DNA replication. It was found that, within the minimal replication origin region, there are complementary sequences which can form a small panhandle structure. The analyses of the results obtained with synthetic oligonucleotides have revealed that the specificity of the replication origin is strand specific and even on a single-stranded template a particular DNA sequence including a 3' terminal C residue for the initiation of PRD1 DNA replication in vitro.

Autoregulation is the tendency for blood flow to remain constant despite a change in arterial perfusion pressure. Flow regulation is achieved by adjustment of arteriolar caliber to the pressure change. The responses of arterioles are most commonly explained by either the metabolic or myogenic hypothesis. According to the metabolic hypothesis, the initial decrease in blood flow that accompanies a reduction in arterial pressure would reduce oxygen delivery and decrease tissue oxygen tension. We reasoned that the contribution of this mechanism to autoregulation of flow would be increased during sympathetic nerve stimulation because the latter causes arteriolar constriction and decreases tissue oxygen tension. However, the elevated vascular tone might also influence the myogenic response (i.e., arteriolar constriction to elevated intravascular pressure). In these experiments, we examined flow and diameter changes in arterioles of the exteriorized cat sartorius muscle during sympathetic nerve stimulation and tested the contribution of both metabolic and myogenic factors. Without sympathetic nerve stimulation, autoregulation was weak, flow fell coincident with reduced perfusion pressure. Sympathetic nerve stimulation caused significant constriction of arterioles, enhancing autoregulation, and increasing flow about 20% to 60% during perfusion pressure reduction from 110 to 80, 60, and 40 mmHg. With sympathetic nerve stimulation, arteriolar dilation to arterial pressure reduction was enhanced. This enhanced dilation was not abolished by 20% oxygen in a suffusate over the muscle, suggesting that it was not due to an enhanced metabolic response. On the other hand, arteriolar constriction to venous pressure elevation (which raises arteriolar intravascular pressure), was increased during sympathetic nerve stimulation, indicating an enhanced myogenic response. Arteriolar dilation to pressure reduction was also enhanced during norepinephrine infusion, showing that prejunctional inhibition of neurotransmitter release was not involved. Vasopressin and BayK8644 had similar effects, indicating the enhanced myogenic response did not require adrenergic or receptor mediated vasoconstriction. The autoregulatory response was also examined for all the orders of arterioles in the network. Third and fourth order arterioles showed significantly more dilation during pressure reduction under both with and without sympathetic nerve stimulation. With sympathetic nerve stimulation, arteriolar dilation during pressure reduction was significantly enhanced in first through fourth order arterioles and also appeared to be enhanced in fifth and sixth orders. Enhanced autoregulation appears due to generalized increase in dilation in all orders.

Nine bacteria were tested for the ability to dehalogenate tetrachloromethane (CT), tetrachloroethene (PCE), and 1,1,1-trichloroethane (TCA) under anaerobic conditions. Three bacteria were able to reductively dehalogenate CT. Dehalogenation ability was not readily linked to a common metabolism or changes in culture redox potential. None of the bacteria tested were able to dehalogenate PCE or TCA. One of the bacteria capable of dehalogenating CT, Shewanella putrefaciens, was chosen as a model organism to study mechanisms of bacterially catalyzed reductive dehalogenation. The effect of a variety of alternate electron acceptors on CT dehalogenation ability by S. putrefaciens was determined. Oxygen and nitrogen oxides were inhibitory but Fe(III), trimethylamine oxide, and fumarate were not. A model of the electron transport chain of S. putrefaciens was developed to explain inhibition patterns. A period of microaerobic growth prior to CT exposure increased the ability of S. putrefaciens to dehalogenate CT. A microaerobic growth period also increased cytochrome concentrations. A relationship between cytochrome content and dehalogenation ability was developed from studies in which cytochrome concentrations in S. putrefaciens were manipulated by changing growth conditions. Inhibitors of electron transport by cytochromes, i.e., carbon monoxide and cyanide, inhibited dehalogenation. Spectrophotometric analyses were done to characterize the cytochromes of S. putrefaciens. Cytochromes c were identified in cytoplasmic, periplasmic, and membrane fractions. Cytochrome b was found only in membrane fractions. Observed results were consistent with a mechanism involving fortuitous CT dehalogenation by bacterial cytochromes in the presence of microbially-produced reductant. Stoichiometry studies using ¹⁴C-CT suggested that CT was first reduced to form a trichloromethyl radical. Reduction of the radical to produce chloroform and reaction of the radical with cellular biochemicals explained observed product distributions. Carbon dioxide or other fully dehalogenated products were not found.

Alzheimer’s Disease is a neurodegenerative disease affecting over five million people in the United States alone, with almost half a million new cases every year. As the population ages, these figures will only increase unless an effective treatment can be found. Amyloid-β is the protein implicated in, and believed to be causal to Alzheimer’s Disease. Amyloid-β has been known to misfold into β-sheets and aggregate into neurotoxic oligomers or relatively inert amyloid fibrils. A variety of aggravating factors have been implicated including an excess of metal ions, various genetic anomalies, and the intracellular depositions of neurofibrillatory tangles (NFT). This PhD thesis advances the understanding of Alzheimer’s disease by demonstrating how the presence of copper ions influences the unbinding force of two amyloid-β peptides affecting the initial aggregation pathway by increasing the force between two amyloid-β peptides. The results provide evidence to the hypothesis that the addition of metals increases the amyloid oligomer content in the brain beyond that which the brain can naturally clear. Current Alzheimer’s pharmacological research is focused on developing amyloid aggregation inhibitors that would slow down the accumulation of amyloid oligomers and thereby slow the onset of Alzheimer’s symptoms. A high throughput method of testing potential aggregation inhibitors that is far more efficient than current testing methods is demonstrated. An aggregation inhibitor was tested that functions by inhibiting the initial self-dimerization of amyloid-β preventing further aggregation. The results show that this inhibitor, named SG1, greatly reduces the experimental yield (a parameter calculated during force spectroscopy experiments) of the force experiments demonstrating a drastically lower amyloid-amyloid affinity. The natural conclusion to these results is that if amyloid-β cannot dimerize, it cannot continue to aggregate. Using dynamic force spectroscopy, the height and width of the energy barrier of amyloid-β dimerization is calculated using a variety of different models. These different models are compared and their relative advantages and disadvantages discussed.

Corticosteroids are key regulatory hormones involved in many aspects of physiology and have long been known to exert rapid and delayed effects. The delayed corticosteroid effects are mediated by transcriptional events downstream of glucocorticoid receptor activation. Conversely, the rapid effects are mediated independently of transcriptional regulation and are thought to involve non-classical steroid receptors and signaling pathways. Lately, research has begun to focus on delineating the rapid and nongenomic actions of glucocorticoids but most of these studies have been on mammalian models. Cortisol, the primary corticosteroid in teleosts, is an established genomic regulator of the physiological response to stress, but very little is known about either the rapid effects or their mechanisms of action in non-mammalian vertebrates. Additionally, nongenomic glucocorticoid action in the liver is poorly characterized in all animal species despite the importance of this organ in regulating glucocorticoid-mediated physiological adjustments during stress adaptation. The primary objective of this thesis is to investigate the rapid glucocorticoid effects, and their mode of action, associated with stressor-induced corticosteroid elevation in fish liver. The overriding hypothesis is that rapid effects of cortisol on acute stress adaptation involve changes to liver membrane order and rapid modulation of stress signaling pathways in rainbow trout (Oncorhynchus mykiss), a well studied teleost model. This hypothesis was tested by examining rapid plasma membrane and intracellular responses following stressor-induced cortisol elevations in vivo, as well as to cortisol treatment in vitro using liver plasma membrane, tissue slices, hepatocytes in suspension, or primary culture of hepatocytes. Steroid hormones are lipophilic molecules and freely incorporate into the plasma membrane. Through noncovalent interactions (hydrogen bonds and Van der Waal forces), glucocorticoids can potentially alter physical properties of the plasma membrane, thus leading to intracellular responses. The effect of stressor-induced cortisol elevations on physical changes to the hepatic plasma membrane was investigated by measuring the microviscosity of the plasma membrane. Plasma membrane fluidity (inverse of microviscosity) is an important determinant of transmembrane protein function, and changes to lipid order can transmit extracellular signals by activating membrane-associated signaling pathways. Fluidity of purified liver plasma membranes was monitored using steady-state fluorescence polarization of 1,6-diphenyl-1,3,5 hexatriene, a well characterized membrane probe. In addition to measuring lipid dynamics, the effect of cortisol on plasma membrane structure and surface properties were also investigated using atomic force microscopy. The effect of cortisol on the activation of key stress signaling pathways, including protein kinase (PKA), protein kinase (PKC), Akt, and mitogen-activated protein kinase (MAPK), was tested in fish liver. Also, as acute stress adaptation is regulated by an integrative hormonal response involving catecholamines (primarily epinephrine), the rapid effect of cortisol action on adrenergic signaling in the liver was evaluated in vitro. Finally, an attempt was made to identify cortisol-binding plasma membrane protein, as glucocorticoids are also thought to mediate rapid effects through a novel membrane glucocorticoid receptor. The results demonstrate for the first time that stressor exposure significantly increases liver plasma membrane fluidity (decreased microviscosity). A role for cortisol in mediating stressor-induced fluidization was confirmed in vitro, as physiological stress levels of this steroid (≥100 ng/ml) significantly increased liver plasma membrane fluidity. In addition to increasing lipid fluidity, acute stress and cortisol treatment altered membrane topography, including changes to membrane microdomains. The stressor-induced cortisol elevation also rapidly modulated major signaling cascades in rainbow trout liver, including PKA, PKC, and ERK1/2 MAPKs. A role for cortisol in the activation of these kinase pathways was confirmed in vitro. Specifically, cortisol rapidly and transiently increases cyclic AMP (cAMP) accumulation and induces the phosphorylation of PKA substrate proteins, including cAMP response element-binding (CREB) protein. In addition to activating PKA signaling, cortisol rapidly induced phosphorylation of PKC and Akt substrate proteins, while stimulating p38 MAPK dephosphorylation in vitro. Moreover, rapid cortisol signaling may stimulate metabolite oxidation in order to maintain the energy balance within liver tissue as cortisol acutely depleted key liver metabolites (including liver glucose), suggesting enhanced turnover without impacting the steady-state adenylate energy charge ratio (measure of the energy status of the cell). Also, rapid effects of cortisol alter the hormonal responsiveness of hepatocytes to adrenergic stimulation, including suppression of epinephrine-stimulated cAMP-CREB activation and glucose production. Preliminary results point to a plasma membrane protein that specifically binds cortisol in trout liver, but this remains to be characterized. Also, in addition the membrane-mediated response, mifepristone, a glucocorticoid receptor (GR)-antagonist, blocked some rapid cortisol effects suggesting the possible involvement of GR signaling pathway. Until now, cortisol has been primarily thought to play a role in the long-term recovery process to acute stress by enhancing plasma glucose levels through the upregulation of liver gluconeogenic capacity. The results presented in this thesis provide evidence for a novel role for rapid cortisol action on the acute metabolic adjustments that support liver function immediately following acute stressor exposure. Particularly, the results lead to the proposal that acute cortisol action stabilizes the energy status of the cell by maintaining ATP levels through increased metabolite turnover, suggesting enhanced metabolic activity of the liver immediately following acute stressor exposure. While the mechanism is unclear, plasma membrane alterations in response to cortisol intercalation may facilitate rapid steroid signaling either through mechanotransduction or by altering activity of plasma membrane proteins. The structural changes to the plasma membrane in response to acute stressor exposure and/or cortisol treatment highlight a novel membrane-mediated mechanism of rapid stress adaptation in hepatic tissue.