Now showing items 7986-8005 of 20330

    • Functional and Population Based Viral Ecology

      Sullivan, Matthew B.; Ignacio Espinoza, Julio C.; An, Lingling; Dieckmann, Carol; Gutenkunst, Ryan; Sullivan, Matthew B. (The University of Arizona., 2015)
      Viruses represent the most abundant biological entities on earth where, they are able to interact with all kingdoms of life. Yet their diversity, ecology and evolutionary aspects are only beginning to be fully elucidated, mainly due to technical limitations. The vast majority of the microbial world remains elusive to culture; more than 90% of genome sequenced viral isolates infect only 5 of the 54 prokaryotic phyla that are currently recognized. In contrast, viral metagenomics bypasses the need for cultures by directly sequencing fragmented genetic material of environmental viral communities. This dissertation uses viral metagenomics by applying well-tested bioinformatic protocols and expanding them to compare and contrast patterns of diversity, richness and specialization of large viral metagenomic datasets, in both local and global scales. First I demonstrate the utility of a functional-based perspective by adopting the protein cluster environment to estimate global viral diversity. Then, I use this PC approach to analyze metagenomes from two ecologically different environments, which by uncovering local gene specialization showcases the adequacy of a gene-centered workflow. Then I continue to expand upon this PC framework to study the Tara Oceans virome analyses of these data reveal patters of diversity that support a seed bank model. Finally, in search of a more meaningful ecological unit, I move from a gene-centered standpoint towards a population-based frame. We adopted a novel metagenomic technique that allowed me to uncover the discontinuity in the genomic sequence space, thus empirically defining a population. This final contribution will allow to sort and count viral communities, the first step to applying ecological and evolutionary theory.
    • Functional Biological Spectroscopy and Multiphoton Imaging Using Endogenous Optical Contrast

      Utzinger, Urs; Kirkpatrick, Nathaniel; Utzinger, Urs; Brewer, Molly A.; Hoying, James B.; Gmitro, Arthur F.; Lynch, Ronald M. (The University of Arizona., 2006)
      Endogenous optical signatures of tissue provide information relevant to biological and biomedical questions through measurements of intracellular and extracellular endogenous contrast. To develop sensitive diagnostic methods using this contrast, the important individual endogenous constituents were first evaluated. In particular, based on intracellular fluorescence consistent with NAD(P)H, FAD, and tryptophan in cell suspensions, fluorescence interrogation techniques were defined to determine cellular metabolism and response to metabolism altering treatments. Additionally, the main contributor to extracellular fluorescence, type I fibrillar collagen, was investigated with an in vitro model of collagen polymerization. By defining the fluorescence of collagen due to cross-linking and the structure of fibrillar collagen at a high resolution with second harmonic generation (SHG), sensitive techniques were developed to assess collagen integrity. Next, with tools developed for measurement of the individual endogenous optical constituents in tissue, two model systems were investigated with multiphoton microscopy in order to evaluate processes important in angiogenesis and ovarian carcinogenesis research. Starting with an in vitro model of angiogenesis, the individual intracellular and extracellular optical signals were combined for real-time imaging of angiogenesis related events. By localizing vessel associated cells with two-photon excited fluorescence (2PEF) and their interaction with collagen fibrils with SHG, the dynamics of matrix remodeling were assessed during sprouting events and neovessel growth. A dramatic reorganization of the collagen fibrils by early sprouts and differential collagen remodeling by neovessels was observed, revealing endothelial/matrix events that have previously been difficult to ascertain. Finally, in a model for investigating ovarian carcinogenesis, multiphoton microscopy of endogenous contrast in viable ovarian biopsies resulted in key features for differentiating normal and abnormal tissue. Features based on the morphology of surface epithelium imaged with 2PEF and the underlying collagen structure and integrity imaged SHG as well as cellular redox values determined by two-channel 2PEF were consistent with loss of organization and metabolic changes in the tissue that may aid in early detection and increased understanding of ovarian cancer. Results from the model applications reveal the utility of techniques sensitive to endogenous tissue signatures for non-invasive diagnostic interrogation of biological processes, specifically related to cellular function and cellular/extracellular matrix interactions.
    • Functional characteristics of heterogeneous Cx40/Cx43 gap junction channel formation

      Burt, Janis M.; Cottrell, Graham Trevor (The University of Arizona., 2001)
      Cells of the cardiovascular system express multiple connexins (Cx) with Cx40 and Cx43 being commonly coexpressed in many tissues. The expression levels of connexins are dynamic and can vary in response to a growth stimulus. It is not clear why cells express multiple connexins, or what advantage such dynamic regulation of expression patterns have on cell function. These issues are further complicated by the ability of some connexins to interact to form heterogeneous gap junction channels, with little being known regarding functional properties of such channels. The purpose of these experiments was threefold: (1) To determine whether Cx40 and Cx43 are capable of interacting to form heteromeric/heterotypic gap junction channels; (2) To characterize the functional properties of Cx40/Cx43 heteromeric/heterotypic channels; and (3) To determine the effect that changing Cx40:Cx43 expression ratio has on functional properties of heteromeric/heterotypic channels. Cell lines were developed that express only Cx43 (Rin43), Cx40 (Rin40), and Cx40 and Cx43 in varying Cx40:Cx43 expression ratios (6B5n, A7r5, A7r540C1, and A7r540C3). The Cx40:Cx43 expression ratios in the 6B5N, A7r5, A7r540C1, and A7r540C3 cells are approximately 1:1, 3:1, 5:1, and 10:1, respectively. Functional properties of the gap junction channels formed between these cells were determined using both electrophysiological and dye coupling techniques. Pairing of Rin43 and Rin40 cells demonstrated that Cx40 and Cx43 are capable of forming homomeric/heterotypic gap junctions with unique voltage-dependent gating and single channel behaviors. Rin43/A7r5 cell pairs displayed voltage-dependent gating and single channel conductance profiles that could only be explained by the presence of heteromeric/heterotypic gap junction channels between these cells. Pairing Rin43 cells with coexpressing cells of high Cx40:Cx43 expression ratio resulted in channel activities that were not predicted by the gating and conductance patterns of Cx40/Cx43 heterotypic channels. However, the dye coupling characteristics of these same cells in coculture demonstrated that the permeability of the channels formed between these cell types reflected that of Cx40 channels. In summary, Cx40 and Cx43 are capable of forming heteromeric/heterotypic gap junction channels. Increasing the Cx40:Cx43 ratio in coexpressing cells results in channels with unique gating and conductance properties, however dye permeability of these cells is predicted by their relative Cx40 content. Therefore, varying Cx40:Cx43 expression ratio provides cells with a mechanism to finely control the types of molecules shared between cells.
    • Functional characterization of an allergy-associated regulatory variant at the human IL13 locus

      Vercelli, Donata; Kiesler, Maria Olga Patricia; Wysocki, Vicki; Restifo, Linda; Martinez, Fernando (The University of Arizona., 2009)
      T helper type--2 (Th2) immunity orchestrates responses against extracellular pathogens under normal conditions and mediates pathogenic responses against innocuous substances when dysregulated, leading to allergic disease. Among the cytokines expressed by Th2 cells, interleukin (IL)--13 has emerged as a critical effector molecule in Th2 responses and common IL13 variants are associated with allergy--related phenotypes in populations of distinct ethnic background. IL13 expression in human T cells is paralleled by extensive IL13 locus remodeling, which results in the appearance of multiple DNase I hypersensitive (HS) sites. Among these, HS4 in the distal IL13 promoter is constitutively present in both naive and polarized Th2 cells, and spans a single nucleotide polymorphism, IL13--1512AC (rs1881457), which is common and strongly associated with total serum IgE levels. This dissertation combines in vitro and ex vivo approaches to characterize the role of HS4 in the regulation of IL13 gene expression and to provide novel insights into the mechanisms that underlie the association between IL13--1512AC and allergic disease.The results showed that HS4 acts as a novel cis--acting element that up--regulates IL13 transcription in activated Th2 cells. The enhancing activity of HS4 mapped within the 3' end of this element and was dependent on binding/recruitment of the transcription factors NF90 and NF45. Moreover, the IL13--1512C risk allele significantly enhanced HS4--dependent IL13 expression by creating a binding site for the transcription factor Oct--1. The increased expression of the --1512C allele was dependent on endogenous levels of Oct--1. Collectively, these results illustrate how a functional variant in an important regulatory element may modulate susceptibility to a common complex disease.
    • Functional characterization of glucocorticoid receptor sequences required for steroid-induced cell death.

      Miesfeld, Roger L.; Dieken, Ellen Sue.; Dieckmann, Carol M.; Haussler, Mark R.; Brower, Danny; Hewlett, Marty (The University of Arizona., 1991)
      Glucocorticoid induction of cell death (apoptosis) in mouse lymphoma S49 cells has long been studied as a molecular genetic model of steroid hormone action. The research described in this dissertation focuses on understanding the transcriptional control of apoptosis in two steroid resistant ntⁱ S49 mutant cell lines (S49.55r and S49.143r). These studies extend earlier biochemical analysis of the mutant ntⁱ glucocorticoid receptor (ntⁱ GR) to the molecular level by isolating and characterizing GR cDNA from the two ntⁱ S49 cell lines, S49.55r and S49.143r, and the wild type (wt) parental line, S49.A2. This analysis revealed that ntⁱ GR transcripts encode intact steroid and DNA binding domains but lack 404 amino-terminal (N-terminal) residues as a result of aberrant RNA splicing between exons 1 and 3. Results from transient co-transfection experiments into CV1 cells using ntⁱ receptor expression plasmids and a glucocorticoid responsive reporter gene demonstrate that the truncated ntⁱ receptor is capable of inducing transcription to only 10% the level of wt GR. Gene fusions containing portions of both the wt and ntⁱ GR coding sequences were constructed and used to functionally map the ntⁱ receptor mutation. It was found that the loss of the N-terminal domain alone is sufficient to cause the observed defect in ntⁱ transcriptional transactivation. In addition, a complementation assay utilizing stable transfection of wt and mutant GR cDNA constructs into a GR-deficient cell line (7r) was developed. By measuring steroid-sensitivity of various 7r derivatives, it was determined that GR is rate-limiting for S49 apoptosis and moreover, that the GR N-terminus is absolutely required for complementation in this system. These data also indicate that at physiological levels of receptor, the GR N-terminus plays a crucial role in controlling lymphocyte apoptosis, even though this portion of the receptor seems to be dispensable for low-level induction of mouse mammary tumor virus (MMTV) transcription. The smallest functionally defined transactivation region in the GR N-terminus has a net negative charge and has been named enh2, tau1 or acidic activation domain (AAD). Results from experiments designed to test whether similar activating sequences from the herpes simplex virus-1 (HSV-1) VP16 protein can substitute for the GR N-terminus demonstrate that 7r cells expressing VP-GR fusions are indeed steroid-sensitive, suggesting that S49 apoptosis requires transcriptional induction of specific genes.
    • Functional characterization of hepatic microsomal and heterologousely expressed rabbit cytochrome P450 2B enzymes

      Grimm, Scott Wayne.; Halpert, James R.; Dyroff, Martin C.; Liebler, Daniel C.; Laird, Hugh E.; Lai, Josephine (The University of Arizona., 1994)
      The objective of the research described in this dissertation was to characterize the function of four closely related cytochrome P450 2B enzymes in the rabbit. Although these enzymes display greater than 97% amino acid sequence identity, their expression is highly variable between different organs and in different individuals. Transient and stable heterologous expression systems were used to study the distinct catalytic properties of each P450 2B enzyme. Cytochrome P450 2B5 was found to have a unique pattern of catalytic activities in comparison to the P450 2B4, 2B-B1, and 2B-Bx forms. The regio- and stereoselectivity of hydroxylation of androstenedione in hepatic microsomes depended upon whether the animal expressed cytochrome P450 2B5. Whereas the catalytic activities of P450 2B5 were characterized by high steroid hydroxylase activities, P450 2B4 had relatively low steroid hydroxylase activity and much higher activity towards the non-steroid substrates than 2B5. Androstenedione 15α-hydroxylation and benzyloxyresorufin O-debenzylation were identified as selective markers of the activity of P450 2B5 and 2B4, respectively. Phencyclidine selectively inactivated P450 2B4 in hepatic microsomes from phenobarbital-induced rabbits as well as the expressed enzyme. The basis for poor inactivation of P450 2B5 by PCP was determined to be the low maximal rate constant for this P450 2B form. N-Aralkylated 1-aminobenzotriazole derivatives were found to be potent inactivators of both P450 2B enzymes and to be much less selective than phencyclidine. These results demonstrate that one or more of the amino acid differences in P450 2B5 are critical to its substrate specificities and selective inactivation. Rabbits which express P450 2B5 have the potential to exhibit different hepatic biotransformation pathways in comparison with animals that lack this enzyme.

      Summerton, James Edward, 1944- (The University of Arizona., 1973)
    • Functional Compensation in Response to Increasing Task Difficulty: Comparing Semantic and Episodic Memory Tasks in Young and Older Adults

      Ryan, Lee T.; Baena, Elsa; Ryan, Lee T.; Glisky, Elizabeth; Nadel, Lynn; Alexander, Gene (The University of Arizona., 2017)
      Previous fMRI studies have demonstrated that older adults who perform as well as young adults on certain cognitive tasks recruit additional brain regions relative to younger adults while performing these tasks. This phenomenon has been interpreted as a compensatory response and may reflect an effort to maintain performance in the face of increasing changes in cognitive difficulty or age-related brain changes in structure and/or function. Whether the compensatory response is specific to older adults or represents a more general response of any individual to increasing task difficulty is unclear. The present fMRI experiment explored age differences in brain activity associated with increases in task difficulty in two tasks, an episodic-retrieval task that is expected to be difficult for older compared to young adults, and a lexical-semantic task that is expected to be more difficult for young compared to older adults. In the lexical-semantic task, participants judged whether pairs of words were synonyms or antonyms. In the episodic task that followed, participants made yes/no memory judgments for the word pairs previously presented. Difficulty was manipulated using word frequency -low frequency words are more difficult in the lexical-semantic judgment task and easier in the episodic task. Young (ages 18-24) and older healthy adults (ages 60-83) were scanned on a 3T GE magnet using a single-shot spiral pulse sequence. Behavioral results showed a double dissociation – older adults were adversely affected by word frequency in the episodic but not the semantic task, while young adults were adversely affected by word frequency in the semantic but not the episodic task. Both groups showed activation in similar task-related and task-general regions regardless of difficulty level). Age-related differences were observed for task-specific and linear increases due to difficulty. Linear increases in fMRI activation were associated with younger adults showing increased task difficulty in bilateral task-related regions during the lexical semantic task, whereas in the episodic retrieval task only activating bilateral posterior cortices. As difficulty increased, older adults showed unilateral brain activations: left regions for the lexical-semantic task and medial and right hemisphere regions for the episodic retrieval task. Most importantly, difficulty load increases paralleled the groups' behavioral results: younger adults showed greater increases in activity in the lexical-semantic task compared to older adults, but not in the episodic retrieval task, whereas older adults showed the opposite pattern, with greater increases in activation only in the episodic task when compared to younger adults. Thus, younger and older adults recruit regions differently in response to increases in difficulty. Our results suggest that increases in fMRI activation as difficulty increases occur as an interaction to deal with task difficulty and the inherent abilities of the individuals, rather than occurring only in older adults, or in older adults across all tasks, regardless of their abilities in that domain.
    • Functional description and formal specification of a generic gateway.

      Martinez, Ralph; Son, Chang Won.; Hill, Fredrick; Kuo, Sy-Yen (The University of Arizona., 1988)
      This dissertation is concerned with the design of a generic gateway which provides an interoperability between dissimilar computer networks. The generic gateway is decomposed with subnetwork dependent blocks and subnetwork independent blocks. The subnetwork dependent block is responsible to communicate with subnetwork nodes. The subnetwork independent block is responsible to interconnect the subnetwork dependent blocks. The communications between subnetwork dependent and independent blocks are done by service access points which defined independently to any specific subnetworks. Formal specification of a generic gateway is provided by LOTOS. The generic gateway specification is tested by a verifiable test method which is proposed in this dissertation. The correctness of the specification has been verified while the specified model is simulated. The major difference between conventional simulation and the verifiable test is in the objective of simulation. In the verifiable test method, the semantical properties are examined during the simulation process. The tester can be either human observer or other process.
    • Functional Evolution of the Cro Protein Family of Transcription Factors

      Cordes, Matthew H; Hall, Branwen; Cordes, Matthew H; Montfort, William; Little, John; McEvoy, Megan; Horton, Nancy; Ghosh, Indraneel (The University of Arizona., 2007)
      Members of multi-specific DNA-binding protein families have evolved to specifically recognize diverse DNA site sequences. This dissertation presents evidence that the Cro protein family of helix-turn-helix transcription factors from lambdoid bacteriophages may share a conserved, limited "code" that partially governs evolution of their binding specificity. A bioinformatic study revealed six conserved sequence correlations between residues at three positions in Cro recognition helices and three base-pairs in putative cognate DNA consensus half-sites (Chapter 2). Three of these pairings correspond to sequence-specific contacts observed at the binding interface of lambda Cro and consensus operator DNA in a previously available co-crystal structure (Albright and Matthews, 1998a). In vitro mutagenesis and functional characterization was used to validate the proposed "code" (Chapter 3). Two out of three "coding" combinations acted as specificity switches in lambda Cro, though variant proteins displayed reduced binding specificity for their predicted target DNA sites. Two crystal structures of a lambda Cro variant are presented in Chapter 4, which provide insight into lambda Cro dimer flexibility. Additionally, a co-crystal structure of N15 Cro bound to consensus site DNA was determined which contains two coding residue pairs at the binding interface (Chapter 5), and a crystal structure of Xfasa1 Cro that enables future investigations into Cro functional evolution (Chapter 6). Although there are several caveats, the data are consistent with a model in which Cro proteins may indeed have evolved new binding specificities in part through simple mutations at their binding interfaces that follow a simple set of evolutionarily conserved "coding" rules. The structural and functional diversity of Cro proteins provides an exciting venue for future research into their evolution.
    • Functional expression of alpha-2 adrenergic receptor subtypes in cultured mammalian cells

      Pepperl, David John.; Regan, John W.; Halpert, James; Lai, Josephine; Lindell, Thomas; Miesfeld, Roger (The University of Arizona., 1994)
      The ɑ₂ adrenergic receptors are among the most extensively studied members of the G-protein coupled receptor superfamily. They have been purified from native tissue and cloned from a number of species. Presently, three pharmacologically distinct subtypes of ɑ₂ adrenergic receptors have been identified, termed the ɑ₂-C10, ɑ₂-C2 and ɑ₂-C4. Although stable expression of these proteins in suitable host cells is commonly used for studying the pharmacology and 2nd messenger coupling of these proteins, stable expression systems are extremely time-consuming. Therefore, one focus of this work was to develop a more efficient approach for studying ɑ₂ adrenergic receptor-2nd messenger coupling. A transient gene expression system should dramatically decrease the time required for studying receptor function. Using a cAMP-dependent reporter plasmid and a responsive cell system, we have demonstrated transient functional expression of ɑ₂ adrenergic receptor subtypes. Agonist activation of these receptor subtypes produces unique intracellular responses, suggesting specific receptor-effector interactions within the transfected cells. To directly address these interactions, stable cell lines expressing the ɑ₂ receptor subtypes were developed. Both the ɑ₂-C4 and ɑ₂-C10 receptor subtypes can be stably-expressed at relatively high levels in these cells. All three subtypes expressed in this cell line exhibited the pharmacology appropriate for their respective subtypes. Moreover, agonist activation of both ɑ₂-C4 and ɑ₂-C10 receptors in these cells produced identical dose-dependent inhibition of cAMP production. These studies have demonstrated that ɑ₂ adrenergic receptors can be expressed in human choriocarcinoma cells, and that agonist activation of these subtypes produces unique intracellular responses. This approach has also demonstrated the potential for regulation of gene expression by ɑ₂ adrenergic receptors. Most importantly however, development of a more rapid functional expression system has dramatically increased our ability to study ɑ₂ adrenergic receptor function. In the future, transient expression in JEG-3 cells should provide a useful tool for examining the effect of mutation on ɑ₂ adrenergic receptor function. Further studies should address functional expression of other G-protein coupled receptors as well as help define the structural basis for ɑ₂adrenergic receptor activity.
    • Functional Forms-Formal Functions: An Account of Coeur d'Alene Clause Structure

      Harley, Heidi; Carnie, Andrew; Bischoff, Shannon T.; Harley, Heidi; Hill, Jane; Carnie, Andrew; Demers, Richard; Karimi, Simin; Willie, Mary Ann (The University of Arizona., 2007)
      Coeur d'Alene, also known as Snchitsu'umshtsn, is a Southern Interior Salishan language no longer learned by children. Descriptive work on the language has been carried out since the early nineteenth-century (Tiet 1904 through 1909 in Boaz and Tiet 1930; Reichard 1927-29, 1938, 1939; Doak 1997); however, a formal account of the basic clause structure of this polysynthetic language has until now not been proposed. This thesis presents such a formal analysis within the Minimalist Program (Chomsky 1995, 1998, 2000, 2001a, 2001b; Lasnik 1999a, 1999b, 2000; among others), employing the tenets of Distributed Morphology (Halle and Marantz 1993; Harley and Noyer 1999; among others). Demonstrating that an analysis of person marking morphemes as bound pronouns (Jelinek 1984) is more "economical" in terms of Chomsky's (1995:367)Elementary Principles of Economy, the thesis goes on to account for the phenomena of lexical affixation (Carlson 1990; Kinkade 1998; Gerdts 2003; among others), in Coeur d'Alene as incorporation. Appealing to Hale and Keyser's (2002) theory of conflation as Head-movement (Harley 2004), an approach to incorporation is proposed which captures Chomsky's (1995) claim that head-movement is phonological while at the same time illustrating that lexical affixes in Coeur d'Alene serve as incorporated arguments. The thesis concludes with an articulation of the left periphery (material above vP here), based on the strict ordering of a series of mood, adverbial, model, and aspectual particles. It is shown that this articulation in Coeur d'Alene patterns with Cinque's (1999) proposed universal hierarchy of functional and adverbial heads. In this way, the basic clause structure of Coeur d'Alene is formally presented
    • Functional neuroanatomy of pleasant and unpleasant emotion

      Schwartz, Gary E.; Lane Richard David (The University of Arizona., 1999)
      To investigate the basic neural circuitry underlying emotion, three brain imaging studies were performed using positron emission tomography and 15O-water. In each study subjects viewed pictures from the International Affective Picture System (IAPS). Study #1 examined the neural correlates of pleasant and unpleasant emotion in 12 healthy women. Compared to viewing neutral stimuli, viewing pleasant and unpleasant pictures were each associated with activation of thalamus, hypothalamus, midbrain and medial prefrontal cortex. Viewing pleasant pictures was also associated with activation of the head of the caudate nucleus and viewing unpleasant pictures was associated with activation of left medial temporal structures (amygdala, hippocampus and parahippocampal gyrus), bilateral extrastriate visual cortex, bilateral temporal poles and cerebellum. Study #2 examined the neural substrates of emotional valence, arousal and attention. Six healthy men were studied in twelve scan conditions generated from a 3 x 2 x 2 factorial design: 3 levels of valence (pleasant, unpleasant and neutral), 2 levels of arousal (high and low) and 2 levels of attention (easy and difficult distraction tasks). Subtraction of the low arousal pleasant and unpleasant conditions from the high arousal pleasant and unpleasant conditions revealed activation in the dorso-medial region of the thalamus and the medial prefrontal cortex. Activation of the medial prefrontal cortex was greater during the low distraction compared to the high distraction conditions. These results suggest that the thalamus and medial prefrontal cortex are activated as a function of the intensity of emotional arousal independent of valence. Study #3 examined the neural substrates of the experiential component of emotion using a selective attention paradigm. Ten healthy men viewed IAPS pictures as they attended either to their subjective emotional responses or the spatial location of the depicted scene. During attention to subjective emotional responses increased neural activity was elicited in rostral anterior cingulate cortex (BA32) and medial prefrontal cortex, right temporal pole, insula and ventral cingulate. Under the same stimulus conditions when subjects attended to spatial aspects of the pictures activation was observed in parieto-occipital cortex bilaterally. The findings indicate that the rostral anterior cingulate cortex participates in representing subjective emotional responses.
    • Functional organization of male-specific olfactory glomeruli in the sphinx moth Manduca sexta

      Hildebrand, John G.; Heinbockel, Thomas, 1963- (The University of Arizona., 1997)
      The macroglomerular complex (MGC) in the antennal lobe of the sphinx moth Manduca sexta is the first brain region for processing sex-pheromonal information. How is the MGC is functionally organized, and how are chemical and physical features of the pheromone encoded by projection neurons (PNs) innervating the MGC (MGC-PNs). For some MGC-PNs with arborizations in the toroid, one of the two major glomeruli of the MGC, bombykal (a key pheromone component) can evoke a mixed (inhibitory/excitatory/inhibitory) response similar to that evoked by the pheromone blend. Likewise, for some neurons with arborizations in the cumulus, C-15 (a mimic of the second key component) can evoke a similar mixed response. The maximal pulse frequency encoded by these component-specific neurons was not increased in the presence of the blend, but seemed to arise through the convergence of two parallel pathways, one excitatory and one inhibitory, both activated by the same olfactory stimulus. Convergence of different synaptic pathways allowed MGC-PNs to resolve intermittent stimuli and thus to relay the temporal structure of the pheromonal signal to higher brain centers. In a subset of MGC-PNs that was excited by antennal stimulation with either of the two components (bombykal-C-15 cells, blend neurons), the ability to encode intermittent stimuli was improved when stimulating with the blend. The temporal character of the responses was dependent on the ratio of the two key components in the blend. Component-specific MGC-PNs responded over a range of increasing pheromone concentration with stronger inhibitory and excitatory postsynaptic potentials and more impulses but the responses were not affected by changing the blend ratio. Two basic response patterns emerged when the ipsilateral antennal flagellum was stimulated at different zones along its proximo-distal axis while the activity of MGC-PNs was recorded. A subset of neurons with broad receptive fields was excited regardless of the zone of the antenna stimulated, whereas another subset responded selectively to stimulation of the basal region of the antenna. A diverse array of MGC-PNs forms a heterogeneous group of parallel output channels that encode features of the pheromone signal that the moth is likely to encounter in the natural stimulus situation.
    • Functional properties of aquaporin-1 ion channels in choroid plexus

      Yool, Andrea J.; Boassa, Daniela (The University of Arizona., 2004)
      Aquaporins (also known as water channels) are members of the Major Intrinsic Protein family. Ion channel function has been shown for several members of the aquaporin family and the related neurogenic gene product Big Brain. Aquaporin-1 (AQP1) is a transmembrane channel that mediates osmotically-driven water flux. Prior work demonstrated that AQP1 channels expressed in Xenopus oocytes mediate a cGMP-dependent cationic current. Based on amino acid sequence alignments with cyclic nucleotide-gated channels and cGMP-selective phosphodiesterases, I found that the efficacy of ion channel activation is decreased by mutations of AQP1 at conserved residues in the C-terminal domain (aspartate D237 and lysine K243). These data provide direct evidence for the involvement of the AQP1 carboxyl terminal domain in cGMP-mediated ion channel activation. Because the proportion of active AQP1 ion channels seen in heterologous expression systems is low, it was of fundamental importance to investigate the functional properties of this channel in a physiological context. Using rat choroid plexus, a brain tissue that secretes cerebral spinal fluid (CSF) and endogenously expresses abundant AQP1, I demonstrated the existence of native AQP1 ion channels that show properties similar to those described previously in the oocyte expression system. They mediate a cGMP-dependent cationic conductance, are blocked by cadmium, and show a single-channel conductance of 166 pS. Given the skull's rigidity, pathological increases in CSF secretion (tumors, hydrocephalus, stroke) can result in brain damage. In the choroid plexus several proteins work in concert to regulate CSF secretion. The findings presented in this dissertation are first to demonstrate that AQP1 mediates a cationic current in response to intracellular signals that regulate CSF secretion such as ANP signaling. Fluxes of water and Na⁺ across confluent choroid plexus cell monolayers showed a decreased flow rate following treatment with ANP, and Cd²⁺ reversed the inhibitory effect. These results suggest that activation and block of the AQP1-mediated ionic current may alter net fluid transport across the choroid plexus barrier, and therefore be physiologically relevant in the regulation of net fluid transport in choroid plexus. This places AQP1 as one of the important targets for clinical intervention in brain volume disorders.
    • Functional regulation of opioid receptor signaling

      Roeske, William R.; Tumati, Suneeta; Roeske, William R.; Varga, Eva V.; Hruby, Victor J.; Vanderah, Todd W.; Sipes, Glenn (The University of Arizona., 2009)
      Studies have shown that long-term opioid agonist (such as morphine) treatment produces antinociceptive tolerance and increased pain sensitivity (hyperalgesia and/or allodynia), limiting the clinical efficacy of morphine. Prolonged opiate administration also upregulates spinal pain neurotransmitter (such as calcitonin gene-related peptide (CGRP)) levels and enhances evoked CGRP release in the dorsal horn of rats. It was suggested that augmented spinal pain neurotransmission may contribute to paradoxical pain sensitization and antinociceptive tolerance. The cellular signal transduction pathways involved in sustained opioid mediated augmentation of spinal pain neurotransmitter are not fully clarified.Sustained morphine treatment was shown to augment the concentrations of inflammatory mediators, such as PGE2 in the spinal cord. Studies have shown that PGE2 stimulates cAMP formation and CGRP release by activation of Gs protein-coupled prostaglandin receptor types in primary sensory neurons. Interestingly, it was found earlier that sustained opioid agonist treatment leads to a Raf-1-dependent sensitization of adenylyl cyclase(s) (AC superactivation), augmenting forskolin-stimulated cAMP formation upon opioid withdrawal (cAMP overshoot). It is well demonstrated that cAMP activates cAMP-dependent protein kinase (PKA), which plays an important role in the modulation of presynaptic neurotransmitter release. Therefore, in this study, we investigate the physiological role of Raf-1 mediated AC superactivation and subsequent PKA activation in A. sustained morphine-mediated augmentation of basal or evoked pain neurotransmitter release in vitro, in cultured primary sensory neurons, and B. in vivo, in sustained morphine mediated paradoxical pain sensitization and antinociceptive tolerance in rats.Our data demonstrates that A. sustained morphine treatment augments both basal and capsaicin-evoked CGRP release from isolated primary sensory neurons in a PKA- and Raf-1- dependent manner. B. sustained morphine treatment- augments of PGE2-evoked CGRP release from these cells. C. selective knockdown of spinal PKA or Raf-1 protein levels by intrathecal PKA- or Raf-1-specific siRNA pretreatment completely attenuates sustained morphine-mediated thermal hyperalgesia, tactile allodynia and greatly reduces antinociceptive tolerance in rats.In conclusion, we suggest that Raf-1-mediated AC superactivation may have a crucial trigger role in sustained morphine-mediated compensatory adaptations in the nervous system. Thus, we expect that pharmacological attenuation of Raf-1-mediated AC superactivation may improve the clinical treatment of chronic and neuropathic pain.
    • Functional Responses of Sonoran Desert Plant Species to Precipitation

      Huxman, Travis E.; Ignace, Danielle Denise; Huxman, Travis E.; Huxman, Travis E.; Enquist, Brian J.; Robichaux, Robert H.; McPherson, Guy R.; Scott, Russell L. (The University of Arizona., 2006)
      Arid and semi-arid ecosystems of the southwestern U.S. are experiencing major changes that have profound impacts for community structure and ecosystem function. First, these ecosystems are experiencing dramatic shifts in vegetation composition as a result of the invasion of non-native species. Second these ecosystems are predicted to undergo substantial shifts in climate regime, which include increases in the variability and frequency of extreme temperature and precipitation events. It is not well understood how these current and predicted changes will affect the physiological performance of different plant types in arid and semi-arid ecosystems. To address the effect of these changes, this dissertation focused on the photosynthetic response of a native and non-native grass species, and dominant shrub species to precipitation across contrasting soil surfaces in southeastern Arizona. The native and non-native grasses were exposed to wet and dry seasonal precipitation and responses to precipitation events ('pulses') were measured over the course of a summer growing season. To gain a mechanistic understanding of these patterns, the biochemical and diffusion limitations to photosynthetic function were measured over the course of a pulse period. Building on this foundation, natural stands of the non-native grass species were exposed to sequences of different sized pulse events. The physiological performance of a dominant shrub species, Larrea tridentata, was measured in order to determine the biochemical and diffusional constraints to photosynthetic function across seasons and contrasting soil surfaces. The results showed that leaf area development of these grass species affects water availability and time lags in photosynthetic response. Initial soil moisture conditions across contrasting soil surfaces influence the magnitude of photosynthetic response in grasses. Large photosynthetic responses of the non-native grass require large and consecutive precipitation pulses. Co-limitation of photosynthesis of Larrea tridentata by diffusion and biochemistry does not illustrate typical trends across seasons and soil surfaces. Overall results demonstrate the importance of determining the mechanisms responsible for observed leaf-level photosynthetic patterns across individual pulse events, seasons, and contrasting soil surfaces. This is especially important for predicting the magnitude of the response of plant communities in arid and semi-arid ecosystems to species invasions and changes in climate.
    • Functional Restoration of Irradiated Salivary Glands Through Modulation of aPKCζ and Nuclear Yap in Salivary Progenitors

      Limesand, Kirsten H.; Martinez Chibly, Agustin Alejandro; Limesand, Kirsten H.; Wilson, Jean; Briehl, Margaret; Burd, Randy (The University of Arizona., 2016)
      Radiotherapy is the primary treatment for patients with head and neck cancer, which account for roughly 60,000 annual diagnoses in the U.S. and approximately 500,000 worldwide. About 90% of these individuals receive radiation therapy, and salivary hypofunction and xerostomia occur in 60-85% of these patients due to irreversible damage to the salivary glands. Current preventative and palliative care fail to improve quality of life, accentuating the need for regenerative therapies. Stem/progenitor-cell based therapies have been proposed to regenerate the irradiated glands; however, the identity of stem and progenitor cells in the adult salivary glands has remained somewhat elusive. Moreover, it is unclear how salivary progenitors respond to radiation and whether they can be stimulated to effectively reinstate salivary function. The second chapter of the present study describes the development of a label-retaining assay in salivary glands using EdU. The label-retaining cells (LRCs) identified in murine salivary glands have proliferative potential in vitro and expressed markers of putative salivary progenitors, such as Keratin 5, Keratin 14, and c-Kit. Interestingly, LRCs were still present 30 days following radiation, when chronic loss of saliva is evident. The significance of these findings lies in the potential of this model to study the mechanisms that prevent salivary progenitors from maintaining salivary gland homeostasis upon exposure to radiation, which will in turn facilitate the development of regenerative therapies for salivary gland dysfunction. In the following chapter, we show that a unique population of murine salivary gland LRCs undergo compensatory proliferation in response to radiation. The initiation of compensatory proliferation is tightly associated with inactivation of the kinase aPKCζ and increased nuclear localization of YAP. This part of the study provides novel insights into the regulation of function of salivary gland progenitors, which can be utilized for the development of therapeutic agents to treat salivary hypofunction. Finally, the last chapter describes how the mechanisms found to initiate compensatory proliferation in acinar LRCs as a response to radiation are involved in the regeneration of salivary glands with IGF-1. Administration of IGF-1 post-radiation restores salivary function in mice, but the mechanisms of regeneration are still unknown. Here, we show that IGF-1 requires aPKCζ to restore saliva production. Further, IGF-1 inhibits nuclear translocation of Yap in an aPKCζ-dependent fashion. We propose that a tightly regulated balance in the levels of aPKCζ and Yap in acinar LRCs has to be maintained in order to restore function following radiation. In conclusion, the findings from this study provide new knowledge in regards to the regulation of function of salivary progenitors during a state of injury (by radiation) and during regeneration (with IGF), and offer potential targets of study for the development of new therapeutics for salivary gland dysfunction. Future studies will determine whether aPKCζ and Yap can be effectively targeted in salivary progenitors to restore salivary function in head and neck cancer patients who receive radiation therapy.
    • The functional role(s) of dual intermediate filament expression in tumor cell migration and invasion.

      Chu, Yi-Wen.; Hendrix, Mary J. C.; Cress, Anne E.; Nagle, Raymond B.; Bernstein, Harris; Gerner, Eugene W. (The University of Arizona., 1993)
      Tumor cell invasion and metastasis are very complicated biological events which involve numerous classes of proteins that participate in controlling each step of the metastatic cascade. Therefore, identifying the key factor(s) that determine(s) how a tumor cell becomes more metastatic is a fascinating issue that challenges the field of cancer biology. Intermediate filaments are cytoskeletal proteins whose expression is highly regulated in a cell-type specific manner; however, recent evidence has indicated that coexpression of two different types of intermediate filaments--specifically keratin(s) (epithelial cell marker) and vimentin (mesenchymal cell marker), in the tumor cells correlates with their invasive and metastatic potential. The focus of this dissertation is to further elucidate the functional role(s) of this dual intermediate filament expression in tumor cells. A dominant negative mutant keratin cDNA was used to transfect a highly metastatic human melanoma cell line, C8161, which contains both vimentin and keratin filaments. The resulting transfected clones showed disrupted keratin filaments by immunofluorescence microscopy. Subsequently, their migratory and invasive ability were reduced, in addition to complete abrogation of metastatic potential. Another strategy involved the transfection of keratin 8 and 18 DNAs into keratin-negative cells (both mouse L fibroblasts and low invasive A375P melanoma), which resulted in clones expressing the dual intermediate filament phenotype, commensurate with increased migratory and invasive ability. Furthermore, these experimental clones have a retarded spreading ability on extracellular matrix compared to the control transfectants. In addition, they do not contain any detectable αᵥβ₃, α₃ or α₆ integrins in the focal contact sites by immunofluorescence staining. Hence, it is postulated that the mechanism responsible for differential spreading ability rests in the unique regulation of specific integrin(s) localized in focal contacts, acting either directly or indirectly, with the intermediate filaments and with extracellular matrix molecules. These results suggest that dual intermediate filament expression is important for the invasive phenotype, and their heretofore assigned role as general maintenance structural proteins has changed to that of dynamic cytoskeletal elements involved in active cellular function(s).