Isovaleric Acid (IVA) is a 5-carbon branched chain fatty acid present in fermented foods and produced by the fermentation of leucine by colonic bacteria. IVA activates G-protein coupled receptors such as FFAR2, FFAR3, and OR51E1 known to be expressed on enteric neurons and enteroendocrine cells. We previously reported that the shorter, straight chain fatty acids acetate, propionate and butyrate, differentially affect colonic propulsion; however, the effect of branched chain fatty acids on gastrointestinal motility is unknown. We hypothesize that IVA relaxes smooth muscle in a cAMP/PKA dependent manner by direct action on smooth muscle cells. IVA will also decrease peristalsis and encourage retention of luminal contents. This thesis investigates the effect of IVA on smooth muscle tension and peristaltic activity in isolated colon and individual smooth muscle cells. Colon segments from C57BL/6J mice were placed in a longitudinal orientation in organ baths in Krebs buffer and fastened to force transducers. Segments were contracted with 10 μM acetylcholine (ACh) and the effects of IVA at several concentrations were measured in the absence and presence of Nitric Oxide Synthase inhibitor L-N-nitroarginine (L-NNA), neuronal action potential inhibitor tetrodotoxin (TTX), and adenylate cyclase inhibitor SQ22536. To study individual live cells, mouse smooth muscle was isolated from colon, suspended in smooth muscle buffer, and after contraction with ACh were relaxed with micromolar concentrations of IVA. For peristalsis studies, whole colonic segments isolated from C57BL/6J were catheterized and placed horizontally in organ baths with circulating Krebs buffer. The colon was clamped on the anal end, and a solution (5 μL per mm of colon length) of either Krebs buffer or 50 mM IVA was delivered from the oral end to the lumen. Video of the peristalsis was then analyzed for diameter, changes in diameter, velocity of diameter changes along the length of the colon, normalized to the anatomical changes in the proximal region. IVA in concentrations of 10 mM to 50 mM relaxed the ACh-induced contraction in a sigmoidal fashion. In separate studies, L-NNA nor TTX affected the ability of IVA to inhibit relaxation. SQ22536 inhibited IVA induced relaxation in longitudinal colon compared to vehicle control. In isolated cells, SQ22536 and PKA inhibitor H-89 inhibited IVA-induced relaxation. In peristalsis studies, 50 mM IVA in Krebs buffer changed the character of the peristaltic action by increasing proximal diameter, inhibiting contractions in the proximal end of the colon, and decreasing overall velocity of peristaltic contractions in the proximal region. The data indicate that the branched chain fatty acid IVA causes a concentration-dependent relaxation of colonic smooth muscle that is direct to the smooth muscle and independent of neuronal activity. This relaxation is cAMP/PKA dependent. In addition to the direct relaxation of smooth muscle, intraluminal IVA decreased overall colonic propulsive activity and encouraged retention of the luminal contents. We conclude that the ingestion and production of branched chain fatty acids could affect overall GI motility and is an area for study in dietary and therapeutic control of bowel activity.

The fitness function developed in this thesis directly links the physicochemical properties of an enzyme to evolutionary fates in a quantitative and predictive manner through a comparative study of empirical and simulated data. The success or failure of organisms during evolution is dictated by changes in molecular structure that give rise to changes in fitness revealed by evolutionary dynamics within a population. While the conceptual link between genotype, phenotype and fitness is clear, the ability to relate these in a quantitative manner remains difficult. I show here that predicting success during adaptation can depend critically upon enzyme kinetic and folding models. We used a 'weak link' method to favor mutations to an essential, but maladapted adenylate kinase gene within a microbial population that resulted in the identification of five mutants that arose nearly simultaneously and competed for success. The unique catalytic role of adenylate kinase in vivo is to maintain adenylate homeostasis by catalyzing the reaction: ATP + AMP [imaginary] ADP. The stabilizing substitutions retained this essential function and were shown to be necessary for viability at higher temperatures. Physicochemical characterization of these mutants demonstrated that, although steady-state enzyme activity is important, success within the population is critically dependent on resistance to denaturation and aggregation thus emphasizing the importance of proper folding in adaptation. In vitro activity is a product of critical catalytic and folding pathways, and hence is a valuable proxy for fitness. A fitness function relating in vitro measurements of enzyme activity and reversible and irreversible unfolding to growth rate must impose an activity threshold above which there is no added fitness benefit in order to reproduce in vivo evolutionary fates in an in silico population. The fitness function thereby links organismal adaptation to the properties of a single gene. Understanding the physical basis for adaptation of an organism is the first step in the development of approaches that can accurately model, and someday predict, the manner in which organisms would respond to new antibiotics and improve upon the current clinical regimens.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 67-80).

published_or_final_version / abstract / toc / Zoology / Master / Master of Philosophy

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [115]-133).

The first type of dynein identified, axonemel dynein (Gibbons and Rowe, 1965), slides adjacent microtubules within the axoneme, generating the force necessary for ciliary and flagellar beating. The outer dynein arm is an important component of the flagellar axoneme, providing up to 60% of the force for flagellar motility. In the absence of the outer arm, cells swim with a slow-jerky motion at about 1/3rd the speed of wild-type cells, and the flagellar beat frequency is markedly reduced. Sixteen genes (ODA1-ODA16) have been identified that are required for outer arm assembly in Chlamydomonas reinhardtii. In addition, PF13, PF22, and FLA14 are required for outer dynein arm assembly, but their phenotypes are pleiotropic, suggesting that they affect additional flagellar components. Of the uncloned genes, ODA5, ODA8, and ODA10 are of particular interest because they do not encode subunits of the outer arm or the outer dynein arm-docking complex (ODA-DC). Mutant alleles of these genes are unable to complement in temporary dikaryons, suggesting that the gene products interact with each other (Kamiya, 1988). Since the genes encoding all of the known components of the outer dynein arm and the ODA-DC have been characterized, it is of great interest to identify the gene products of these additional, uncloned ODA alleles. The first chapter provides an introduction to the Chlamydomonasflagellum, the dyneins in general, the outer dynein arm in particular, and mutations that impinge on the assembly and regulation of this important axonemal structure. The second chapter addresses the identification and isolation of genomic DNA containing the ODA5 gene. Utilizing a NIT1-tagged oda5-insertional mutant, I identified sequences flanking the site of the inserted NIT1 gene. These sequences were used to isolate wild-type genomic clones spanning the ODA5 gene. When transformed into the oda5 mutant, the wild-type clones rescued the mutant phenotype. These results demonstrated the successful isolation of the ODA5 gene. The third chapter describes the identification of the ODA5 gene and its corresponding cDNA. The rescuing genomic fragments were sequenced. Gene modeling was used to predict intron-exon splice sites. Primers to predicted exons were designed and used to obtain the ODA5 cDNA. The gene structure of Oda5 was analyzed and its predicted amino acid sequence deduced. Secondary structure predictions indicate that Oda5p is likely to contain a series of coiled-coil domains, followed by a poly-glycine sequence and a short, highly charged region. Northern analysis demonstrated that ODA5 gene expression is upregulated by deflagellation, a hallmark of many flagellar mRNAs. Data in CHAPTER IV further characterize the Oda5 protein and its association with the axoneme. Oda5p localizes to the flagellum, consistent with the enhancement in mRNA levels in response to deflagellation. Within the flagellum, Oda5p is an axonemal component that is released from the axoneme upon high salt extraction, as are the ODA-DC and the outer dynein arm. However, Oda5p does not associate with this super-complex in the high salt extract as determined by sucrose gradient sedimentation. Oda5p assembles onto the axoneme independently of the outer dynein arm and the ODA-DC,demonstrating it does not require these complexes for localization. Furthermore, Oda5p assembles onto the axoneme in the oda8, but not the oda10 mutant, demonstrating a role for the Oda10 protein in localization of Oda5p. These data provide the first biochemical evidence for an interaction between Oda5p and Oda10p. CHAPTER V reveals the discovery of a previously unrecognized phenotype exhibited in both oda5 and oda10 mutant strains: a defect in the assembly of a previously unknown flagellar adenylate kinase (AK). The protein levels of this flagellar AK are reduced in oda5 mutant axonemes, as determined by quantitative mass spectrometry. Direct enzymatic assays confirmed a reduction in AK activity in both oda5 and oda10 mutant axonemes, providing a second line of biochemical evidence supporting a complex containing Oda5p and OdalOp. The sequence of the flagellar AK gene and its cDNA were determined. CHAPTER VI details our efforts to identify the ODA10 gene. Genomic clones were isolated, which contain sequences at, or near, the ODA10 locus. Analysis of the genomic clones yielded no insights into the identity of the ODA10 gene. The inability of these clones to rescue the Oda10-motility phenotype indicates that these clones most likely do not contain an intact ODA10 gene. And lastly, CHAPTER VII discusses future experimentation that can be done based on the data provided by the current study.

abstract: In a typical living cell, millions to billions of proteins—nanomachines that fluctuate and cycle among many conformational states—convert available free energy into mechanochemical work. A fundamental goal of biophysics is to ascertain how 3D protein structures encode specific functions, such as catalyzing chemical reactions or transporting nutrients into a cell. Protein dynamics span femtosecond timescales (i.e., covalent bond oscillations) to large conformational transition timescales in, and beyond, the millisecond regime (e.g., glucose transport across a phospholipid bilayer). Actual transition events are fast but rare, occurring orders of magnitude faster than typical metastable equilibrium waiting times. Equilibrium molecular dynamics (EqMD) can capture atomistic detail and solute-solvent interactions, but even microseconds of sampling attainable nowadays still falls orders of magnitude short of transition timescales, especially for large systems, rendering observations of such "rare events" difficult or effectively impossible. Advanced path-sampling methods exploit reduced physical models or biasing to produce plausible transitions while balancing accuracy and efficiency, but quantifying their accuracy relative to other numerical and experimental data has been challenging. Indeed, new horizons in elucidating protein function necessitate that present methodologies be revised to more seamlessly and quantitatively integrate a spectrum of methods, both numerical and experimental. In this dissertation, experimental and computational methods are put into perspective using the enzyme adenylate kinase (AdK) as an illustrative example. We introduce Path Similarity Analysis (PSA)—an integrative computational framework developed to quantify transition path similarity. PSA not only reliably distinguished AdK transitions by the originating method, but also traced pathway differences between two methods back to charge-charge interactions (neglected by the stereochemical model, but not the all-atom force field) in several conserved salt bridges. Cryo-electron microscopy maps of the transporter Bor1p are directly incorporated into EqMD simulations using MD flexible fitting to produce viable structural models and infer a plausible transport mechanism. Conforming to the theme of integration, a short compendium of an exploratory project—developing a hybrid atomistic-continuum method—is presented, including initial results and a novel fluctuating hydrodynamics model and corresponding numerical code. / Dissertation/Thesis / Doctoral Dissertation Physics 2017

Plus de 200 facteurs pré-ribosomiques sont impliqués dans la maturation des ribosomes. La majorité de ces facteurs sont essentiels à la survie cellulaire, mais la fonction précise de la plupart d’entre eux demeure inconnue. Une des dernières étapes de maturation de la petite sous-unité du ribosome est le clivage du pré-ARNr 20S en ARNr 18S mature. Ce clivage est réalisé par l'endonucléase Nob1 et nécessite également la présence de la NTPase Fap7 ainsi que d’une pléthore d’autres facteurs pré-ribosomiques. La fonction de Fap7 est particulièrement intrigante, car l'homologue humain hCINAP possède une activité adénylate kinase, activité enzymatique qui n’est généralement pas liée à la biogenèse des particules ribonucléoprotéiques. En outre, la fonction de Fap7 est intimement liée à son interaction avec la protéine ribosomique Rps14. La partie C-terminale de Rps14 est essentielle pour le clivage au niveau du site D et est située à proximité de l’extrémité 3’ de l’ARNr 18S dans le ribosome mature. La suppression de cette protéine provoque le syndrome 5q qui est phénotypiquement proche de l’anémie de Diamond-Blackfan. Ces deux protéines interviennent également au niveau d’une voie de régulation de p53 qui est dérégulée dans de nombreux cancers. La combinaison d’études structurales par cristallographie aux rayons X, d’études enzymatiques sur des protéines recombinantes ainsi que des tests de maturation in vitro réalisés sur des pré-ribosomes purifiés, nous a permis de mieux appréhender la fonction de Fap7 au sein de la sous-unité pré-40S du ribosome. Nous avons également montré que l'interaction Fap7-Rps14 est impliquée dans un changement conformationnel majeur au cœur des pré-ribosomes et que cette réorganisation est nécessaire afin d'exposer le site D pour le clivage par l’endonucléase Nob1. / Over 200 pre-ribosomal factors involved in the maturation of ribosomes. Most of these factors are essential to cell survival, but the precise function of most of these factors remains elusive. One of the last steps of maturation of the small subunit of the ribosome is the cleavage of 20S pre-rRNA in 18S rRNA in the cytoplasm. This cleavage is carried out by the endonuclease Nob1 and also requires the presence of other factors such as the methyltransferase Dim1, and a plethora of NTPases including the Rio protein kinases, Prp43 and its cofactor Pfa1, the Ltv1 GTPase and the Fap7 NTPase. The function of Fap7 is especially intriguing since the human homologue bears Adenylate activity, an enzymatic activity not usually linked to ribonucleoprotein biogenesis. In addition, the function of Fap7 is intimately linked its interaction with the Rps14 ribosomal protein. The Rps14 C-terminal is essential of D site cleavage and is located in proximity to the 18S C-terminus in the mature ribosome. The deletion of this protein causes the 5q syndrome that is phenotypically close to Diamond Blackfan anemia. The link between the enzymatic activity of Fap7 and its role in ribosome biogenesis remains enigmatic. Using a combination of structural studies by X-ray crystallography, small angle X-ray scattering (SAXS) in solution, enzymatic studies on purified proteins, and in vitro D site cleavage reaction assays on purified pre-ribosomes, we were able to uncover the function of Fap7 within pre-40S ribosomes. We show that the Fap7/Rps14 interaction is involved in a major conformational change at the heart of the pre-ribosomes and that this structural rearrangement is necessary to expose the D-site for cleavage by the endonuclease Nob1.

Chronic pressure overload (PO) is associated with cardiac hypertrophy and altered autonomic control of cardiac function, in which the latter may involve adaptations in central and/or peripheral cardiac neural control mechanisms. To evaluate the specific remodeling of the intrinsic cardiac nervous system following pressure overload, the descending thoracic aorta artery of the guinea pig was constricted ∼20%, and the animals recovered for 9 wk. Thereafter, atrial neurons of the intrinsic cardiac plexus were isolated for electrophysiological and immunohistochemical analyses. Intracellular voltage recordings from intrinsic cardiac neurons demonstrated no significant changes in passive membrane properties or action potential depolarization compared with age-matched controls and sham-operated animals, but afterhyperpolarization duration was increased in PO animals. Neuronal excitability, as determined by the number of action potentials produced with depolarizing stimuli, was differentially increased in phasic neurons derived from PO animals in response to exogenously applied histamine compared with sham and age-matched controls. Conversely, pituitary adenylate cyclase-activating polypeptide-induced increases in intrinsic cardiac neuron evoked AP frequency were similar between control and PO animals. Immunohistochemical analysis demonstrated a two-fold increase in the percentage of neurons immunoreactive for neuronal nitric oxide synthase in PO animals compared with control. The density of mast cells within the intrinsic cardiac plexus from PO animals was also increased twofold compared with preparations from control animals. These results indicate that congestive heart failure associated with chronic pressure overload induces a differential remodeling of intrinsic cardiac neurons and upregulation of neuronal responsiveness to specific neuromodulators.

Human schistosomiasis popularly known as bilharzias in many regions of Africa is a freshwater snail-transmitted disease caused by parasitic flatworms known as schistosomes. The growth and development of schistosomes typically requires developmental stages in multiple hosts and transmission stages in freshwater. These life cycle environments present a plethora of stressors. Certain gene families including heat shock proteins (HSPs/Hsps) and universal stress proteins (USPs) help schistosomes to respond to unfavourable conditions. The availability of genomes sequences information for Schistosoma japonicum, Schistosoma mansoni and Schistosoma haematobium provide unique research resources to apply bioinformatics analysis of its associated USPs to predict regulatory features from sequence analysis. The objectives of the research were to (i) Infer the biochemical and environmental regulation of universal stress proteins of Schistosoma species; (ii) Identify biological function relevant protein sequence and structure features for prioritized universal stress proteins from Schistosoma species; (iii) Determine the distinctive structural features of a predicted regulator of Schistosoma adenylate cyclase activity that has possible influence on the functioning of universal stress proteins. The findings revealed that (i) schistosomes USPs are hydrophilic and very reactive in the water environment or in aqueous phase, which seems adaptive with their immediate environment and developmental stages; (ii) The functions of Smp_076400 and Sjp_0058490 (Q86DW2) are regulated by conserved binding site residues and metallic ions ligands (Ca2+, Mg2+ and Zn2+), particularly Ca2+ predicted to bind to both USPs; (iii) The S. mansoni life cycle and stress resistance pathway protein (Smp_059340.1) is regulated by Ser53, Thr188, Gly210 and Asp207 residues. The overall scope has highlighted the role of bioinformatics in predicting exploitable regulatory features of schistosome universal stress proteins and biological pathways that might lead to identification of putative functional biomarkers of common environmental diseases. The findings of this research can be applicable to other areas of environmental health and environmental genomics. / Environmental Sciences / (D. Litt et Phil. (Environmental Sciences)