Inhibition de l'apoptose par inversion du rapport [Na⁺]ᵢ/[K⁺]ᵢ : preuve de l'existence de facteur(s) de transcription sensible(s) à la [Na⁺]ᵢ et rôle de la mortaline

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Récepteurs aux estrogènes et remodelage cardiaque chez l'animal adulte ayant subi un environnement foetal défavorable

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Membrane protein production in the Yeast, S. cerevisiae

The first crystal structures of recombinant mammalian membrane proteins were solved in 2005 using protein that had been produced in yeast cells. One of these, the rabbit Ca2+-ATPase SERCA1a, was synthesized in Saccharomyces cerevisiae. All host systems have their specific advantages and disadvantages, but yeast has remained a consistently popular choice in the eukaryotic membrane protein field because it is quick, easy and cheap to culture, whilst being able to post-translationally process eukaryotic membrane proteins. Very recent structures of recombinant membrane proteins produced in S. cerevisiae include those of the Arabidopsis thaliana NRT1.1 nitrate transporter and the fungal plant pathogen lipid scramblase, TMEM16. This chapter provides an overview of the methodological approaches underpinning these successes.

Dynamic Compartmentalization of Persistent UPEC in the Superficial Bladder Epithelium

Urinary tract infections (UTIs) are typically caused by bacteria that colonize different regions of the urinary tract, mainly the bladder and the kidney. Approximately 25% of women that suffer from UTIs experience a recurrent infection within 6 months of the initial bout, making UTIs a serious economic burden resulting in more than 10 million hospital visits and $3.5 billion in healthcare costs in the United States alone. Type-1 fimbriated Uropathogenic E. coli (UPEC) is the major causative agent of UTIs, accounting for almost 90 % of bacterial UTIs. The unique ability of UPEC to bind and invade the superficial bladder epithelium allows the bacteria to persist inside epithelial niches and survive antibiotic treatment. Persistent, intracellular UPEC are retained in the bladder epithelium for long periods, making them a source of recurrent UTIs. Hence, the ability of UPEC to persist in the bladder is a matter of major health and economic concern, making studies exploring the underlying mechanism of UPEC persistence highly relevant.

In my thesis, I will describe how intracellular Uropathogenic E.coli (UPEC) evade host defense mechanisms in the superficial bladder epithelium. I will also describe some of the unique traits of persistent UPEC and explore strategies to induce their clearance from the bladder. I have discovered that the UPEC virulence factor Alpha-hemolysin (HlyA) plays a key role in the survival and persistence of UPEC in the superficial bladder epithelium. In-vitro and in-vivo studies comparing intracellular survival of wild type (WT) and hemolysin deficient UPEC suggested that HlyA is vital for UPEC persistence in the superficial bladder epithelium. Further in-vitro studies revealed that hemolysin helped UPEC persist intracellularly by evading the bacterial expulsion actions of the bladder cells and remarkably, this virulence factor also helped bacteria avoid t degradation in lysosomes.

To elucidate the mechanistic basis for how hemolysin promotes UPEC persistence in the urothelium, we initially focused on how hemolysin facilitates the evasion of UPEC expulsion from bladder cells. We found that upon entry, UPEC were encased in “exocytic vesicles” but as a result of HlyA expression these bacteria escaped these vesicles and entered the cytosol. Consequently, these bacteria were able to avoid expulsion by the cellular export machinery.

Since bacteria found in the cytosol of host cells are typically recognized by the cellular autophagy pathway and transported to the lysosomes where they are degraded, we explored why this was not the case here. We observed that although cytosolic HlyA expressing UPEC were recognized and encased by the autophagy system and transported to lysosomes, the bacteria appeared to avoid degradation in these normally degradative compartments. A closer examination of the bacteria containing lysosomes revealed that they lacked V-ATPase. V-ATPase is a well-known proton pump essential for the acidification of mammalian intracellular degradative compartments, allowing for the proper functioning of degradative proteases. The absence of V-ATPase appeared to be due to hemolysin mediated alteration of the bladder cell F-actin network. From these studies, it is clear that UPEC hemolysin facilitates UPEC persistence in the superficial bladder epithelium by helping bacteria avoid expulsion by the exocytic machinery of the cell and at the same time enabling the bacteria avoid degradation when the bacteria are shuttled into the lysosomes.

Interestingly even though UPEC appear to avoid elimination from the bladder cell their ability to multiple in bladder cells seem limited.. Indeed, our in-vitro and in-vivo experiments reveal that UPEC survive in superficial bladder epithelium for extended periods of time without a significantly change in CFU numbers. Indeed, we observed these bacteria appeared quiescent in nature. This observation was supported by the observation that UPEC genetically unable to enter a quiescence phase exhibited limited ability to persist in bladder cells in vitro and in vivo, in the mouse bladder.

The studies elucidated in this thesis reveal how UPEC toxin, Alpha-hemolysin plays a significant role in promoting UPEC persistence via the modulation of the vesicular compartmentalization of UPEC at two different stages of the infection in the superficial bladder epithelium. These results highlight the importance of UPEC Alpha-hemolysin as an essential determinant of UPEC persistence in the urinary bladder.

Inhibition de l'apoptose par inversion du rapport [Na⁺]ᵢ/[K⁺]ᵢ : preuve de l'existence de facteur(s) de transcription sensible(s) à la [Na⁺]ᵢ et rôle de la mortaline

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Récepteurs aux estrogènes et remodelage cardiaque chez l'animal adulte ayant subi un environnement foetal défavorable

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Differential impacts of elevated CO2 and acidosis on the energy budget of gill and liver cells from Atlantic cod

Ocean acidification impacts fish and other marine species through increased seawater PCO2 levels (hypercapnia). Knowledge of the physiological mechanisms mediating effects in various tissues of fish is incomplete. Here we tested the effects of extracellular hypercapnia and acidosis on energy metabolism of gill and liver cells of Atlantic cod. Exposure media mimicked blood conditions in vivo, either during normo- or hypercapnia and at control or acidic extracellular pH (pHe). We determined metabolic rate and energy expenditure for protein biosynthesis, Na+/K+-ATPase and H+-ATPase and considered nutrition status by measurements of metabolic rate and protein biosynthesis in media with and without free amino acids (FAA). Addition of FAA stimulated hepatic but not branchial oxygen consumption. Normo- and hypercapnic acidosis as well as hypercapnia at control pHe depressed metabolic stimulation of hepatocytes. In gill cells, acidosis depressed respiration independent of PCO2 and FAA levels. For both cell types, depressed respiration was not correlated with the same reduction in energy allocated to protein biosynthesis or Na+/K+-ATPase. Hepatic energy expenditure for protein synthesis and Na+/K+- ATPase was even elevated at acidic compared to control pHe suggesting increased costs for ion regulation and cel- lular reorganization. Hypercapnia at control pHe strongly reduced oxygen demand of branchial Na+/K+-ATPase with a similar trend for H+-ATPase. We conclude that extracellular acidosis triggers metabolic depression in gill and metabolically stimulated liver cells. Additionally, hypercapnia itself seems to limit capacities for metabolic usage of amino acids in liver cells while it decreases the use and costs of ion regulatory ATPases in gill cells.

Impacts of seawater acidification on mantle gene expression patterns of the Baltic Sea blue mussel: implications for shell formation and energy metabolism, link to supplementary material

Marine organisms have to cope with increasing CO2 partial pressures and decreasing pH in the oceans. We elucidated the impacts of an 8-week acclimation period to four seawater pCO2 treatments (39, 113, 243 and 405 Pa/385, 1,120, 2,400 and 4,000 µatm) on mantle gene expression patterns in the blue mussel Mytilus edulis from the Baltic Sea. Based on the M. edulis mantle tissue transcriptome, the expression of several genes involved in metabolism, calcification and stress responses was assessed in the outer (marginal and pallial zone) and the inner mantle tissues (central zone) using quantitative real-time PCR. The expression of genes involved in energy and protein metabolism (F-ATPase, hexokinase and elongation factor alpha) was strongly affected by acclimation to moderately elevated CO2 partial pressures. Expression of a chitinase, potentially important for the calcification process, was strongly depressed (maximum ninefold), correlating with a linear decrease in shell growth observed in the experimental animals. Interestingly, shell matrix protein candidate genes were less affected by CO2 in both tissues. A compensatory process toward enhanced shell protection is indicated by a massive increase in the expression of tyrosinase, a gene involved in periostracum formation (maximum 220-fold). Using correlation matrices and a force-directed layout network graph, we were able to uncover possible underlying regulatory networks and the connections between different pathways, thereby providing a molecular basis of observed changes in animal physiology in response to ocean acidification.

Differential acid-base regulation in various gills of the green crab Carcinus maenas: Effects of elevated environmental pCO2

Euryhaline decapod crustaceans possess an efficient regulation apparatus located in the gill epithelia, providing a high adaptation potential to varying environmental abiotic conditions. Even though many studies focussed on the osmoregulatory capacity of the gills, acid-base regulatory mechanisms have obtained much less attention. In the present study, underlying principles and effects of elevated pCO2 on acid-base regulatory patterns were investigated in the green crab Carcinus maenas acclimated to diluted seawater. In gill perfusion experiments, all investigated gills 4-9 were observed to up-regulate the pH of the hemolymph by 0.1-0.2 units. Anterior gills, especially gill 4, were identified to be most efficient in the equivalent proton excretion rate. Ammonia excretion rates mirrored this pattern among gills, indicating a linkage between both processes. In specimen exposed to elevated pCO2 levels for at least 7 days, mimicking a future ocean scenario as predicted until the year 2300, hemolymph K+ and ammonia concentrations were significantly elevated, and an increased ammonia excretion rate was observed. A detailed quantitative gene expression analysis revealed that upon elevated pCO2 exposure, mRNA levels of transcripts hypothesized to be involved in ammonia and acid-base regulation (Rhesus-like protein, membrane-bound carbonic anhydrase, Na+/K+-ATPase) were affected predominantly in the non-osmoregulating anterior gills.

Seawater carbonate chemistry, mass, length and gene expression of Sepia officinalis during experiments, 2011

The specific transporters involved in maintenance of blood pH homeostasis in cephalopod molluscs have not been identified to date. Using in situ hybridization and immuno histochemical methods, we demonstrate that Na+/K+-ATPase (soNKA), a V-type H+-ATPase (soV-HA), and Na+/HCO3- cotransporter (soNBC) are co-localized in NKA-rich cells in the gills of Sepia officinalis. mRNA expression patterns of these transporters and selected metabolic genes were examined in response to moderately elevated seawater pCO2 (0.16 and 0.35 kPa) over a time-course of six weeks in different ontogenetic stages. The applied CO2 concentrations are relevant for ocean acidification scenarios projected for the coming decades. We determined strong expression changes in late stage embryos and hatchlings, with one to three log2-fold reductions in soNKA, soNBCe, socCAII and COX. In contrast, no hypercapnia induced changes in mRNA expression were observed in juveniles during both short- and long-term exposure. However a transiently increased demand of ion regulatory demand was evident during the initial acclimation reaction to elevated seawater pCO2. Gill Na+/K+-ATPase activity and protein concentration were increased by approximately 15% in during short (2-11 day), but not long term (42 day) exposure. Our findings support the hypothesis that the energy budget of adult cephalopods is not significantly compromised during long-term exposure to moderate environmental hypercapnia. However, the down regulation of ion-regulatory and metabolic genes in late stage embryos, taken together with a significant reduction in somatic growth, indicates that cephalopod early life stages are challenged by elevated seawater pCO2.

Seawater carbonate chemistry, Mg/Ca, Sr/Ca, oxygen production and calcification rate during experiments with coral Pocillopora damicornis, 2011

The Sr/Ca of aragonitic coral skeletons is a commonly used palaeothermometer. However skeletal Sr/Ca is typically dominated by weekly-monthly oscillations which do not reflect temperature or seawater composition and the origins of which are currently unknown. To test the impact of transcellular Ca2+ transport processes on skeletal Sr/Ca, colonies of the branching coral, Pocillopora damicornis, were cultured in the presence of inhibitors of Ca-ATPase (ruthenium red) and Ca channels (verapamil hydrochloride). The photosynthesis, respiration and calcification rates of the colonies were monitored throughout the experiment. The skeleton deposited in the presence of the inhibitors was identified (by 42Ca spike) and analysed for Sr/Ca and Mg/Ca by secondary ion mass spectrometry. The Sr/Ca of the aragonite deposited in the presence of either of the inhibitors was not significantly different from that of the solvent (dimethyl sulfoxide) control, although the coral calcification rate was reduced by up to 66% and 73% in the ruthenium red and verapamil treatments, respectively. The typical precision (95% confidence limits) of mean Sr/Ca determinations within any treatment was <±1% and differences in skeletal Sr/Ca between treatments were correspondingly small. Either Ca-ATPase and Ca channels transport Sr2+ and Ca2+ in virtually the same ratio in which they are present in seawater or transcellular processes contribute little Ca2+ to the skeleton and most Ca is derived from seawater transported directly to the calcification site. Variations in the activities of Ca-ATPase and Ca-channels are not responsible for the weekly-monthly Sr/Ca oscillations observed in skeletal chronologies, assuming that the specificities of Ca transcellular transport processes are similar between coral genera.

Seawater carbonate chemistry and Strongylocentrotus purpuratus body length and gene expression pattern changes during experiments, 2011

Extensive use of fossil fuels is leading to increasing CO2 concentrations in the atmosphere and causes changes in the carbonate chemistry of the oceans which represents a major sink for anthropogenic CO2. As a result, the oceans' surface pH is expected to decrease by ca. 0.4 units by the year 2100, a major change with potentially negative consequences for some marine species. Because of their carbonate skeleton, sea urchins and their larval stages are regarded as likely to be one of the more sensitive taxa. In order to investigate sensitivity of pre-feeding (2 days post-fertilization) and feeding (4 and 7 days post-fertilization) pluteus larvae, we raised Strongylocentrotus purpuratus embryos in control (pH 8.1 and pCO2 41 Pa e.g. 399 µatm) and CO2 acidified seawater with pH of 7.7 (pCO2 134 Pa e.g. 1318 µatm) and investigated growth, calcification and survival. At three time points (day 2, day 4 and day 7 post-fertilization), we measured the expression of 26 representative genes important for metabolism, calcification and ion regulation using RT-qPCR. After one week of development, we observed a significant difference in growth. Maximum differences in size were detected at day 4 (ca. 10 % reduction in body length). A comparison of gene expression patterns using PCA and ANOSIM clearly distinguished between the different age groups (Two way ANOSIM: Global R = 1) while acidification effects were less pronounced (Global R = 0.518). Significant differences in gene expression patterns (ANOSIM R = 0.938, SIMPER: 4.3% difference) were also detected at day 4 leading to the hypothesis that differences between CO2 treatments could reflect patterns of expression seen in control experiments of a younger larva and thus a developmental artifact rather than a direct CO2 effect. We found an up regulation of metabolic genes (between 10 to 20% in ATP-synthase, citrate synthase, pyruvate kinase and thiolase at day 4) and down regulation of calcification related genes (between 23 and 36% in msp130, SM30B, SM50 at day 4). Ion regulation was mainly impacted by up regulation of Na+/K+-ATPase at day 4 (15%) and down regulation of NHE3 at day 4 (45%). We conclude that in studies in which a stressor induces an alteration in the speed of development, it is crucial to employ experimental designs with a high time resolution in order to correct for developmental artifacts. This helps prevent misinterpretation of stressor effects on organism physiology.

Boron isotope composition and seawater characteristics of cold water scleratinian corals

The boron isotope systematics has been determined for azooxanthellate scleractinian corals from a wide range of both deep-sea and shallow-water environments. The aragonitic coral species, Caryophyllia smithii, Desmophyllum dianthus, Enallopsammia rostrata, Lophelia pertusa, and Madrepora oculata, are all found to have relatively high d11B compositions ranging from 23.2 per mil to 28.7 per mil. These values lie substantially above the pH-dependent inorganic seawater borate equilibrium curve, indicative of strong up-regulation of pH of the internal calcifying fluid (pH(cf)), being elevated by ~0.6-0.8 units (Delta pH) relative to ambient seawater. In contrast, the deep-sea calcitic coral Corallium sp. has a significantly lower d11B composition of 15.5 per mil, with a corresponding lower Delta pH value of ~0.3 units, reflecting the importance of mineralogical control on biological pH up-regulation. The solitary coral D. dianthus was sampled over a wide range of seawater pH(T) and shows an approximate linear correlation with Delta pH(Desmo) = 6.43 - 0.71 pH(T) (r**2 = 0.79). An improved correlation is however found with the closely related parameter of seawater aragonite saturation state, where Delta pH(Desmo) = 1.09 - 0.14 Omega(arag) (r**2 = 0.95), indicating the important control that carbonate saturation state has on calcification. The ability to up-regulate internal pH(cf), and consequently Omega(cf), of the calcifying fluid is therefore a process present in both azooxanthellate and zooxanthellate aragonitic corals, and is attributed to the action of Ca2+ -ATPase in modulating the proton gradient between seawater and the site of calcification. These findings also show that the boron isotopic compositions (d11Bcarb) of aragonitic corals are highly systematic and consistent with direct uptake of the borate species within the biologically controlled extracellular calcifying medium.

Structural and Functional Characterization of the Human Tribbles Homologue 2 Pseudokinase

The Tribbles Homologues are a family of three eukaryotic pseudokinases (Trb1, Trb2, Trb3) that act as allosteric inhibitors and regulatory scaffold sites in pathways governing adipogenesis, cell proliferation and insulin signaling. The Tribbles Homologues have the same overall tertiary structure of the eukaryotic protein kinase domain, but lack multiple residues necessary to catalysis in the nucleotide-binding P-loop and the Mg2+-coordinating DFG motif. Trb1 has been shown conclusively to be incapable of binding ATP, whereas a recent study presents evidence that Trb2 autophosphorylates independently of Mg2+ in vitro. This finding is surprising given the high degree of sequence similarity between the two proteins (71%), and suggests unique nucleotide binding and phosphotransfer mechanisms. The goal of this project was to investigate whether Trb2 possesses kinase activity or not and determine its structural basis. A method for the high-yield recombinant expression and purification of stable Trb2 was developed. Trb2 nucleotide binding and autophosphorylation could not be detected across multiple experimental approaches, including thermal shift assays, MANT-ATP fluorescence, radiolabeled phosphate incorporation, and nonspecific ATPase activity assays. Further characterization also revealed that Trb2 forms homomultimers with possible functional consequences, and extensive crystallization screening has yielded multiple promising conditions that could produce diffraction-quality crystals with further optimization. This project explores the difficulties in functionally characterizing putatively active pseudokinases, and proposes a structural basis for the conserved pseudokinase features of the Tribbles homologues.

RECQ5 promotes recombination and mutagenesis at targeted nicks through disruption of RAD51 filaments

Thesis (Ph.D.)--University of Washington, 2016-08