Investigating the Impact of Nonenzymatic Lysine Acetylation on the Function of the Bacterial Ribosome

An under-investigated target of lysine acetylation is the bacterial ribosome. Although lysine acetylations on the ribosome are common and conversed in diverse bacterial species, little work has been done to understand how lysine acetylation affects the bacterial ribosome. The goal of this work is to determine if lysine acetylation has functional impact on the bacterial ribosome.I have identified in vitro and in vivo effects of nonenzymatic, AcP-dependent lysine acetylation on translation and the ribosome. In vitro acetylation of transcription-translation reactions causes a translation-specific defect that is unaffected by the addition of the CobB deacetylase. This suggests certain AcP-sensitive residues that are part of the translation machinery can inhibit translation, and these acetylations are not reversible. In vivo, I have demonstrated that high acetylation bacterial cultures have a ribosome population that favors the presence of dissociated 30S and 50S subunits over intact 70S ribosomes in stationary phase. This is true for cultures that are acetylation high due to manipulation and cultures that are acetylation high due to media manipulation. This suggests that the impact of nonenzymatic lysine acetylation on the ribosome is linked to central carbon metabolism, due to the relationship between AcP levels and carbon flux. I have also demonstrated that there is some contribution by the CobB deacetylase to the subunit skew pattern. However, complications caused by the sensitivity of the pattern to growth conditions have stymied efforts to determine if the shift is caused primarily by CobB-sensitive acetylated lysines or a mixture of CobB-sensitive and -insensitive acetylated lysines. Finally, preliminary mass spectrometry data of 30S, 50S, and 70S fractions from wild-type E. coli grown in a rich medium until stationary phase have allowed me to identify 18 acetylated lysine that are only observed in the subunit fractions. Of these acetylations, acetylated lysines on uS7 and bL12 have functional roles that make them promising targets for future studies into the mechanistic effects of lysine acetylation on the ribosome

The deubiquitylase TRABID coordinates sex-specific neurodegeneration in drosophila melanogaster

Ubiquitin-related post translation modification (PTM) plays an important role in cellular signalling. TRABID is a deubiquitylase that preferentially hydrolyses Lys29 and 33 ubiquitin chains and is known to affect inflammation and consequently, neurodegeneration as the latter is considered functionally linked to the former. However, sex differences are widely observed in both inflammation as well as neurodegeneration phenotypes. In this study, I investigated sex dimorphism, in ubiquitination at the intersection of inflammation and neurodegeneration. I demonstrated Drosophila TRABID has similar enzymatic activity as human TRABID and generated an enzymatically inactive TRABID (trbdC518A) in Drosophila flies. I discovered that trbdC518A has a sexually dimorphic impact in the insect’s locomotor activity, sleep pattern, lifespan, cerebral cells count, brain volume and NF-κB signalling. When blocking female development in astrocytes or immunocompetent cells, sex dimorphisms in locomotor activity and NF-κB signalling are partially eliminated. Further evidence ubiquitinomics and proteomics of fly heads, provided insight into the sex-specific impact of trbdC518A

Global analysis of gene expression in response to L-Cysteine deprivation in the anaerobic protozoan parasite Entamoeba histolytica

<p>Abstract</p> <p>Background</p> <p><it>Entamoeba histolytica</it>, an enteric protozoan parasite, causes amebic colitis and extra intestinal abscesses in millions of inhabitants of endemic areas. <it>E. histolytica </it>completely lacks glutathione metabolism but possesses L-cysteine as the principle low molecular weight thiol. L-Cysteine is essential for the structure, stability, and various protein functions, including catalysis, electron transfer, redox regulation, nitrogen fixation, and sensing for regulatory processes. Recently, we demonstrated that in <it>E. histolytica</it>, L-cysteine regulates various metabolic pathways including energy, amino acid, and phospholipid metabolism.</p> <p>Results</p> <p>In this study, employing custom-made Affymetrix microarrays, we performed time course (3, 6, 12, 24, and 48 h) gene expression analysis upon L-cysteine deprivation. We identified that out of 9,327 genes represented on the array, 290 genes encoding proteins with functions in metabolism, signalling, DNA/RNA regulation, electron transport, stress response, membrane transport, vesicular trafficking/secretion, and cytoskeleton were differentially expressed (≥3 fold) at one or more time points upon L-cysteine deprivation. Approximately 60% of these modulated genes encoded proteins of no known function and annotated as hypothetical proteins. We also attempted further functional analysis of some of the most highly modulated genes by L-cysteine depletion.</p> <p>Conclusions</p> <p>To our surprise, L-cysteine depletion caused only limited changes in the expression of genes involved in sulfur-containing amino acid metabolism and oxidative stress defense. In contrast, we observed significant changes in the expression of several genes encoding iron sulfur flavoproteins, a major facilitator super-family transporter, regulator of nonsense transcripts, NADPH-dependent oxido-reductase, short chain dehydrogenase, acetyltransferases, and various other genes involved in diverse cellular functions. This study represents the first genome-wide analysis of transcriptional changes induced by L-cysteine deprivation in protozoan parasites, and in eukaryotic organisms where L-cysteine represents the major intracellular thiol.</p

Hemodynamics and Endothelial Cell Biology in Cardiovascular Diseases

Atherosclerotic plaques develop preferentially in curved and branching arteries in-vivo. Lipids and inflammatory cells accumulation in the intimal layer of the arterial wall is considered as the main driving mechanism in the disease progression. Evidences suggest that this focal distribution of plaques may result from the combination of systemic risk factors including high plasma cholesterol, smoking, diabetis, hypertension or genetic pre-disposition and local hemodynamic risk factors such as low and oscillatory flows. The exact mechanism of the biological and biomechanical interactions between the endothelium, blood flow and the growing lesion underneath still remains unclear. This thesis is a study on the relationship between biomechanical factors found in proatherogenic flow and endothelial inflammation. The thesis focuses in particular on the effect of secondary flows on wall shear stress and mass transport distribution. To that end, we have combined different techniques from flow imaging, 3D flow reconstruction, vascular biology and mathematical simulation of biological network. In particular, shear stress is involved in the regulation of the pro-inflammatory transcription factor nuclear factor -kB (NF-kB) and the vasoregulator Nitric Oxide. The role of endothelial Nitric Oxide and wall shear stress on NF-kB activation is still controversial. We investigated here the hypothesis that NO negatively regulates NF- kB activation in flow chamber with sheared endothelial cells and using a mathematical model of the NF-kB-NO pathway. Understanding the underlying relationship between hemodynamic factors and inflammatory cells transport to the wall may contribute to the development of better therapies or interventional practices to treat patients with atherosclerotic diseases

Desulfovibrio vulgaris defenses against oxidative and nitrosative stresses

Dissertation presented to obtain the Ph.D degree in Biochemistry, Microbial BiochemistryThe work presented in this dissertation aimed to unravel the defense mechanisms of the anaerobic sulfate reducing bacterium Desulfovibrio (D.) vulgaris Hildenborough against oxidative and nitrosative stresses. Desulfovibrio spp. are usually found in anaerobic niches in soil, marine and fresh waters and sediments, but also in zones periodically exposed to oxygen. Ecologically, Desulfovibrio spp.(...

NITRIC OXIDE-RELEASING ALGINATES AS A DUAL-ACTION THERAPEUTIC FOR CYSTIC FIBROSIS

Cystic fibrosis (CF) is an inherited disease characterized by impaired mucociliary clearance of thick mucus and persistent bacterial infection in the lungs. Nitric oxide (NO), an endogenously produced radical with integral roles in the mammalian immune response to foreign pathogens, holds promise as a CF therapy. Inhalation of exogenous NO-releasing biopolymers represents an attractive therapeutic strategy as NO release can be sustained for hours, allowing for near continuous delivery. Alginate is particularly promising for this application due to its water solubility, mucus-altering ability, and biocompatibility. Herein, we evaluated the utility of NO-releasing alginates as a dual-action (i.e., mucolytic and antibacterial) CF therapeutic. Low (~5 kDa) and high (~300 kDa) molecular weight alginate were first modified with a series of small molecule alkyl amines via carbodiimide chemistry to store and release NO, with release kinetics dependent on the precursor amine structure. The liberated NO showed bactericidal activity against Pseudomonas aeruginosa and Staphylococcus aureus with pathogen eradication efficiency dependent on both molecular weight and NO-release kinetics. Toxicity against human respiratory epithelial cells proved negligible at NO-releasing alginate concentrations required to achieve biofilm eradication. The excessive production of thick mucus in CF leads to airway obstruction and provides a suitable environment for bacteria colonization. The impact of NO-releasing alginates on the viscoelasticity of human bronchial epithelial mucus was investigated using parallel plate rheology. The efficacy of the NO-releasing alginates was shown to be dependent on molecular weight, NO-release kinetics, and dose. Nitric oxide-releasing alginates also achieved greater reduction in mucus rheology than both NO-releasing chitosan, a biocompatible cationic polymer, and N-acetyl cysteine, a conventional mucolytic agent. Biofilm-forming pathogens, the major contributor to mortality in CF, are difficult to treat due to additional protection provided by both the biofilm exopolysaccharide matrix and mucus in the airways. The antibiofilm efficacy of NO-releasing alginates were evaluated against P. aeruginosa, Burkholderia cepacia complex, S. aureus, and methicillin-resistant S. aureus biofilms under CF-relevant conditions. The NO-releasing alginates were highly antibacterial against the four pathogens, with biocidal efficacy dependent on NO-release kinetics. Relative to tobramycin and vancomycin, the NO-releasing alginates proved to be more effective regardless of growth conditions.Doctor of Philosoph

Effects of zinc transporters on Cryptococcus gattii virulence

Zinc is an essential nutrient for all living organisms because it is a co-factor of several important proteins. Furthermore, zinc may play an essential role in the infectiousness of microorganisms. Previously, we determined that functional zinc metabolism is associated with Cryptococcus gattii virulence. Here, we characterized the ZIP zinc transporters in this human pathogen. Transcriptional profiling revealed that zinc levels regulated the expression of the ZIP1, ZIP2 and ZIP3 genes, although only the C. gattii zinc transporter Zip1 was required for yeast growth under zinc-limiting conditions. To associate zinc uptake defects with virulence, the most studied cryptococcal virulence factors (i.e., capsule, melanin and growth at 37 °C) were assessed in ZIP mutant strains; however, no differences were detected in these classical virulence-associated traits among the mutant and WT strains. Interestingly, higher levels of reactive oxygen species were detected in the zip1Δ and in the zip1Δ zip2Δ double mutants. In line with these phenotypic alterations, the zip1Δ zip2Δ double mutant displayed attenuated virulence in a murine model of cryptococcosis. Together, these results indicate that adequate zinc uptake is necessary for cryptococcal fitness and virulence

Elucidating Biological Mechanisms of Host Response to Nitric Oxide-Releasing Glucose Biosensors

Despite the utility of continuous glucose monitors (CGM) in improving health outcomes for type I diabetic persons, these implants have a limited duration due to the foreign body response (FBR). Though nitric oxide (NO)-releasing glucose biosensors have been demonstrated to reduce the FBR and extend implant lifetime, the biological mechanisms impacted by NO release to trigger these therapeutic outcomes have not been explored in detail. Herein, NO’s effects on phenotype and activity of biological systems were investigated to identify biomarkers and NO’s mechanisms of actions. As macrophages deplete enough local glucose from an implant site to create reductions in sensor accuracy, glucose consumption was analyzed as a function of both NO and inflammatory state. Using 2-NBDG uptake as a measure of glucose consumption, preliminary data demonstrated 500 µM SNAP causing increases in glucose consumption (~10% or ~60% increase in pro- or anti-inflammatory macrophages, respectively) and 5 µM SNAP resulting in a ~50% reduction in glucose consumption for pro-inflammatory macrophages and no change for the anti-inflammatory counterparts.Using a euglycemic porcine model, mechanisms and biomarkers affected by NO release were evaluated through protein and gene expression analyses on tissue explanted from an NO-releasing implantation site. With NO release, chemokine levels were found to be reduced at 7-days post-implantation and pro-inflammatory mediators were found to be broadly reduced by NO exposure. Genetic analysis supported previously reported decreases in leukocyte infiltration with NO and discovered variations in the leukocyte composition present at the implantation site. The activity of matrix metalloproteinases (MMPs) was investigated as a function of both inflammatory state and NO release. Reductions in MMP activity were observed at lower NO doses, showing the potential of NO release to reduce inflammation associated with increased MMP levels. Significant increases were observed in macrophages exposed to 0.02 mg/mL DPTA/NO at endotoxin levels ≥ 2.5 µg/mL. As meaningful differences were not observed at 0.2 mg/mL DPTA/NO or from 0.02-0.2 mg/mL DETA/NO, a slower releasing NO donor, these data imply that there is a narrow therapeutic window for NO reducing MMP activity which merits further investigation.Doctor of Philosoph

SYNTHESIS AND ANTICANCER ACTION OF NITRIC OXIDE-RELEASING LIPOSOMES

The implementation of nitric oxide (NO)-based therapeutics has been met with formidable challenges relating to NO’s gaseous, reactive nature and difficulties associated with controlled delivery. Although macromolecular vehicles have been developed for applications in NO release, a common limitation associated with these systems is exposure of the NO donor to the surrounding medium, resulting in unintended NO release. To overcome this issue, liposomes were investigated as new vehicles for NO delivery whereby the NO donor is encapsulated within the aqueous core, protected from the external solution by a lipid membrane. Liposomes with encapsulated N-diazeniumdiolate NO donors were first synthesized using a reverse-phase evaporation protocol. Encapsulation efficiencies for several molecular NO donors were in the range of 33–41%. Relative to the unencapsulated (free) NO donor, NO-release half-lives at pH 7.4 were up to 7-times greater upon encapsulation, yet the NO-releasing liposomes still exhibited their unique pH-sensitive release properties. The liposomes retained ~80% of the encapsulated NO concentrations after 3 months of storage at 4°C, indicating excellent stability. In order to determine if the liposomes held merit as therapeutic agents, cytotoxicity against human pancreatic cancer cells were performed that demonstrated the liposomal NO donors required less NO to kill versus the free NO donor (183 μM and 2.4 mM, respectively). The ability to tune NO-release kinetics of these liposomes was further studied. It was possible to vary the NO-release kinetics by altering the encapsulated NO donor molecule or the phospholipid composing the bilayer (independently or in combination). Phospholipid headgroup surface area was determined to be a main factor in controlling NO-release half-lives. As the surface area of the lipid headgroup was decreased from 0.660 nm2 to 0.420 nm2, a concomitant increase in NO-release half-life was also observed. The composition of the lipid bilayer is known to affect in vivo properties, so NO-release kinetics were also measured in serum and whole blood. Half-lives in serum were equivalent to those measured in buffer, while those measured in blood were ~60% faster. An investigation into the cytotoxicity of slow (t1/2 > 72 h) versus fast (t1/2 ~ 2.5 h) NO-releasing liposomes demonstrated how the biological consequences were dependent on the NO-release rate. Fast NO-releasing liposomes yielded consistently higher LD50 values (>230 μM NO), relative their slow-releasing counterparts (<230 μM NO), across 9 different cancer cell lines encompassing 3 different types of cancer (breast, colorectal, and pancreatic). The fast-release system was able to eradicate 50% of the cells much quicker (~36 h vs. 72 h for slow-release system). Flow cytometry studies suggest that this faster killing is due to a more rapid intracellular build-up of NO, which was observed for both the free and encapsulated NO donors. Western blotting revealed that both the slow and fast NO-release systems could induce apoptosis, albeit to different degrees.Doctor of Philosoph