Ocr is unusual among proteins in its ability to form a transparent gel at high ammonium sulphate concentrations. This transition was investigated using a combination of spectroscopic, microscopic and rheological techniques. It occurs sharply at a concentration of 3.2M ammonium sulphate and is not observed with other types of salt. Rheological measurements showed that rather than precipitating under such conditions, ocr forms a weak viscoelastic gel. Far UV circular dichroism spectra reveal that ocr does not denature in the gel phase, while near UV CD spectra suggest the formation of long, helical structures. Well resolved fibrils were observed using atomic force microscopy. They were over 1µm in length and varied between 2.6nm to 10.4nm in height, corresponding to the thickness and length of the ocr dimer. Ocr is a highly charged protein (-56e at pH 8) and is shaped like a banana. We argue that it is stabilized in specifically aggregated structures at large salt concentrations by these physical properties. Electrostatic repulsions between proteins are screened by salts, allowing proteins to approach close enough to aggregate. The charge on ocr is high enough to resist such precipitation. However, at 3.2M ammonium sulphate we suggest that the salt molecules bridge neighbouring ocr dimers via hydrogen bonds, connecting amino acid carboxyl groups with the ammonium groups of the salt. The banana-shaped dimers stack on top of each other, forming long helical fibrils that intertwine into a semi flexible network.

<p>Bacterial colonization of the upper respiratory tract is the first step in the pathogenesis of nontypeable Haemophilus influenzae (NTHi) disease. Examination of the determinants of NTHi colonization process has been hampered by the lack of an appropriate animal model. To address this, we have developed a model of NTHi colonization in adult rhesus macaques that involves intranasal inoculation of 1x105 CFU and results in persistent colonization of the upper respiratory tract for at least three weeks with no signs of disease, mimicking asymptomatic colonization of humans. Using this model, we assessed the contributions to colonization of the HMW1 and HMW2 adhesive proteins. In competition experiments, the parent strain expressing both HMW1 and HMW2 was able to efficiently out-compete an isogenic mutant strain expressing neither HMW1 nor HMW2. In experiments involving inoculation of single isogenic derivatives of NTHi strain 12, the strains expressing HMW1 or HMW2 or both were able to colonize efficiently, while the strain expressing neither HMW1 nor HMW2 colonized inefficiently. Furthermore, colonization resulted in antibody production against HMW1 and HMW2 in one-third of the animals, demonstrating that colonization can be an immunizing event. In conclusion, we have established that NTHi is capable of colonizing the upper respiratory tract of rhesus macaques, in some cases associated with stimulation of an immune response. The HMW1 and HMW2 adhesive proteins play a major role in the process of colonization.</p><p>After establishing that the HMW1 and HMW2 proteins are colonization factors we further investigated the determinants of HMW1 function. HMW1 is encoded in the same genetic locus as two other proteins, HMW1B and HMW1C, with which HMW1 must interact in order to be functional. Interaction with HMW1C in the cytoplasm results in the glycosylation of HMW1. By employing homologues of HMW1C that glycosylate HMW1 in slightly different patterns we show that the pattern of modification is critical to HMW1 function. Structural analysis showed a change in protein structure when the pattern of HMW1 modification differed. We also identified two specific sites which must be glycosylated for HMW1 to function properly. These point mutations did not have a significant effect on protein structure, suggesting that glycosylation at those specific sites is instead necessary for interaction of HMW1 with its receptor. HMW1B is an outer membrane pore through which HMW1 is transported to reach the bacterial cell surface. We observed that HMW1 isolated from the cytoplasm has a different structure than HMW1 isolated from the bacterial cell surface. By forcing HMW1 to be secreted in a non-HMW1B dependent manner, we show that secretion alone is not sufficient for HMW1 to obtain a functional structure. This leads us to hypothesize that there is something specific in the interaction between HMW1 and HMW1B that aids in proper HMW1 folding.</p><p>The NTHi HMW1C glycosyltransferase mediates unconventional N-linked glycosylation of HMW1. In this system, HMW1 is modified in the cytoplasm by sequential transfer of hexose residues. To determine if this mechanism of N-linked glycosylation is employed by species other than NTHi, we examined Kingella kingae and Aggregatibacter aphrophilus homologues of HMW1C. We found both homologues to be functional glycosyltransferases and identified their substrates as the K. kingae Knh and the A. aphrophilus EmaA trimeric autotransporter proteins. LC-MS/MS analysis revealed multiple sites of N-linked glycosylation on Knh and EmaA. Without glycosylation, Knh and EmaA failed to facilitate wild type levels of bacterial autoaggregation or adherence to human epithelial cells, establishing that glycosylation is essential for proper protein function.</p> / Dissertation

Hydrothermal vent communities exhibit great taxonomic novelty with 88% of species, 51% of genera, and 21% of families new to science. Given the severe physiological barriers to invasion presented by hydrothermalism and the energetic independence of the community due to in situ primary production by chemoautotrophic bacteria, it has been previously proposed that hydrothermal vents may have acted as a refugia for groups of metazoan animals that originated during the Cambrian, Paleozoic, or Mesozoic. The alternate explanation is of rapid change of recent immigrants from the adjacent deep-sea and false taxonomic inflation. Six major groups of hydrothermal vent endemic gastropods exhibit high taxonomic novelty and a lack of known fossils. Discovery of these hydrothermal vent endemic groups has resulted in dramatic changes in how we view the evolution and phylogeny of the Gastropoda, particularly in regards to the novel anatomy of the Neomphalina (Neomphalidae + Peltospiridae). Recent cladistic examinations of gastropod phylogeny using anatomical and morphological characters disagree on the placement and monophyly of the Neomphalina or find few characters supporting their position in the overall gastropod phylogeny. In this dissertation, a molecular systematic investigation of gastropod phylogeny was performed to examine the antiquity of the vent endemic Neomphalina. Twenty-three new D1 domain and thirty new D6 domain DNA sequences of the 28S ribosomal RNA gene were obtained from fresh-frozen and formalin-ethanol preserved specimens. These were combined with previously published molluscan 28S ribosomal RNA sequences for a total of 159 sequences. Gastropod phylogeny was examined using both parsimony and distance-based analyses. The 28S ribosomal RNA gene exhibited saturation of substitutions beyond 15% divergence between sequences, estimated using Kimura’s two-parameter model. Alone, either domain exhibited poor resolution of gastropod phylogeny but together (32 genera only) monophyly of the Neritimorpha, Neomphalina, Vetigastropoda, Patellogastropoda, Caenogastropoda (including Viviparus, Ampullaria, and Campanile), and Heterobranchia (Euthyneura plus Valvata) was supported by bootstrap values. Relationships among these groups could not be resolved due to saturation of substitutions. Evidence of elevated evolutionary rates in the Patellogastropoda conformed to previous studies and confounded analyses. Regardless, the hydrothermal vent Neomphalina exhibited divergence values and phylogenetic novelty equivalent to the other early-Paleozoic radiations, supporting its consideration as a vent refugial phylogenetic relic. 28S ribosomal RNA sequences cannot resolve Cambrian or early Paleozoic radiations of the Gastropoda and use of diverse specimens limits reliability of sub-ordinal relationships due to long-branch attraction. Sequences of 28S ribosomal RNA are best used to examine within-order gastropod relationships due to saturation of substitutions at higher levels and among-order evolutionary rate variation. / Graduate

<p>Foam cell formation from arterial intima macrophages and defective clearance of apoptotic foam cells drive the progression of the inflammatory disease atherosclerosis. The role of innate immune protein C1q in autoimmune disease and pathogen defense is well characterized, however its role in atherosclerosis remains largely uninvestigated. Prior studies have characterized the complement independent role of C1q in polarizing macrophages towards an anti-inflammatory phenotype during uptake of apoptotic cells and modified lipoproteins. To further understand the role of C1q in programming human monocyte-derived macrophages during foam cell formation, we used RNA-sequencing to elucidate pathways that are modulated by C1q during clearance of atherogenic lipoproteins. Expression of genes in JAK-STAT, PPAR, apoptotic, and TLR signaling pathways were modulated by C1q in this study. In addition, C1q suppressed STAT1 and PPAR transcriptional activity. This study identifies potential molecular mechanisms that support a beneficial role for C1q in early atherosclerosis. </p>

<p> The struggle for survival between phages and their bacterial hosts is best exemplified by the diverse mechanisms bacteria utilize to block phage infection, and the methods phages use to surmount them. Mycobacteriophage DS6A is unique amongst the more than 8000 identified mycobacteriophages in that its host range is restricted to mycobacteria that are members of the <i><u> My</u>cobacterium <u>t</u>u<u>b</u>erculosis </i> <u>C</u>omplex <i>(MTBC)</i>. However, the molecular mechanism for this specificity remains unknown. To study the relationship DS6A has to both MTBC and non-tuberculous mycobacteria (NTMs), we generated two novel recombinant DS6A shuttle phasmids containing fluorescent reporter mVenus. These phages were utilized to clearly demonstrate that 50 years of previous scientific dogma was incorrect, and DS6A can in fact infect NTM mycobacteria to a wide range of degrees. Work teasing out the mechanisms of resistance is underway. Additionally, we identified the chaperone trigger factor (Tig; Rv2462c) to be required for a productive DS6A infection in <i> Mtb</i>. Interestingly, no host range mutants have been generated that can overcome this resistance to plaguing. Deleting <i>tig</i> did not affect the lysis profile of any of the other >30 mycobacteriophages able to infect <i>Mtb</i>. Susceptibility of <i>Mtb</i> &Delta;<i> tig</i> to DS6A infection was rescued by complementation of tig on an integrating plasmid. DS6A fluorophage infection of <i>Mtb</i> &Delta;<i> tig</i> induced high levels of detectable fluorescence, suggesting Tig is not required for the adsorption of phage or introduction of DS6A DNA. Additionally assays determined that Tig was not involved in transcriptional or translational activation. However, deleting <i>tig</i> did yield an additional phenotype in <i>Mtb</i>; significant attenuation in both immunocompetent and immunocompromised mice. Lipidome and secretome assays showed stark differences between the lipid makeup and secreted protein profiles of the tig mutant versus the WT that could explain the cause of attenuation. For Mtb, the loss of <i> tig</i> is a double edged sword; total resistance to DS6A is afforded in the clonal population, but at the cost of virulence in eukaryotic hosts. </p>

<p>A common complication among sickle cell disease (SCD) patients is the development of renal disease. Paradoxically, the incidence of chronic kidney disease (CKD) increases as patient survival improves, and as such the development of sickle cell disease nephropathy (SCDN) has become an emergent health concern in SCD. For individuals with sickle cell anemia (SCA), albuminuria rates are as high as 68% in adult patients and as many as 18% of these patients progress to end-stage renal disease (ESRD). The detection of SCDN relies on relatively late markers of the disease process, namely proteinuria and reduced glomerular filtration rate (GFR), delaying identification of at-risk SCD patients prior to organ damage. Thus, early detection of those at risk is required to reduce morbidity and mortality among SCD patients. </p><p>In order to accomplish this, we have used a tiered approach employing genetic association strategy in patient populations and functional examination in relevant zebrafish model systems. We demonstrated previously that MYH9 and APOL1, in linkage disequilibrium on chromosome 22, are strong, independent predictors of risk for proteinuria in SCD. This region, particularly two major risk variants (named G1 and G2) in APOL1, has been replicated widely in non-SCD nephropathy and represents one of the strongest genetic signals for a complex human phenotype. Using the zebrafish system, we discovered a functional role for APOL1 in the developing zebrafish kidney and uncovered a complex genetic architecture, in which the G2 allele exerts adverse functions on the kidney and kidney cell types. Critically, we also found that APOL1 and MYH9 interact genetically, particularly in the context of anemic stress, which we also observed in a SCD patient population. </p><p>However, variants at the MYH9/APOL1 locus appear to only explain a part of the disease risk, suggesting that additional genetic factors may be contributing to renal outcome in SCD patients. As such, we performed an unbiased interrogation of the genome (GWAS) in order to uncover putative new nephropathy genes for genetic evaluation. Using a host of genetic methods to identify both common and rare variation present in SCDN individuals, we identified seven candidate loci associated with renal outcome in SCD. Again, using zebrafish models, we provide relevant functional evidence for a subset of these genetic candidates by assessing their effect on glomerular filtration barrier integrity. </p><p>Collectively, these genes and markers may indicate novel genetic mechanisms contributing to SCD nephropathy, and may further diagnostic paradigms for identifying those patients most at risk. In addition, these results stand to make significant progress in identifying novel therapeutics for SCDN.</p> / Dissertation

The thesis compares the effects of temperature and substrate on the growth of fish pathogen Saprolegnia diclina and the saprophyte Saprolegnia ferax. Studies revealed optimal growth of both species occurs at 20-25'C depending upon the substrates used. Growth is restricted at higher and lower temperatures. For both species optimum growth was recorded on medium containing 1% casein and 1% glucose, but high levels of lipid and glucose inhibited growth. However, S. _diclina exhibits a higher growth rate than S. ferax at high lipid concentrations. S. diclina produces more proteolytic activity per unit weight at 100C than S. ferax, regardless of substrate. S. diclina also demonstrated greatest lipase activity at 10°C, particularly in the presence of casein or lipid. These factors may facilitate colonisation of fish tissues by S. diclina at low temperatures. Carp maintained at 10° C showed greater infection by S. diclina, than carp kept at 20°C. Evidence is presented for a lack of antibody production in infected carp maintained at 10°C. Fish kept at 20°C only produced antibody to Saprolegnia antigens when they were coupled to erythrocyte carriers. During infection phagocytic, macrophages and neutrophils increased; there was a decrease in the numbers of mucous secreting goblet cells in the skin, and lymphoid organs showed increased pigment deposition. Infected fish showed evidence of physiological stress including decreased levels of erythrocytes, haemoglobin, liver glycogen and protein, and an increase in liver lipid. Ascorbic acid levels decreased in interrenal tissue. Histological and scanning E. M. studies of skin lesions provide new information about changes in the surface during UDN disease. Key words: Saprolggnia infection and UDN Temperature, substrates

No description available.

No description available.

The effect of processing on the antioxidant activity of sulphur-containing compounds, with particular reference to nickel dialkyldithiophosphates and their corresponding di sulphides, were studied in polyolefins under melt, thermal and photo-oxidative conditions. These compounds were evaluated both at low (normal) and high (concentrates) concentrations. In general, the dithiophosphates were found to be very efficient melt stabilisers at normal concentrtion levels, and compare quite favourably with the best commercially available systems. The nickel dithiophosphates were also found to be very efficient thermal stabilisers for polyolefins, but their activity is highly dependent on the alkyl substituent in the molecule. The corresponding disulphides on the other hand showed very little activity under thermal oxidative conditions, and this was attributed to their inefficiency in scavenging alkyl peroxyl radicals since both compounds possess similar peroxidolytic activity. Furthermore, the nickel dithiophosphates were found to be excellent photo stabilisers for mildly-processed polyolefins while the corresponding disulphides only offer slight protection to the polymer. Oxidative processing of the disulphide, however, results in a dramatic improvement in their photo antioxidant activity. Thionophospho-ric acid, a major oxidation product of dithiophosphates, was also shown to have photo antioxidant activity similar to that of the disulphides. A combination of a U.V. absorber with the nickel complex and/or the disulphide resulted in a synergistic stabiliser system which was further augmented by oxidative processing. Moreover, the dilute analogues of such multicomponent stabiliser concentrates also showed excellent melt, thermal and photo-stabilising activity. The mechanistic studies carried out on the nickel complex and the corresponding disulphide clearly identified the thionophosphoric acid a a major transformation product although various triesters were formed as reaction intermediates. The mechanisms of the antioxidant action of the dithiophosphates, which is believed to involve a cyclical process similar to that shown for simple alkyl sulphides and nitroxyls, are discussed.