The repercussions of climate change are felt worldwide, but Arctic and subarctic regions, where climate warming is expected to be amplified, are especially vulnerable. An episode of mass fish mortality in the Sutton River in the Hudson Bay Lowlands (HBL) of Northern Ontario has elicited the interest of the scientific community. Several lakes were sampled over three years in an effort to better understand and document the changes that may be occurring in these lakes. This study uses sediment cores to assess the history of mercury (Hg) deposition and to assess changes occurring in autochthonous productivity in these lakes. Sediments deposited after the onset of the industrial revolution contained significantly higher concentrations of Hg, with the highest concentrations found in the most recently deposited sediments. Hg concentrations in these pristine lakes rival those of lakes in heavily urbanized areas, indicating that they are in fact subjected to atmospheric deposition of Hg. There was a large variation in [Hg] of the surface sediments of 13 lakes; underscoring the importance of in situ processes in the fate of atmospherically deposited Hg. Methylmercury (MeHg) concentrations were not correlated with total mercury concentrations (THg), demonstrating how THg is a poor predictor of MeHg; the bioaccumulative neurotoxic form of mercury. The S2 fraction of Rock-Eval® Pyrolysis, C:N ratios and ∂13C signatures were used as proxies of autochthonous carbon and all indicated that the lakes have become increasingly productive, presumably due to warmer water temperatures and longer ice-free seasons. Additionally, I use molecular techniques to detect and quantify the merA gene in the sediment; a proxy of bacterial mercury resistance involved in redox transformations. In Aquatuk, Hawley and North Raft Lakes, I observed a subsurface increase in merA genes in the sediment core, independently of a control gene and the [THg]. While I have not been able to explain the driving variables of this subsurface increase, I believe that the role of merA within remote lake sediments deserves further work. Lastly, microcosms were used to measure the production of volatile elemental mercury (Hg(0)) from surface sediments of Aquatuk Lake. I used a combination of analytical and molecular techniques to show that the production of Hg(0) is biogenic and tested the effect of nutrients, pH and ionic strength on the Hg(0) production rates. Ionic strength alone had the greatest impact on Hg(0) production rates, with increased Hg(0) production as ionic strength increases.

Bartonella, a group of Gram negative facultative intracellular bacteria, are known to cause diseases, such as Cat Scratch Disease, Trench Fever and Carrion’s Disease, that involve angiogenesis during the infective cycle. B. bacilliformis, the etiological agent of Carrion’s Disease, causes a bi-phasic infection resulting in the formation of blood-filled angiogenic proliferative cutaneous nodules called verruga peruana. The work presented here was undertaken to characterize the mechanism by which these nodules are produced. Previous work in our laboratory suggested that the Bartonella henselae genome contains a homologue to the virB operon, a set of genes coding for a Type IV Secretion System (TFSS) that has been implicated in the pathogenesis of other α-2-proteobacteria. We identified virB operons in two additional Bartonella pathogens, B. quintana and B. clarridgeiae. No corresponding operon sequences were detected in B. bacilliformis DNA, however. This finding suggests that virB gene products are not required for verruga peruana formation. To continue our search for factors involved in B. bacilliformis-induced angiogenesis, we conducted a microarray analysis of differential gene expression in infected and uninfected endothelial cells. The results suggest similarities between later stage (36 hours) B. bacilliformis infection and that of HHV-8, the causative agent of Kaposi’s Sarcoma, particularly in relation to the host immune response. Finally, our research focused on the secreted factors that B. bacilliformis produces during its host infective cycle. Our data suggest that the B. bacilliformis homologue to the molecular chaperone GroEL not only induces angiogenesis in endothelial cells, but also protects endothelial cell tubule from the degradation seen when these cells are in the presence of live B. bacilliformis. In summary, the induction of verruga peruana nodules via B. bacilliformis may be the result of multiple factors over the course of persistent infection. Early infection may cause vascular damage, which induces VEGF and hypoxia factors. As infection persists, bacterial secretion of a unique GroEL may result in continued angiogenesis and the ensuing activation of immune cells, producing a localized environment of continual incomplete angiogenesis in areas of cutaneous infection.

Group A Streptococcus (GAS) is a common pathogen of the human skin and mucosal surfaces and is capable of producing a variety of diseases. This dissertation investigates the function of a metalloregulator named MtsR in GAS physiology and disease process. An mtsR mutant was constructed and analyzed. Consistent with MtsR role in iron uptake regulation, the mtsR mutant accumulates more iron (80 ± 22.5%) than the wild type strain. Inactivation of mtsR results in constitutive transcription of the sia (Streptococcal Iron Acquisition) operon, which is negatively regulated by iron in the parent strain. We identified the promoter that controls the expression of the sia operon (Pshr) and used it as a model to study MtsR interaction with DNA. Electrophoretic mobility gel shift assays (EMSAs) demonstrated that MtsR binds to the shr upstream region specifically and in an iron and manganese dependent manner. DNase I footprint analysis revealed that MtsR protects a 69 bp segment in Pshr that includes 2 inverted repeats, overlapping the core promoter elements. A global transcriptional analysis determined that MtsR modulates the expression of 64 genes, of which 44 were upregulated and 20 were downregulated in the mtsR mutant. MtsR controls genes with diverse functions including immune evasion, colonization, dissemination, metal homeostasis, nucleic acid and amino acid metabolism, and protein stability. MtsR functions as a dual regulator as it binds to the promoters of the repressed genes ska, aroE, and nrdF.2, as well as the upstream region of the positively regulated genes mga, emm49, and pyrF. A 16 bp MtsR-binding consensus region was identified in all of the promoters that are directly regulated by MtsR. In conclusion, we have demonstrated that MtsR is a global regulator in GAS that controls the expression of vital virulence factors and genes involved in metal transport, virulence and metabolic pathways.

Gene regulation mediated by transcription factors (TFs) is essential for all organisms. The functionality of TFs can largely be described by the fraction of time they occupy their regulatory binding sites on the chromosome. DNA-binding proteins have been shown to find their targets through facilitated diffusion in vitro. In its simplest form this means that the protein combines a random 3D search in the cytoplasm with 1D sliding along DNA. This has been proposed to speed up target location. It is difficult to mimic the in vivo conditions for gene regulation in biochemistry experiments; i.e. the ionic strength, chromosomal structure, and the presence of other DNA-binding macromolecules.    In this thesis single molecule imaging assays for live cell measurements were developed to study the kinetics of the Escherichia coli transcription factor LacI. The low copy number LacI, in fusion with a fluorescent protein (Venus) is detected as a localized near-diffraction limited spot when being DNA-bound for longer than the exposure time. An allosteric inducer is used to control binding and release. Using this method we can measure the time it takes for LacI to bind to different operator sequences. We then extend the assay and show that LacI slides in to and out from the operator site, and that it is obstructed by another DNA-binding protein positioned next to its target. We present a new model where LacI redundantly passes over the operator many times before binding.    By combining experiments with molecular dynamics simulations we can characterize the details of non-specific DNA-binding. In particular, we validate long-standing assumptions that the non-specific association is diffusion-controlled. In addition it is seen that the non-specifically bound protein diffuses along DNA in a helical path.    Using microfluidics we design a chase assay to measure in vivo dissociation rates for the LacI-Venus dimer. Based on the comparison of these rates with association rates and equilibrium binding data we suggest that there might be a short time following TF dissociation when transcription initiation is silenced. This implies that the fraction of time the operator is occupied is not enough to describe the regulatory range of the promoter.

No description available.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 133-156).

College level science is frequently taught as a recitation of facts in a lecture hall, and the students are expected to gain understanding and insight with their own study. Interactive learning is more effective than lecture based learning and more memorable for the students. Teaching with hands on models has been shown to specifically be beneficial in a college level molecular biology context. Included here is a guide for the instructor leading her through topic selection, activity development, and presentation to the class, as well as five complete and tested lesson plans with notes on alteration made and the reasons for them. / text

For pathogenic microorganisms, heme uptake and degradation is a critical mechanism for iron acquisition that enables multiplication and survival within hosts they invade. While the bacterial proteins involved in heme transport had been identified at the initiation of our investigation, the fate of heme once it reached the cytoplasm was largely uncharacterized. Here we report the first crystal structures of two members of the heme utilization operon from the human pathogen Escherichia coli O157:H7. These are the heme oxygenase ChuS in its apo and heme-complexed forms, and the apo form of heme binding protein ChuX. Surprisingly, despite minimal sequence similarity between the N- and C-terminal halves, the structure of ChuS is a structural repeat. Furthermore, the ChuS monomer forms a topology that is similar to the homodimeric structure of ChuX. Based on spectral analysis and carbon monoxide measurement by gas chromatography, we demonstrated that ChuS is a heme oxygenase, the first to be identified in any E. coli strain. We also show that ChuS coordinates heme in a unique fashion relative to other heme oxygenases, potentially contributing to its enhanced activity. As ChuS and ChuX share structural homology, we extended the structural insight gained in our analysis of ChuS to purport a hypothesis of heme binding for ChuX. Furthermore, we demonstrated that ChuX may serve to modulate cytoplasmic stores of heme by binding heme and transferring it to other hemoproteins such as ChuS. Based on sequence and structural comparisons, we designed a number of site-directed mutations in ChuS and ChuX to probe heme binding sites and mechanisms in each. ChuS and ChuX mutants were analyzed through reconstitution experiments with heme and functional analyses, including enzyme catalysis by ChuS and mutants, and in culture development during heme challenge experiments by ChuX and mutants. Taken together, our results suggested that ChuX acts upstream of ChuS, and regulates heme uptake through ChuX-mediated heme binding and release. ChuS can degrade heme as a potential iron source or antioxidant, thereby contributing directly to E. coli O157:H7 pathogenesis. Functional implications that may be revealed from sequence and structure based information will be addressed as they pertained to our evaluation of ChuS and ChuX. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2007-04-27 11:34:50.272

Borrelia burgdorferi s. l. is a spirochete which causes the multisystemic disorder Lyme disease. As the borreliae lack toxin production, the pathogenicity is thought to involve, at least in part, molecules from the outer surface. Most Lyme disease Borrelia strains express two major outer surface lipoproteins, OspA (31 kD) and OspB (34 kD), on their surface. However, some strains lack the expression of OspA and OspB, but express a smaller 21 to 25 kD OspC protein instead. This thesis focuses on the importance of these proteins in the pathogenesis of Lyme disease. Biochemical and immunochemical studies of the OspA and OspB proteins from strains of various geographic origins show considerable differences in the apparent molecular weights and in their reactivities to monoclonal antibodies. The cloning and sequencing of the ospAB opérons from strains of different origins has demonstrated that the heterogeneity is found also at the DNA level Comparison of the ospAB sequences allows the classification of the strains into three types, which coincide with the recent species designations, B. burgdorferi sensu stricto, B. afzelii and B. garinii The genes are located on a linear plasmid about 50 kb in size, and are cotranscribed as a single message. The expression of the osp operon in different strains was studied by Western blot and Northern blot analysis. The ospAB operon of strains expressing varying amounts of the Osp proteins was cloned and sequenced. The DNA sequence was found to be &gt;99% identical. The regulation appears to be primarily at the transcriptional level. In patients who have received incomplete treatment, B. burgdorferi have been isolated several years after the onset of the disease. As mentioned above, the ospAB loci of different strains show considerable heterogeneity, and it has been speculated that the spirochetes evade the host’s immune system by antigenic variation of the Osp proteins. In a mouse model system it was shown that no variation of the osp genes occurs over the course of an infection, and that other escape mechanisms must be used. The OspB proteins in particular have been shown to be very heterogeneous in different isolates. The MAb 84C recognizes a wide variety of B. burgdorferi strains, and the binding epitope was mapped to a conserved region in the carboxyl terminus of the OspB protein with putative structural and/or functional importance. It is well known that antibodies can kill bacteria in the presence of complement and phagocytes. Some antibodies seem to have a bactericidal effect by themselves. H6831 is a monoclonal antibody recognizing the OspB protein of some B. burgdorferi strains. The bactericidal action of univalent FAb fragments from H6831 was further characterized, and the binding epitope was mapped to a very heterogeneous region of the carboxyl end of the OspB protein. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1994, härtill 5 uppsatser</p> / digitalisering@umu

The repercussions of climate change are felt worldwide, but Arctic and subarctic regions, where climate warming is expected to be amplified, are especially vulnerable. An episode of mass fish mortality in the Sutton River in the Hudson Bay Lowlands (HBL) of Northern Ontario has elicited the interest of the scientific community. Several lakes were sampled over three years in an effort to better understand and document the changes that may be occurring in these lakes. This study uses sediment cores to assess the history of mercury (Hg) deposition and to assess changes occurring in autochthonous productivity in these lakes. Sediments deposited after the onset of the industrial revolution contained significantly higher concentrations of Hg, with the highest concentrations found in the most recently deposited sediments. Hg concentrations in these pristine lakes rival those of lakes in heavily urbanized areas, indicating that they are in fact subjected to atmospheric deposition of Hg. There was a large variation in [Hg] of the surface sediments of 13 lakes; underscoring the importance of in situ processes in the fate of atmospherically deposited Hg. Methylmercury (MeHg) concentrations were not correlated with total mercury concentrations (THg), demonstrating how THg is a poor predictor of MeHg; the bioaccumulative neurotoxic form of mercury. The S2 fraction of Rock-Eval® Pyrolysis, C:N ratios and ∂13C signatures were used as proxies of autochthonous carbon and all indicated that the lakes have become increasingly productive, presumably due to warmer water temperatures and longer ice-free seasons. Additionally, I use molecular techniques to detect and quantify the merA gene in the sediment; a proxy of bacterial mercury resistance involved in redox transformations. In Aquatuk, Hawley and North Raft Lakes, I observed a subsurface increase in merA genes in the sediment core, independently of a control gene and the [THg]. While I have not been able to explain the driving variables of this subsurface increase, I believe that the role of merA within remote lake sediments deserves further work. Lastly, microcosms were used to measure the production of volatile elemental mercury (Hg(0)) from surface sediments of Aquatuk Lake. I used a combination of analytical and molecular techniques to show that the production of Hg(0) is biogenic and tested the effect of nutrients, pH and ionic strength on the Hg(0) production rates. Ionic strength alone had the greatest impact on Hg(0) production rates, with increased Hg(0) production as ionic strength increases.