Organophosphonates are characterized by a very stable carbon-phosphorus bond. Eshcherichia coli and many other strains of bacteria possess a multi-enzyme system called carbon-phosphorus (C-P) lyase that enables these organisms to cleave the C-P bond of organophosphonates when inorganic phosphate is scarce in the local environment. Genetic studies have demonstrated that C-P lyase is encoded by the fourteen-gene phn operon, phnCDEFGHIJKLMNOP. However, the mechanism for C-P bond cleavage is still unclear. We have expressed, purified, and characterized phnP from this operon. PhnP is a phosphodiesterase which will hydrolyze both bis-(p-nitrophenyl) phosphate and 2’:3’-cyclic nucleotides as substrates. In collaboration with Dr. Zongchao Jia and Katarenya Podzelinska (Queen’s Biochemistry), we have crystallized phnP and solved the crystal structure at a resolution of 1.3 Å. The structure displayed similarity to zinc-dependent metallo-β-lactamase family proteins. However, phnP displays unique structural features with two metal binding sites per monomer: the active site containing potentially two manganese ions, and a ‘structural’ site coordinating one zinc ion. Potential active site residues were identified and corresponding point mutations were generated by site-directed mutagenesis. Studies based on the importance of these residues and the knowledge from our high resolution structure will help elucidate the mechanism of phnP as well as its function in the C-P lyase pathway. Furthermore, we performed a broad range of ligand screening for phnH, another key member from the C-P lyase pathway, by ITC experiments, co-crystallization and high throughput ligand screening. However, all the trials for the identification of the true physiological substrate for phnH proved unsuccessful. Although the mechanism of C-P bond cleavage by C-P lyase still remains unclear, we synthesized a fluorescently labelled organophosphonate (FPn) and utilized it for probing the in vivo degradation of the C-P bond by wild type E. coli and various mutants. Analysis by TLC and mass spectrometry demonstrated the production of the expected alkane product. With this promising fluorescent probe, potential intermediates and substrates can be identified for individual C-P lyase enzymes. Taken together, our studies on the C-P lyase pathway will contribute to elucidate the still unknown mechanism of the cleavage of the stable C-P bond. / Thesis (Master, Chemistry) -- Queen's University, 2008-08-29 12:11:55.179

The nucleus is a highly compartmentalized organelle where specific cellular activities are confined to discrete domains. One such domain is the promyelocytic leukemia nuclear body (PML NB). PML NBs are protein-based structures that make numerous contacts with neighboring chromatin domains. To elucidate the function of PML NBs, research has been focused on identifying the protein complement of PML NBs. More than 60 proteins have been shown to localize to PML NBs, implicating the bodies in numerous cellular activities such as transcription regulation, apoptosis, tumor suppression, and the antiviral response. This approach has not yielded a general model for PML NB function. Instead I have chosen to focus on the chromatin contacts made with PML NBs. Using live-cell microscopy, my observations support the hypothesis that changes in chromatin topology affect the structural integrity of PML NBs. Moreover, I have developed a technique, called immunoTRAP, which allows for the extraction of chromatin specifically associated with PML NBs. Analysis of these chromatin associations reveal that specific genes associate with PML NBs and these associations are cell type specific. Therefore, PML NBs make specific contacts with neighboring chromatin domains and these contacts are integral to PML NB morphology. Thus making PML NBs a meeting place for a specific set of genomic loci.

The nucleus is a highly compartmentalized organelle where specific cellular activities are confined to discrete domains. One such domain is the promyelocytic leukemia nuclear body (PML NB). PML NBs are protein-based structures that make numerous contacts with neighboring chromatin domains. To elucidate the function of PML NBs, research has been focused on identifying the protein complement of PML NBs. More than 60 proteins have been shown to localize to PML NBs, implicating the bodies in numerous cellular activities such as transcription regulation, apoptosis, tumor suppression, and the antiviral response. This approach has not yielded a general model for PML NB function. Instead I have chosen to focus on the chromatin contacts made with PML NBs. Using live-cell microscopy, my observations support the hypothesis that changes in chromatin topology affect the structural integrity of PML NBs. Moreover, I have developed a technique, called immunoTRAP, which allows for the extraction of chromatin specifically associated with PML NBs. Analysis of these chromatin associations reveal that specific genes associate with PML NBs and these associations are cell type specific. Therefore, PML NBs make specific contacts with neighboring chromatin domains and these contacts are integral to PML NB morphology. Thus making PML NBs a meeting place for a specific set of genomic loci.

No description available.

This dissertation is a study of the relationships between nitrogen (N) availability and spiraling (the paired processes of nutrient cycling and advective transport) in stream ecosystems. Anthropogenic activities have greatly increased rates of N loading to aquatic ecosystems. However, streams may be important sites for retention, removal, and transformation of N. In order to identify controls on NO3-N spiraling in anthropogenically impacted streams, I examined relationships among NO3-N spiraling and a suite of chemical, physical, and biological variables in streams spanning a gradient of N concentration. Across all streams, gross primary production (GPP) accounted for most NO3-N demand. Uptake of NO3-N was also related to GPP but was limited by N availability when N concentrations were low. A combination of GPP and NO3-N explained 80% of the variance in uptake. In chapter 3, I conducted a series of short-term nutrient releases in which streamwater NO3-N concentration was incrementally elevated to identify conditions leading to saturation of uptake capacity. Four of six study streams showed signs of N limitation whereas there was no significant change in uptake with increasing NO3-N amendment in two streams, suggesting N saturation. Proximity to saturation was generally correlated to N concentration but was also predicted by the ratio of N:P. My results suggest complex relationships between N spiraling and availability that depend on resident biota and other limiting factors. In chapter 4, I examined nutrient spiraling methodology by comparing differences between ambient and amendment-derived NO3-N spiraling metrics. I quantified spiraling metrics during a short-term NO3-N amendment and under ambient conditions using a stable isotope (15NO3-N) tracer. Uptake lengths measured during amendments were consistently longer than ambient uptake lengths. Amendment-derived NO3-N uptake velocity and uptake were underestimated relative to ambient conditions. Using a technique to estimate ambient uptake length extrapolated from the relationship between uptake length and nutrient amendment concentration for a series of amendments at different concentrations, I found that extrapolated uptake lengths were generally better predictors of ambient uptake lengths than amendment-derived uptake lengths but the technique was less effective in high N streams that showed signs of weak N limitation. / Ph. D.

The pentameric ligand gated ion channel super family plays a central role in fast synaptic communication between neurons and at the neuromuscular junction. Extensive studies on the prototypic pLGIC, the Torpedo nicotinic acetylcholine receptor (nAChR) have revealed an exquisite lipid sensitivity, with the nAChR adopting a novel uncoupled conformation in membranes lacking activating anionic and neutral lipids. The lipid-exposed transmembrane alpha-helix, M4, in each homologous subunit likely acts as a lipid sensor. One model proposes that activating lipids promote M4 “binding” to the adjacent alpha-helices, M1 and M3, to enhance interactions between the M4 C-terminus and the Cys-loop of the agonist-binding domain, with such interactions promoting coupling between the agonist site and channel gate. The first part of my thesis indirectly tests this hypothesis by exploring the effects of membrane hydrophobic thickness on nAChR function. Specifically, I tested the hypothesis that thicker membranes, which should promote alignment of M4 parallel to M1/M3 and thus helix-helix interactions, favor a coupled conformation. Although I found that the nAChR is uncoupled in all membranes tested, regardless of hydrophobic thickness, thicker membranes promote transitions from uncoupled to ultimately the desensitized state over the minutes to hours time frame. In contrast to anionic lipids, which influence function primarily via a conformational selection mechanism, membrane hydrophobic thickness influences function via a kinetic mechanism - thick membranes lower the activation energy between uncoupled and coupled conformations to promote conformational transitions. In the second part of my thesis, I used the two prokaryotic homologs, GLIC and ELIC, to explore how amino acid interactions at the interface between M4 and M1/M3 influence channel activity. Alanine scanning mutagenesis of this interface shows that disruption of almost any interaction in GLIC leads to a loss of folding and/or function, while analogous mutations in ELIC typically lead to no change or produce gains in function. Sequence comparisons with other members of the pLGIC superfamily suggest that the transmembrane domains of GLIC and ELIC represent two distinct archetypes. Each archetype may strike a different balance between the need for strong M4 binding to M1/M3 to promote folding and pentamer assembly, and the need for weaker interactions that allow for greater conformational flexibility during function.

We investigated the influence of multiple disturbances on ecosystem structure and function in a headwater stream adjacent to an abandoned arsenic mine using an upstream (reference) and downstream (mine-influenced) comparative reach approach. In this study, floods were addressed as a pulse disturbance, and the abandoned arsenic mine was characterized as a press disturbance. Chronically elevated levels of arsenic were specifically addressed as a ramp disturbance. Stream ecosystem structure and biogeochemical functioning were characterized monthly over a period from July to December 2004 by determining benthic organic matter standing stocks, ecosystem metabolism, and by using solute additions to examine differences in phosphorus uptake and hydrology over the monitoring period. Influences of the press disturbance were evident in the mine-influenced reach where arsenic concentrations (254 ± 39 µg/L) were >30 higher than in the reference reach (8 ± 1 µg/L). However, in almost all cases the presence of the abandoned arsenic mine appeared to exert little influence on reach-scale measures of ecosystem structure and function (e.g., organic matter standing crops, phosphorus uptake). Conversely, floods (i.e., pulse disturbances) influenced organic matter standing stocks and hydrologic interactions between the stream and transient storage zones in both study reaches. Interactions between press and pulse disturbances were evident in several cases and illustrated by phosphorus uptake responses. Phosphorus uptake was best predicted by coarse particulate organic matter standing stocks in the reference reach. However, in the reach exposed to the press disturbance (i.e., mine-influenced reach), both coarse particulate organic matter standing stocks and characteristics of the pulse disturbance regime (i.e., number of days post-flood) were significant predictors of phosphorus uptake. Within the mine-influenced reach, arsenic concentrations increased from 16–600 µg/L and were addressed as a ramp disturbance. Analysis of phosphorus uptake in the mine-influenced reach across a gradient of arsenic concentrations correlated with Michaelis-Menton models of enzyme kinetics in the presence of a competitive inhibitor. These results suggest that arsenic appears to competitively inhibit phosphorus uptake by microbial assemblages in the mine-influenced reach. Results from this study highlight the fact that ecotoxilogical studies at the ecosystem scale should consider not only contaminant influences, but rather place its implications within the extant disturbance regime generated from both natural and anthropogenic sources. / Master of Science

Os receptores acoplados à proteína G (GPCRs) são proteínas integrais de membrana caracterizados por possuírem sete alfa-hélices transmembranares. Esses receptores são importantes alvos de estudos biomédicos e aproximadamente 40% dos medicamentos atualmente comercializados agem sobre estes receptores. O receptor de Angiotensina II do tipo 1 (AT1) é um GPCR e o principal mediador do Sistema Renina-Angiotensina que tem como principal efetor o octopeptídeo Angiotensina II (AngII). Recentemente foi descrito que as mutações A244S e I103T-A244S no receptor AT1 podem estar relacionadas com a predisposição à fibrilação atrial. Neste trabalho foi realizada a construção, caracterização bioquímica e farmacológica destes mutantes, bem como do mutante I103T, com o objetivo de compreender como a funcionalidade desses receptores mutantes poderiam contribuir para a predisposição à fibrilação atrial. Os mutantes I103T e I103T-A244S revelaram ser mais eficientes e potentes que o receptor selvagem em aumentar os níveis de cálcio intracelular em resposta à AngII. Todos os mutantes estudados apresentaram baixa eficiência quanto à ativação da via das MAPKs e apresentaram comportamento diferente do receptor selvagem quando bloqueados com o antagonista Losartan, seletivo para o receptor AT1 e muito usado na clínica como medicamento anti-hipertensivo. Esses dados ressaltam a relevância do estudo tanto em termos de melhor compreender as bases moleculares da relação entre as mutações e a doença, bem como possível prevenção ao uso de medicamentos que possam interagir e agir diferentemente em receptores com essas mutações. / G-protein coupled receptors (GPCRs) are integral membrane proteins characterized by having seven transmembrane alpha-helices. These receptors are important targets of biomedical studies and approximately 40% of currently marketed drugs act on such receptors. The angiotensin II type 1 receptor (AT1) is a GPCR and the main mediator of the Renin-Angiotensin System whose main effector is the octapeptide Angiotensin II (Ang II). It was recently described that I103T and A244S mutations in the AT1 receptor may be related to the susceptibility to atrial fibrillation. In this study we carried out the construction of these mutants and their biochemical and functional characterization. The I103T and I103T/A244S mutants were shown to be more efficient and potent than the wild-type receptor on the increase of intracellular calcium levels. All mutants showed lower efficcacy for MAPK pathway activation and showed different behavior when compared to the wild-type receptor after antagonism with Losartan. These data highlight the relevance of the present study concerning a better understanding of the molecular basis of cardiovascular diseases and showing that conventional therapies for certain diseases may lead to adverse effects on patients carrying point mutations on the receptor sequence.

The biological function of RNA is often linked to an ability to adopt one or more mutually exclusive conformational states or isomers, a characteristic that distinguishes this biomolecule from proteins. Two examples of conformationally inconverting RNAs were structurally investigated. The first is found in the 3' untranslated region (UTR) of the coronavirus mouse hepatitis virus (MHV). A proposed molecular switch between mutually exclusive stable stem loop and pseudoknot conformations was investigated using thermal unfolding methods, NMR spectroscopy, sedimentation velocity ultracentrifugation and fluorescence resonance energy transfer (FRET) spectroscopy. Utilizing a "divide and conquer" approach we establish that the independent subdomains are folded as predicted by the proposed model and that a pseudoknotted conformation is accessible. Using the subdomains as spectral markers for the investigation of the intact 3' UTR RNA, we show that the 3' UTR is indeed a superposition of a double stem conformation and a pseudoknotted conformation in the presence of KCl and MgCl2. In the absence of added salt however, the 3' UTR adopts exclusively the double stem conformation. Analysis of the pseudoknotted stem reveals only a marginally stable folded state (deltaG25 = 0.5 kcal mol-1, tm = 31 oC) which makes it likely that a viral or host encoded protein(s) is required to stabilize the pseudoknotted conformation. A second conformationally interconverting RNA system investigated is an RNA element that stimulates -1 programmed ribosomal frameshifting in the human Ma3 gene. Structural analysis of the frameshifting element reveals a dynamic equilibrium between a functionally inactive double stem loop conformation and the active pseudoknotted conformation. Thermal melting and NMR spectroscopy reveal that the double stem loop is the predominant conformation in the absence of added KCl or MgCl2. The addition of KCl and MgCl2 results in the formation of a pseudoknot conformation. This conformation is dominant in solution only when the competing double stem loop conformation is abrogated by mutation. Functional studies of the Ma3 pseudoknot reveal that abrogation of double stem conformation increases frameshift stimulation by 2-fold and indicates that the pseudoknot is the active conformation.

Abstract Both qualitative and quantitative limitations of two-dimensional (2DE) imaging have lead to the emergence of the three-dimensional echo (3DE) technique. Until recently the development of 3DE technology has been slow due to inferior image quality and longer processing and acquisition times compared with 2DE. However, in recent times 3DE technology has advanced to “real-time” (RT3D) and has become feasible as a standard clinical tool. The overall hypothesis of the studies undertaken in this thesis is that the increased accuracy of left ventricular (LV) assessment using RT3D justifies its use in clinical decision-making based on both single and sequential measurements in routine echocardiographic practice. It shows the increased accuracy and feasibility of 3DE using online and offline techniques and opacification, and evaluates the prognostic implications of LV measurements globally and regionally using 3DE compared with 2DE. Further, it assesses the accuracy of LV assessment using 3DE and demonstrates its superior accuracy to 2DE in cross-sectional studies. The thesis initially reviews the use of physiologic parameters used for clinical decision making and looks at the principles of incremental value in diagnostic testing. The assessment of ventricular structure and function is discussed as well as the current problems with clinical echocardiography, which have lead to the evolution and development of 3DE. The second chapter describes the methodologies used in this thesis, with particular emphasis on new technologies used in the studies. Chapter 3 looks at the role of experience as a barrier to incorporation of 3DE into standard practice. We tested attendees before and after an intensive interactive training course to evaluate the learning curve. An interactive teaching course with rehearsal and direct mentoring appears to overcome this limitation and may improve the uptake of the 3DE technique. Chapters 4 to 6 validate the use of 3DE by comparing it to 2DE and magnetic resonance imaging (MRI) with respect to online and offline techniques, the use of left ventricular opacification (LVO) and the serial follow-up of patients. The availability of automated on-line software may increase the feasibility of real-time 3DE for LV volume calculation in clinical practice. Chapter 4 looks at the comparison of off- and on-line approaches with magnetic resonance imaging. Online measurement of LV volumes was found to be feasible and more accurate than with 2DE, however the offline approach was more accurate - although more time-consuming. Chapter 5 involves two studies which sought to validate 3DE in a multicentre setting. They examine the variability of LV measurements across multiple sites and to validate 3DE against MRI. The first study found that the semi-automated measurement of 3DE reduces the variation of LV volume between centers. The second study provides information on the role of different potential sources of error and provides guidelines for future users on how to minimize these errors as well as how to interpret their findings. The next chapter further validates 3DE with use of LVO as a technique to improve the accuracy of LV volume measurements. We sought to examine the accuracy of non-contrast and contrast enhanced 2DE and 3DE for calculation of LV volumes and ejection fraction (EF), relative to cardiac magnetic resonance imaging. 2DE with LVO was analogous to 3DE in accurate categorization of LV function. However, 3DE with LVO was feasible and superior to other non-contrast and contrast enhanced techniques in patients with previous infarction. Chapter 7 examines the technical details of regional volume measurements. The use of a fixed external frame of reference in analyses of regional wall motion in the apical four-chamber view is prone to a systematic error and the use of a floating-axis analysis corrects for this, but this is based on landmarks by MRI (apex, annulus) and center of mass by 3DE. The axis is likely to be influenced by reverse remodeling after intervention and therefore we sought to evaluate 3DE regional volume assessment vs. cardiac MRI over follow-up. The use of a center-line based on center of mass with RT3D may contribute to problems with use of this modality to track regional volumes over time. Although global 3DE volumes compare well with MRI volumes, new developments in image quality and automated software will be needed before changes in regional volumes can be reliably followed with 3DE. 2DE ESE is limited by sampling in standard imaging planes and suboptimal acquisition may cause poor matching of pre- and post-stress images. 3DE may avoid these orientation problems and contraction front mapping (CFM) may also provide a method for defining the temporal homogeneity of contraction. Chapter 8 examines the incremental benefit of 3DE CFM to 2DE and 3DE, using coronary angiography as the reference standard. It found that analysis of the temporal distribution of contraction may be an alternate means than 3DE wall motion assessment for identification of ischemia for ESE. Another significant validation needed for 3DE is not only for the LV but for the right ventricle (RV). The non-geometric nature of the RV makes it difficult to measure. Chapter 9 shows that 3DE is superior to 2DE for follow-up of RV function by validation vs. cardiac MRI. It was found that 3DE was indeed more accurate than 2DE approaches and reduces test-retest variation of RV volumes and EF measurements in follow-up RV assessment. Chapter 10 investigates the accuracy and reproducibility of the use of 3DE for serial follow-up of LV measurements. Echocardiographic follow-up of LV volumes is difficult because of the test-retest variation of 2DE. In this follow-up study, sequential 3DE measurements of change of volumes and EF were similar to those obtained using MRI, but 2DE overestimated change in EDV. As MRI is expensive, of limited availability and cannot be performed on patients with implanted devices, 3DE appears to be an effective long-term follow-up imaging tool for when sequential measurement of LV volumes is sought to guide management decisions. Chapter 11 investigates whether 3DE is more predictive of outcome than 2DE. In this outcome study, 3DE measurements of ESV and EF showed a stronger prediction of outcomes than 2DE. 3DE now appears to be the measurement of choice for when LV volumes and EF are sought to guide management decisions. In conclusion, the work reported in this thesis demonstrates that 3DE increases the accuracy of LV assessment and justifies its use in clinical decision-making based on both single and sequential measurements in routine echocardiographic practice.