Investigation of molecular mechanisms of LKB1 function in Dictyostelium development and stress responses and the function of superoxide dismutase C (SodC) in chemotaxis

The serine/threonine kinase LKB1 is a regulator of critical events including development and stress responses in metazoans. The current study was undertaken to determine the function of LKB1 in Dictyostelium . During multicellular development and in response to stress insult, an apparent increase in the DdLKB1 kinase activity was observed. Depletion of DdLKB1 with a knockdown construct led to aberrant development; a severe reduction in prespore cell differentiation and a precocious induction of prestalk cells, which were reminiscent of cells lacking GSK3, a well known cell-fate switch. Furthermore, DdLKB1 depleted cells displayed lower GSK3 activity than wild type cells in response to cAMP stimulation during development and failed to activate AMPK, a well known LKB1 target in mammals, in response to cAMP and stress insults. These results suggest that DdLKB1 positively regulates both GSK3 and AMPK during Dictyostelium development, and DdLKB1 is necessary for AMPK activation during stress response regulation. No apparent GSK3 activation was observed in response to stress insults. Spatial and temporal regulation of phosphatidylinositol-(3,4,5)-triphosphate (PIP3) along the membrane of polarized cells is important for efficient chemotaxis. A REMI screen for PIP3 suppressors in the absence of stimulation led to the identification of SodC as PIP3 regulator. Consistent with their higher PIP3 levels, sodC− cells showed defects in chemotaxis and exhibited higher intra-cellular superoxide levels. Protein localization studies along with observations from GPI specific PI-PLC treatment of wild-type cells suggested that SodC is a GPI anchored outer-membrane protein. SodC showed superoxide dismutase activity in vitro, and motility defects of sodC− cells can be rescued by expressing the intact SodC but not by the mutant SodC, which has point mutations that affect its dismutase function. Treatment of sodC− cells with LY294002, a pharmacological inhibitor of PI3K, partially rescued the polarization and chemoattractant sensing defects but not motility defects. Consistent with increased intracellular superoxide levels, sodC − cells also exhibited higher basal Ras activity, an upstream regulator of PI3K, which can be suppressed by a cell permeable superoxide scavenger, XTT, indicating that SodC is important in regulation of intracellular superoxide levels thereby regulating the Ras activity and PIP3 levels at the membrane.

Studying Key Amino Acids in the Function of Ras Interference 1 (Rinl) SH2 and Vps9 Domains

Receptor mediated endocytosis effectively removes the "ears" with which a cell would "hear" a signal conveyed by extracellular signaling molecules, but does not necessarily block the signaling pathway in which the endocytosed receptor participates. In the process of signal attenuation, this newly formed vesicle is fused with a phagosome and the receptor molecules are degraded. Receptor mediated endocytosis as a way to attenuate epidermal growth factor (EGF) and insulin signaling will be the focus here. Ras Interference 1 (Rin 1) is a multifunctional protein involved in intracellular membrane trafficking and receptor mediated endocytosis through its Rab5 Guanine Exchange Factor and SH2 domains. The goal of this investigation is to determine the role of key amino acids involved in the interaction of Rinl with Epidermal Growth Factor Receptor and Rab5. To elucidate this role, a number of point mutations have been created and the effects of each mutation on Rin 1 function will be investigated. Key amino acids in the SH2 and Vps9 Domain were identified and effects of mutations on rate of endocytosis were observed.

Carbon (d13c) and Nitrogen (d15n) Isotopic Discrimination in Mangroves in Florida Coastal Everglades as a Function of Environmental Stress

Isotope signatures of mangrove leaves can vary depending on discrimination associated with plant response to environmental stressors defined by gra­dients of resources (such as water and nutrient limitation) and regulators (such as salinity and sul­fide toxicity). We tested the variability of man­grove isotopic signatures (d13C and d15N) across a stress gradient in south Florida, using green leaves from four mangrove species collected at six sites. Mangroves across the landscape studied are stressed by resource and regulator gradients repre­sented by limited phosphorus concentrations com­bined with high sulfide concentrations, respec­tively. Foliar d13C ratios exhibited a range from ­ 24.6 to –32.7‰, and multiple regression analysis showed that 46% of the variability in mangrove d13C composition could be explained by the differ­ences in dissolved inorganic nitrogen, soluble reac­tive phosphorus, and sulfide porewater concentra­tions. 15N discrimination in mangrove species ranged from –0.1 to 7.7‰, and porewater N, salin­ity, and leaf N:Pa ratios accounted for 41% of this variability in mangrove leaves. The increase in soil P availability reduced 15N discrimination due to higher N demand. Scrub mangroves (<1.5 m tall) are more water-use efficient, as indicated by higher d13C; and have greater nutrient use efficiency ratios of P than do tall mangroves (5 to 10 m tall) existing in sites with greater soil P concentrations. The high variability of mangrove d13C and d15N across these resource and regulator gradients could be a con­founding factor obscuring the linkages between mangrove wetlands and estuarine food webs. These results support the hypothesis that landscape fac­tors may control mangrove structure and function, so that nutrient biogeochemistry and mangrove-based food webs in adjacent estuaries should ac­count for watershed-specific organic inputs.

Perceptions of alibi evidence as a function of the strength of incriminating *evidence

The current study investigated the exculpatory value of alibi evidence when presented together with various types of incriminating evidence. Previous research has reported that alibi evidence could weaken the effects of DNA evidence and eyewitness identification. The present study assessed the effectiveness of alibi evidence in counteracting defendant's confession (experiment 1) and eyewitness evidence (experiment 2). In experiment 1, three levels of alibi evidence (none, weak, strong) were combined with three levels of confession evidence (voluntary, elicited under low pressure, elicited under high pressure). Results indicated significant main effects of confession and alibi and an alibi by confession interaction. Of participants exposed to high-pressure confession, those in the strong alibi condition rendered lower guilt estimates than those in the no alibi condition. In experiment 2, three levels of alibi were combined with two levels of eyewitness evidence (bad view, good view). A main effect of alibi was obtained, but no interaction between alibi and eyewitness evidence. ^ An explanation of this pattern is based in part on the Story Model (Pennington & Hastie, 1992) and a novel “culpability threshold” model of juror decision-making. The Story Model suggests that jurors generate verdict stories (interpretations of events consistent with a guilty or not guilty verdict) based on trial evidence. If the evidence in favor of guilt exceeds jurors' threshold for perceiving culpability, jurors will fail to properly consider exonerating evidence. However, when the strength of incriminating evidence does not exceed the jurors' threshold, they are likely to give appropriate consideration to exculpatory evidence in their decisions. ^ Presentation of a reliable confession in Experiment 1 exceeded jurors' culpability threshold and rendered alibi largely irrelevant. In contrast, presentation of a high-pressure confession failed to exceed jurors' culpability threshold, so jurors turned to alibi evidence in their decisions. Similarly, in the second experiment, eyewitness evidence (in general) was not strong enough to surpass the culpability threshold, and thus jurors incorporated alibi evidence in their decisions. A third study is planned to further test this “culpability threshold” model, further explore various types of alibi evidence, and clarify when exculpatory evidence will sufficiently weaken the prosecution's “story.” ^

A Method for Testing Object Localization as a Function of Eod Amplitude in the Weakly Electric Fish Brachyhypopomus Pinnicaudatus

I would like to thank Dr. Philip Stoddard for his patience and guidance throughout the past four years. He has not only taught me about behavior and electricity, but he has also taught me how to think scientifically. Vielka Salazar for making herself available to answer my questions and to help me with my projects. Montserrat Alfaro for providing me with support under times of frustration. Fabian A. Pal, who has often made himself available when I needed help to finish my projects, for being supportive, and for believing in me and my abilities. Most importantly, I would like to thank my parents who have shown tremendous support and patience during the past years. I would also like to thank the Honors Committee, specially Dr. Richards for taking the time to review my thesis and helping me modify it. Finally, I would like to thank the MARC program for providing me with financial assistance and the opportunity to perform this project.

Investigation of molecular mechanisms of lkb1 function in dictyostelium development and stress responses and the function of superoxide dismutase c (sodc) in chemotaxis

The serine/threonine kinase LKB1 is a regulator of critical events including development and stress responses in metazoans. The current study was undertaken to determine the function of LKB1 in Dictyostelium. During multicellular development and in response to stress insult, an apparent increase in the DdLKB1 kinase activity was observed. Depletion of DdLKB1 with a knockdown construct led to aberrant development; a severe reduction in prespore cell differentiation and a precocious induction of prestalk cells, which were reminiscent of cells lacking GSK3, a well known cell-fate switch. Furthermore, DdLKB1 depleted cells displayed lower GSK3 activity than wild type cells in response to cAMP stimulation during development and failed to activate AMPK, a well known LKB1 target in mammals, in response to cAMP and stress insults. These results suggest that DdLKB1 positively regulates both GSK3 and AMPK during Dictyostelium development, and DdLKB1 is necessary for AMPK activation during stress response regulation. No apparent GSK3 activation was observed in response to stress insults. Spatial and temporal regulation of phosphatidylinositol-(3,4,5)-triphosphate (PIP3) along the membrane of polarized cells is important for efficient chemotaxis. A REMI screen for PIP3 suppressors in the absence of stimulation led to the identification of SodC as PIP3 regulator. Consistent with their higher PIP3 levels, sodC- cells showed defects in chemotaxis and exhibited higher intra-cellular superoxide levels. Protein localization studies along with observations from GPI specific PI-PLC treatment of wild-type cells suggested that SodC is a GPI anchored outer-membrane protein. SodC showed superoxide dismutase activity in vitro, and motility defects of sodC- cells can be rescued by expressing the intact SodC but not by the mutant SodC, which has point mutations that affect its dismutase function. Treatment of sodC- cells with LY294002, a pharmacological inhibitor of PI3K, partially rescued the polarization and chemoattractant sensing defects but not motility defects. Consistent with increased intracellular superoxide levels, sodC- cells also exhibited higher basal Ras activity, an upstream regulator of PI3K, which can be suppressed by a cell permeable superoxide scavenger, XTT, indicating that SodC is important in regulation of intracellular superoxide levels thereby regulating the Ras activity and PIP3 levels at the membrane.

On-screen pre-deblurring of digital images using the wavefront aberration function of the human eye to improve computer access for the visually impaired

Traditional Optics has provided ways to compensate some common visual limitations (up to second order visual impairments) through spectacles or contact lenses. Recent developments in wavefront science make it possible to obtain an accurate model of the Point Spread Function (PSF) of the human eye. Through what is known as the "Wavefront Aberration Function" of the human eye, exact knowledge of the optical aberration of the human eye is possible, allowing a mathematical model of the PSF to be obtained. This model could be used to pre-compensate (inverse-filter) the images displayed on computer screens in order to counter the distortion in the user's eye. This project takes advantage of the fact that the wavefront aberration function, commonly expressed as a Zernike polynomial, can be generated from the ophthalmic prescription used to fit spectacles to a person. This allows the pre-compensation, or onscreen deblurring, to be done for various visual impairments, up to second order (commonly known as myopia, hyperopia, or astigmatism). The technique proposed towards that goal and results obtained using a lens, for which the PSF is known, that is introduced into the visual path of subjects without visual impairment will be presented. In addition to substituting the effect of spectacles or contact lenses in correcting the loworder visual limitations of the viewer, the significance of this approach is that it has the potential to address higher-order abnormalities in the eye, currently not correctable by simple means.

Multilevel structural equation models for longitudinal data where predictors are measured more frequently than outcomes : an application to the effects of stress on the cognitive function of nurses

Funded by Chief Scientist Office, Scotland. Grant Number: CZH/4/394 Economic and Social Research Council grant as part of the National Centre for Research Methods. Grant Number: RES-576-25-0032

The function of the Saccharomyces Cerevisiae ribonucleotide reductase second [beta] subunit in DNA repair

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

Metabolism Regulates the Fate and Function of T Lymphocytes

Proper balancing of the activities of metabolic pathways to meet the challenge of providing necessary products for biosynthetic and energy demands of the cell is a key requirement for maintaining cell viability and allowing for cell proliferation. Cell metabolism has been found to play a crucial role in numerous cell settings, including in the cells of the immune system, where a successful immune response requires rapid proliferation and successful clearance of dangerous pathogens followed by resolution of the immune response. Additionally, it is now well known that cell metabolism is markedly altered from normal cells in the setting of cancer, where tumor cells rapidly and persistently proliferate. In both settings, alterations to the metabolic profile of the cells play important roles in promoting cell proliferation and survival.

It has long been known that many types of tumor cells and actively proliferating immune cells adopt a metabolic phenotype of aerobic glycolysis, whereby the cell, even under normoxic conditions, imports large amounts of glucose and fluxes it through the glycolytic pathway and produces lactate. However, the metabolic programs utilized by various immune cell subsets have only recently begun to be explored in detail, and the metabolic features and pathways influencing cell metabolism in tumor cells in vivo have not been studied in detail. The work presented here examines the role of metabolism in regulating the function of an important subset of the immune system, the regulatory T cell (Treg) and the role and regulation of metabolism in the context of malignant T cell acute lymphoblastic leukemia (T-ALL). We show that Treg cells, in order to properly function to suppress auto-inflammatory disease, adopt a metabolic program that is characterized by oxidative metabolism and active suppression of anabolic signaling and metabolic pathways. We found that the transcription factor FoxP3, which is highly expressed in Treg cells, drives this phenotype. Perturbing the metabolic phenotype of Treg cells by enforcing increased glycolysis or driving proliferation and anabolic signaling through inflammatory signaling pathways results in a reduction in suppressive function of Tregs.

In our studies focused on the metabolism of T-ALL, we observed that while T-ALL cells use and require aerobic glycolysis, the glycolytic metabolism of T-ALL is restrained compared to that of an antigen activated T cell. The metabolism of T-ALL is instead balanced, with mitochondrial metabolism also being increased. We observed that the pro-anabolic growth mTORC1 signaling pathway was limited in primary T-ALL cells as a result of AMPK pathway activity. AMPK pathway signaling was elevated as a result of oncogene induced metabolic stress. AMPK played a key role in the regulation of T-ALL cell metabolism, as genetic deletion of AMPK in an in vivo murine model of T-ALL resulted in increased glycolysis and anabolic metabolism, yet paradoxically increased cell death and increased mouse survival time. AMPK acts to promote mitochondrial oxidative metabolism in T-ALL through the regulation of Complex I activity, and loss of AMPK reduced mitochondrial oxidative metabolism and resulted in increased metabolic stress. Confirming a role for mitochondrial metabolism in T-ALL, we observed that the direct pharmacological inhibition of Complex I also resulted in a rapid loss of T-ALL cell viability in vitro and in vivo. Taken together, this work establishes an important role for AMPK to both balance the metabolic pathways utilized by T-ALL to allow for cell proliferation and to also promote tumor cell viability by controlling metabolic stress.

Overall, this work demonstrates the importance of the proper coupling of metabolic pathway activity with the function needs of particular types of immune cells. We show that Treg cells, which mainly act to keep immune responses well regulated, adopt a metabolic program where glycolytic metabolism is actively repressed, while oxidative metabolism is promoted. In the setting of malignant T-ALL cells, metabolic activity is surprisingly balanced, with both glycolysis and mitochondrial oxidative metabolism being utilized. In both cases, altering the metabolic balance towards glycolytic metabolism results in negative outcomes for the cell, with decreased Treg functionality and increased metabolic stress in T-ALL. In both cases, this work has generated a new understanding of how metabolism couples to immune cell function, and may allow for selective targeting of immune cell subsets by the specific targeting of metabolic pathways.

Occurrence and Function of Hoogsteen Base Pairs in Nucleic Acids

Nucleic acids (DNA and RNA) play essential roles in the central dogma of biology for the storage and transfer of genetic information. The unique chemical and conformational structures of nucleic acids – the double helix composed of complementary Watson-Crick base pairs, provide the structural basis to carry out their biological functions. DNA double helix can dynamically accommodate Watson-Crick and Hoogsteen base-pairing, in which the purine base is flipped by ~180° degrees to adopt syn rather than anti conformation as in Watson-Crick base pairs. There is growing evidence that Hoogsteen base pairs play important roles in DNA replication, recognition, damage or mispair accommodation and repair. Here, we constructed a database for existing Hoogsteen base pairs in DNA duplexes by a structure-based survey from the Protein Data Bank, and structural analyses based on the resulted Hoogsteen structures revealed that Hoogsteen base pairs occur in a wide variety of biological contexts and can induce DNA kinking towards the major groove. As there were documented difficulties in modeling Hoogsteen or Watson-Crick by crystallography, we collaborated with the Richardsons’ lab and identified potential Hoogsteen base pairs that were mis-modeled as Watson-Crick base pairs which suggested that Hoogsteen can be more prevalent than it was thought to be. We developed solution NMR method combined with the site-specific isotope labeling to characterize the formation of, or conformational exchange with Hoogsteen base pairs in large DNA-protein complexes under solution conditions, in the absence of the crystal packing force. We showed that there are enhanced chemical exchange, potentially between Watson-Crick and Hoogsteen, at a sharp kink site in the complex formed by DNA and the Integration Host Factor protein. In stark contrast to B-form DNA, we found that Hoogsteen base pairs are strongly disfavored in A-form RNA duplex. Chemical modifications N1-methyl adenosine and N1-methyl guanosine that block Watson-Crick base-pairing, can be absorbed as Hoogsteen base pairs in DNA, but rather potently destabilized A-form RNA and caused helix melting. The intrinsic instability of Hoogsteen base pairs in A-form RNA endows the N1-methylation as a functioning post-transcriptional modification that was known to facilitate RNA folding, translation and potentially play roles in the epitranscriptome. On the other hand, the dynamic property of DNA that can accommodate Hoogsteen base pairs could be critical to maintaining the genome stability.

Function of the PDLIM2 protein in oncogenic Wnt signalling pathways

Aberrant regulation of the Wnt signalling pathway is a recurrent theme in cancer biology. Hyper activation due to oncogenic mutations and paracrine activity has been found in both colon cancer and breast cancer, and continues to evolve as a central mechanism in oncogenesis. PDLIM2, a cytoskeletal PDZ protein, is an IGF-1 regulated gene that is highly expressed in cancer cell lines derived from metastatic tumours. Suppression of PDLIM2 inhibits polarized cell migration, reverses the Epithelial to Mesenchymal transition (EMT) phenotype, suppresses the transcription of β-catenin target genes, and regulates gene expression of key transcription factors in EMT. This thesis investigates the mechanism by which PDLIM2 contributes to the maintenance of Wnt signalling in cancer cells. Here we show that PDLIM2 is a critical regulator of the Wnt pathway by regulating β-catenin at the adherens juctions, as also its transcriptional activity by the interaction of PDLIM2 with TCF4 at the nucleus. Evaluation of PDLIM2 in macrophages and co-culture studies with cancer cells and fibroblasts showed the influence exerted on PDLIM2 by paracrine cues. Thus, PDLIM2 integrates cytoskeleton signalling with gene expression by modulating the Wnt signalling pathway and reconciling microenvironmental cues with signals in epithelial cells. Negative correlation of mRNA and protein levels in the triple negative breast cancer cell BT549 suggests that PDLIM2 is part of a more complex mechanism that involves transcription and posttranslational modifications. GST pulldown studies and subsequent mass spectrometry analysis showed that PDLIM2 interacts with 300 proteins, with a high biological function in protein biosynthesis and Ubiquitin/proteasome pathways, including 13 E3 ligases. Overall, these data suggest that PDLIM2 has two distinct functions depending of its location. Located at the cytoplasm mediates cytoskeletal re-arrangements, whereas at the nucleus PDLIM2 acts as a signal transduction adaptor protein mediating transcription and ubiquitination of key transcription factors in cancer development.

The function of the Saccharomyces Cerevisiae ribonucleotide reductase second [beta] subunit in DNA repair

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

Sea-surface temperature reconstruction and calibration-function of sediment cores from the Southern Ocean

We provide a new multivariate calibration-function based on South Atlantic modern assemblages of planktonic foraminifera and atlas water column parameters from the Antarctic Circumpolar Current to the Subtropical Gyre and tropical warm waters (i.e., 60°S to 0°S). Therefore, we used a dataset with the abundance pattern of 35 taxonomic groups of planktonic foraminifera in 141 surface sediment samples. Five factors were taken into consideration for the analysis, which account for 93% of the total variance of the original data representing the regional main oceanographic fronts. The new calibration-function F141-35-5 enables the reconstruction of Late Quaternary summer and winter sea-surface temperatures with a statistical error of ~0.5°C. Our function was verified by its application to a sediment core extracted from the western South Atlantic. The downcore reconstruction shows negative anomalies in sea-surface temperatures during the early-mid Holocene and temperatures within the range of modern values during the late Holocene. This pattern is consistent with available reconstructions.

Unraveling the function of bacterial-type phosphoenolpyruvate carboxylase in vascular plants

Two distinct phosphoenolpyruvate carboxylase (PEPC) isozymes occur in vascular plants and green algae: plant-type PEPC (PTPC) and bacterial-type PEPC (BTPC). PTPC polypeptides typically form a tightly regulated cytosolic Class-1 PEPC homotetramer. BTPCs, however, appear to be less widely expressed and to exist only as catalytic and regulatory subunits that physically interact with co-expressed PTPC subunits to form hetero-octameric Class-2 PEPC complexes that are highly desensitized to Class-1 PEPC allosteric effectors. Yeast two-hybrid studies indicated that castor plant BTPC (RcPPC4) interacts with all three Arabidopsis thaliana PTPC isozymes, and that it forms stronger interactions with AtPPC2 and AtPPC3, suggesting that specific PTPCs are preferred for Class-2 PEPC formation. In contrast, Arabidopsis BTPC (AtPPC4) appeared to interact very weakly with AtPPC2 and AtPPC3, suggesting that BTPCs from different species may have different physical properties, hypothesized to be due to sequence dissimilarities within their ~10 kDa intrinsically disordered region. Recent RNA-seq and microarray data were analyzed to obtain a better understanding of BTPC expression patterns in different tissues of various monocot and dicot species. High levels of BTPC transcripts, polypeptides and Class-2 PEPC complexes were originally discovered in developing castor seeds, but the analysis revealed a broad range of diverse tissues where abundant BTPC transcripts are also expressed, such as the developing fruits of cucumber, grape, and tomato. Marked BTPC expression correlated well with the presence of ~116 kDa immunoreactive BTPC polypeptides, as well as Class-2 PEPC complexes in the immature fruit of cucumbers and tomatoes. It is therefore hypothesized that in vascular plants BTPC and thus Class-2 PEPC complexes maintain anaplerotic PEP flux in tissues with elevated malate levels that would potently inhibit ‘housekeeping’ Class-1 PEPCs. Elevated levels of malate can be used by biosynthetically active sink tissues such as immature tomatoes and cucumbers for rapid cell expansion, drought or salt stressed roots for osmoregulation, and developing seeds and pollen as a precursor for storage lipid and protein biosynthesis.