Mechanisms of Bicarbonate Use Influence the Photosynthetic Carbon Dioxide Sensitivity of Tropical Seagrasses

The photosynthetic bicarbonate () use properties of three widely distributed tropical seagrasses were compared using a series of laboratory experiments. Photosynthetic rates of Thalassia testudinum, Halodule wrightii, and Syringodium filiforme were monitored in an enclosed chamber while being subjected to shifts in pH and dissolved inorganic carbon. Specific mechanisms of seagrass use were compared by examining the photosynthetic effects of the carbonic anhydrase inhibitor acetazolamide (AZ). All seagrasses increased photosynthetic rates with reduced pH, suggesting a large effect of dissolved aqueous carbon dioxide (CO2(aq)). However, there was considerable interspecific variation in pH response. T. testudinum was highly sensitive, increasing photosynthetic rates by 100% as the pH was reduced from 8.2 to 7.4, whereas rates in H. wrightii and S. filiforme increased by only 20% over a similar range, and displayed prominent photosynthetic plateaus, indicating an increased capacity for use. Additional incubations that manipulated [] under constant [CO2(aq)] support these findings, as only H. wrightii and S. filiforme increased photosynthetic rates with increasing []. T. testudinum responded to AZ addition, indicating that carbonic anhydrase enzymes facilitate limited use. H. wrightii and S. filiforme showed no response to AZ, suggesting alternate, more efficient mechanisms of use. Estimated kinetic parameters, Ks(CO2) and Vmax, revealed interspecific variation and further support these conclusions. Variation in photosynthetic pH responses and AZ sensitivity indicate distinctions in the carbon use properties of seagrasses exposed to similar environmental conditions. These results suggest that not all seagrasses will similarly respond to future increases in CO2(aq) availability. Attention towards potential shifts in competitive interactions within multispecific seagrass beds is warranted.

Novel insights into the mechanisms of regulation of tyrosine kinase receptors by Ras Interference 1

Receptor-tyrosine kinases (RTKs) are membrane bound receptors characterized by their intrinsic kinase activity. RTK activities play an essential role in several human diseases, including cancer, diabetes and neurodegenerative diseases. RTK activities have been regulated by the expression or silencing of several genes as well as by the utilization of small molecules. Ras Interference 1 (Rin1) is a multifunctional protein that becomes associated with activated RTKs upon ligand stimulation. Rin1 plays a key role in receptor internalization and in signal transduction via activation of Rab5 and association with active form of Ras. This study has two main objectives: (1) It determines the role of Rin1 in the regulation of several RTKs focusing on insulin receptor. This was accomplished by studying the Rin1-insulin receptor interaction using a variety of biochemical and morphological assays. This study shows a novel interaction between the insulin receptor and Rin1 through the Vps9 domain. Two more RTKs (epidermal growth factor receptor and nerve growth factor receptor) also interacted with the SH2 domain of Rin1. The effect of the Rin1-RTK interaction on the activation of both Rab5 and Ras was also studied during receptor internalization and intracellular signaling. Finally, the role of Rin1 was examined in two differentiation processes (adipogenesis and neurogenesis). Rin1 showed a strong inhibitory effect on 3T3-L1 preadipocyte differentiation but it seems to show a modest effect in PC12 neurite outgrowth. These data indicate a selective function and specific interaction of Rin1 toward RTKs. (2) It examines the role of the small molecule Dehydroleucodine (DhL) on several key signaling molecules during adipogenesis. This was accomplished by studying the differentiation of 3T3-L1 preadipocytes exposed to different concentrations of DhL in different days of the adipocyte formation process. The results indicate that DhL selectively blocked adipocyte formation, as well as the expression of PPARγ, and C/EBP&agr;. However, DhL treatment did not affect Rin1 or Rab5 expression and their activities. Taken together, the data indicate a potential molecular mechanism by which proteins or small molecules regulate selective and specific RTK intracellular membrane trafficking and signaling during cell growth and differentiation in normal and pathological conditions.

Resistance Performances: (Re)constructing Spaces of Resistance and Contention in the 2010-2011 University of Puerto Rico Student Movement

On the night of April 20, 2010, a group of students from the University of Puerto Rico (UPR), Río Piedras campus, met to organize an indefinite strike that quickly broadened into a defense of accessible public higher education of excellence as a fundamental right and not a privilege. Although the history of student activism in the UPR can be traced back to the early 1900s, the 2010-2011 strike will be remembered for the student activists’ use of new media technologies as resources that rapidly prompted and aided the numerous protests. This activist research entailed a critical ethnography and a critical discourse analysis (CDA) of traditional and alternative media coverage and treatment during the 2010 -2011 UPR student strike. I examined the use of the 2010-2011 UPR student activists’ resistance performances in constructing local, corporeal, and virtual spaces of resistance and contention during their movement. In particular, I analyzed the different tactics and strategies of resistance or repertoire of collective actions that student activists used (e.g. new media technologies) to frame their collective identities via alternative news media’s (re)presentation of the strike, while juxtaposing the university administration’s counter-resistance performances in counter-framing the student activists’ collective identity via traditional news media representations of the strike. I illustrated how both traditional and alternative media (re)presentations of student activism developed, maintained, and/or modified students activists’ collective identities. As such, the UPR student activism’s success should not be measured by the sum of demands granted, but by the sense of community achieved and the establishment of networks that continue to create resistance and change. These networks add to the debate surrounding Internet activism and its impact on student activism. Ultimately, the results of this study highlight the important role student movements have had in challenging different types of government policies and raising awareness of the importance of an accessible public higher education of excellence.

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.

From theory-inspired to theory-based interventions : A protocol for developing and testing a methodology for linking behaviour change techniques to theoretical mechanisms of action

This research is funded by UK Medical Research Council grant number MR/L011115/1

From Theory-Inspired to Theory-Based Interventions : A Protocol for Developing and Testing a Methodology for Linking Behaviour Change Techniques to Theoretical Mechanisms of Action

This research is funded by UK Medical Research Council grant number MR/L011115/1. We would like to thank the 105 experts in behaviour change who have committed their time and offered their expertise for study 2 of this research. We are also very grateful to all those who sent us peer-reviewed behaviour change intervention descriptions for study 1. Finally, we would like thank Dr. Emma Beard and Dr. Dan Dediu for their statistical input and to all the researchers, particularly Holly Walton, who have assisted in the coding of papers for study 1.

Regional Magma Plumbing and emplacement mechanisms of the Faroe-Shetland Sill Complex : Implications for magma transport and petroleum systems within sedimentary basins

Peer reviewed

Regional Magma Plumbing and emplacement mechanisms of the Faroe-Shetland Sill Complex : Implications for magma transport and petroleum systems within sedimentary basins

Peer reviewed

Resistance and symbiosis : Québec discourses of resistance in the context of postcolonial theory

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Synthesis & retention mechanisms of novel mixed mode stationary phase for detection of specific polar low molecular weight biological samples

The thesis primarily reports the synthesis, characterization and application of novel mixed mode stationary phases for Hydrophilic Interaction Liquid Chromatography (HILIC). HILIC is a rapidly emerging chromatographic mode that is finding great applicability in the analysis of polar organic molecules. In addition, there is a chapter on the analysis of Bisphenol A and related species using capillary electrophoresis (CE) coupled with boron-doped diamond electrodes for electrochemical detection. The synthesis and characterization of the novel mixed mode stationary phases prepared in this work is an important contribution to the field as the materials prepared exhibited better performance than similar materials obtained commercially. In addition a more thorough characterization of the materials (e.g.,thermogravimetric analysis, various NMR modes, elemental analysis, etc.) and resulting columns (e.g., H) than is typically encountered. The application of these new materials to the analysis of sugars using evaporative light scattering is also novel. In CE studies, electrochemical detection is sufficiently rare that the work is also novel.

Mechanisms of Cdc42 Polarization in Yeast

Polarization is important for the function and morphology of many different cell types. The keys regulators of polarity in eukaryotes are the Rho-family GTPases. In the budding yeast Saccharomyces cerevisiae, which must polarize in order to bud and to mate, the master regulator is the highly conserved Rho GTPase, Cdc42. During polarity establishment, active Cdc42 accumulates at a site on the plasma membrane characterizing the “front” of the cell where the bud will emerge. The orientation of polarization is guided by upstream cues that dictate the site of Cdc42 clustering. However, in the absence of upstream cues, yeast can still polarize in a random direction during symmetry breaking. Symmetry breaking suggests cells possess an autocatalytic polarization mechanism that can amplify stochastic fluctuations of polarity proteins through a positive feedback mechanism.

Two different positive feedback mechanisms have been proposed to polarize Cdc42 in budding yeast. One model posits that Cdc42 activation must be localized to a site at the plasma membrane. Another model posits that Cdc42 delivery must be localized to a particular site at the plasma membrane. Although both mechanisms could work in parallel to polarize Cdc42, it is unclear which mechanism is critical to polarity establishment. We directly tested the predictions of the two positive feedback models using genetics and live microscopy. We found that localized Cdc42 activation is necessary for polarity establishment.

While this explains how active Cdc42 localizes to a particular site at the plasma membrane, it does not address how Cdc42 concentrates at that site. Several different mechanisms have been proposed to concentrate Cdc42. The GDI can extract Cdc42 from membranes and selective mobilize GDP-Cdc42 in the cytoplasm. It was proposed that selectively mobilizing GDP-Cdc42 in combination with local activation could locally concentrate total Cdc42 at the polarity site. Although the GDI is important for rapid Cdc42 accumulation at the polarity site, it is not essential to Cdc42 concentration. It was proposed that delivery of Cdc42 by actin-mediated vesicle can act as a backup pathway to concentrate Cdc42. However, we found no evidence for an actin-dependent concentrating pathway. Live microscopy experiments reveal that prenylated proteins are not restricted to membranes, and can enter the cytoplasm. We found that the GDI-independent concentrating pathway still requires Cdc42 to exchange between the plasma membrane and the cytoplasm, which is supported by computational modeling. In the absence of the GDI, we found that Cdc42 GAP became essential for polarization. We propose that the GAP limits GTP-Cdc42 leak into the cytoplasm, which would be prohibitive to Cdc42 polarization.

Exploring Mechanisms of Bacterial Adaptation to Seasonal Temperature Change

This research examines three potential mechanisms by which bacteria can adapt to different temperatures: changes in strain-level population structure, gene regulation and particle colonization. For the first two mechanisms, I utilize bacterial strains from the Vibrionaceae family due to their ease of culturability, ubiquity in coastal environments and status as a model system for marine bacteria. I first examine vibrio seasonal dynamics in temperate, coastal water and compare the thermal performance of strains that occupy different thermal environments. Our results suggest that there are tradeoffs in adaptation to specific temperatures and that thermal specialization can occur at a very fine phylogenetic scale. The observed thermal specialization over relatively short evolutionary time-scales indicates that few genes or cellular processes may limit expansion to a different thermal niche. I then compare the genomic and transcriptional changes associated with thermal adaptation in closely-related vibrio strains under heat and cold stress. The two vibrio strains have very similar genomes and overall exhibit similar transcriptional profiles in response to temperature stress but their temperature preferences are determined by differential transcriptional responses in shared genes as well as temperature-dependent regulation of unique genes. Finally, I investigate the temporal dynamics of particle-attached and free-living bacterial community in coastal seawater and find that microhabitats exert a stronger forcing on microbial communities than environmental variability, suggesting that particle-attachment could buffer the impacts of environmental changes and particle-associated communities likely respond to the presence of distinct eukaryotes rather than commonly-measured environmental parameters. Integrating these results will offer new perspectives on the mechanisms by which bacteria respond to seasonal temperature changes as well as potential adaptations to climate change-driven warming of the surface oceans.

Neural Mechanisms of Young Adult Sexual Decision-Making and Risk Behavior

Sexual risk behavior among young adults is a serious public health concern; 50% will contract a sexually transmitted infection (STI) before the age of 25. The current study collected self-report personality and sexual history data, as well as neuroimaging, experimental behavioral (e.g., real-time hypothetical sexual decision making data), and self-report sexual arousal data from 120 heterosexual young adults ages 18-26. In addition, longitudinal changes in self-reported sexual behavior were collected from a subset (n = 70) of the participants. The primary aims of the study were (1) to predict differences in self-report sexual behavior and hypothetical sexual decision-making (in response to sexually explicit audio-visual cues) as a function of ventral striatum (VS) and amygdala activity, (2) test whether the association between sexual behavior/decision-making and brain function is moderated by gender, self-reported sexual arousal, and/or trait-level personality factors (i.e., self-control, impulsivity, and sensation seeking) and (3) to examine how the main effects of neural function and interaction effects predict sexual risk behavior over time. Our hypotheses were mostly supported across the sexual behavior and decision-making outcome variables, such that neural risk phenotypes (heightened reward-related ventral striatum activity coupled with decreased threat-related amygdala activity) were associated with greater lifetime sexual partners at baseline measured and over time (longitudinal analyses). Impulsivity moderated the relationship between neural function and self-reported number of sexual partners at baseline and follow up measures, as well as experimental condom use decision-making. Sexual arousal and sensation seeking moderated the relationship between neural function and baseline and follow up self-reports of number of sexual partners. Finally, unique gender differences were observed in the relationship between threat and reward-related neural reactivity and self-reported sexual risk behavior. The results of this study provide initial evidence for the potential role for neurobiological approaches to understanding sexual decision-making and risk behavior. With continued research, establishing biomarkers for sexual risk behavior could help inform the development of novel and more effective individually tailored sexual health prevention and intervention efforts.

Molecular Mechanisms of Airway Epithelial Progenitor Cell Maintenance and Repair.

The lungs are vital organs whose airways are lined with a continuous layer of epithelial cells. Epithelial cells in the distal most part of the lung, the alveolar space, are specialized to facilitate gas exchange. Proximal to the alveoli is the airway epithelium, which provides an essential barrier and is the first line of defense against inhaled toxicants, pollutants, and pathogens. Although the postnatal lung is a quiescent organ, it has an inherent ability to regenerate in response to injury. Proper balance between maintaining quiescence and undergoing repair is crucial, with imbalances in these processes leading to fibrosis or tumor development. Stem and progenitor cells are central to maintaining balance, given that they proliferate and renew both themselves and the various differentiated cells of the lung. However, the precise mechanisms regulating quiescence and repair in the lungs are largely unknown. In this dissertation, ionizing radiation is used as a physiologically relevant injury model to better understand the repair process of the airway epithelium. We use in vitro and in vivo mouse models to study the response of a secretory progenitor, the club cell, to various doses and qualities of ionizing radiation. Exposure to radiation found in space environments and in some types of radiotherapy caused clonal expansion of club cells specifically in the most distal branches of the airway epithelium, indicating that the progenitors residing in the terminal bronchioles are radiosensitive. This clonal expansion is due to an increase in p53-dependent apoptosis, senescence, and mitotic defects. Through the course of this work, we discovered that p53 is not only involved in radiation response, but is also a novel regulator of airway epithelial homeostasis. p53 acts in a gene dose-dependent manner to regulate the composition of airway epithelium by maintaining quiescence and regulating differentiation of club progenitor cells in the steady-state lung. The work presented in this dissertation represents an advance in our understanding of the molecular mechanisms underlying maintenance of airway epithelial progenitor cells as well as their repair following ionizing radiation exposure.