The redoxomics of PTEN: walking a fine line between damage and signaling:mass spectrometry-based approaches to study the effect of oxidation on PTEN function, structure, and protein-protein interactions

The research described in this PhD thesis focuses on proteomics approaches to study the effect of oxidation on the modification status and protein-protein interactions of PTEN, a redox-sensitive phosphatase involved in a number of cellular processes including metabolism, apoptosis, cell proliferation, and survival. While direct evidence of a redox regulation of PTEN and its downstream signaling has been reported, the effect of cellular oxidative stress or direct PTEN oxidation on PTEN structure and interactome is still poorly defined. In a first study, GST-tagged PTEN was directly oxidized over a range of hypochlorous acid (HOCl) concentration, assayed for phosphatase activity, and oxidative post-translational modifications (oxPTMs) were quantified using LC-MS/MS-based label-free methods. In a second study, GSTtagged PTEN was prepared in a reduced and reversibly H2O2-oxidized form, immobilized on a resin support and incubated with HCT116 cell lysate to capture PTEN interacting proteins, which were analyzed by LC-MS/MS and comparatively quantified using label-free methods. In parallel experiments, HCT116 cells transfected with a GFP-tagged PTEN were treated with H2O2 and PTENinteracting proteins immunoprecipitated using standard methods. Several high abundance HOCl-induced oxPTMs were mapped, including those taking place at amino acids known to be important for PTEN phosphatase activity and protein-protein interactions, such as Met35, Tyr155, Tyr240 and Tyr315. A PTEN redox interactome was also characterized, which identified a number of PTEN-interacting proteins that vary with the reversible inactivation of PTEN caused by H2O2 oxidation. These included new PTEN interactors as well as the redox proteins peroxiredoxin-1 (Prdx1) and thioredoxin (Trx), which are known to be involved in the recycling of PTEN active site following H2O2-induced reversible inactivation. The results suggest that the oxidative modification of PTEN causes functional alterations in PTEN structure and interactome, with fundamental implications for the PTEN signaling role in many cellular processes, such as those involved in the pathophysiology of disease and ageing.

The relationship of fear of intimacy, body image avoidance and body mass index

Issues of body image and ability to achieve intimacy are connected to body weight, yet remain largely unexplored and have not been evaluated by gender. The underlying purpose of this research was to determine if avoidant attitudes and perceptions of one's body may hold implications toward its use in intimate interactions, and if an above average body weight would tend to increase this avoidance. The National Health and Nutrition Examination Survey (NHANES, 1999-2002) finds that 64.5% of US adults are overweight, with 61.9% of women and 67.2% of men. The increasing prevalence of overweight and obesity in men and women shows no reverse trend, nor have prevention and treatment proven effective in the long term. The researcher gathered self-reported age, gender, height and weight data from 55 male and 58 female subjects (determined by a prospective power analysis with a desired medium effect size (r=.30) to determine body mass index (BMI), determining a mean age of 21.6 years and mean BMI of 25.6. Survey instruments consisted of two scales that are germane to the variables being examined. They were (1) Descutner and Thelen of the University of Missouri‘s (1991) Fear-of-Intimacy scale; and (2) Rosen, Srebnik, Saltzberg, and Wendt's (1991) Body Image Avoidance Questionnaire. Results indicated that as body mass index increases, fear of intimacy increases (p<0.05) and that as body mass index increases, body image avoidance increases (p<0.05). The relationship that as body image avoidance increases, fear of intimacy increases was not supported, but approached significance at (p<0.07). No differences in these relationships were determined between gender groups. For age, the only observed relationship was that of a difference between scores for age groups [18 to 22 (group 1) and ages 23 to 34 (group 2)] for the relationship of body image avoidance and fear of intimacy (p<0.02). The results suggest that the relationship of body image avoidance and fear of intimacy, as well as age, bear consideration toward the escalating prevalence of overweight and obesity. An integrative approach to body weight that addresses issues of body image and intimacy may prove effective in prevention and treatment.

Changes in mass and nutrient content of wood during decomposition in a south Florida mangrove forest

1. Large pools of dead wood in mangrove forests following disturbances such as hurricanes may influence nutrient fluxes. We hypothesized that decomposition of wood of mangroves from Florida, USA (Avicennia germinans, Laguncularia racemosa and Rhizophora mangle), and the consequent nutrient dynamics, would depend on species, location in the forest relative to freshwater and marine influences and whether the wood was standing, lying on the sediment surface or buried. 2. Wood disks (8–10 cm diameter, 1 cm thick) from each species were set to decompose at sites along the Shark River, either buried in the sediment, on the soil surface or in the air (above both the soil surface and high tide elevation). 3. A simple exponential model described the decay of wood in the air, and neither species nor site had any effect on the decay coefficient during the first 13 months of decomposition. 4. Over 28 months of decomposition, buried and surface disks decomposed following a two-component model, with labile and refractory components. Avicennia germinans had the largest labile component (18 ± 2% of dry weight), while Laguncularia racemosa had the lowest (10 ± 2%). Labile components decayed at rates of 0.37–23.71% month−1, while refractory components decayed at rates of 0.001–0.033% month−1. Disks decomposing on the soil surface had higher decay rates than buried disks, but both were higher than disks in the air. All species had similar decay rates of the labile and refractory components, but A. germinans exhibited faster overall decay because of a higher proportion of labile components. 5. Nitrogen content generally increased in buried and surface disks, but there was little change in N content of disks in the air over the 2-year study. Between 17% and 68% of total phosphorus in wood leached out during the first 2 months of decomposition, with buried disks having the greater losses, P remaining constant or increasing slightly thereafter. 6. Newly deposited wood from living trees was a short-term source of N for the ecosystem but, by the end of 2 years, had become a net sink. Wood, however, remained a source of P for the ecosystem. 7. As in other forested ecosystems, coarse woody debris can have a significant impact on carbon and nutrient dynamics in mangrove forests. The prevalence of disturbances, such as hurricanes, that can deposit large amounts of wood on the forest floor accentuates the importance of downed wood in these forests.

Online solid phase extraction liquid chromatography tandem mass spectrometry (SPE-LC-MS/MS) method for the determination of sucralose in reclaimed and drinking waters and its photo degradation in natural waters from South Florida

Background Sucralose has gained popularity as a low calorie artificial sweetener worldwide. Due to its high stability and persistence, sucralose has shown widespread occurrence in environmental waters, at concentrations that could reach up to several μg/L. Previous studies have used time consuming sample preparation methods (offline solid phase extraction/derivatization) or methods with rather high detection limits (direct injection) for sucralose analysis. This study described a faster and sensitive analytical method for the determination of sucralose in environmental samples. Results An online SPE-LC–MS/MS method was developed, being capable to quantify sucralose in 12 minutes using only 10 mL of sample, with method detection limits (MDLs) of 4.5 ng/L, 8.5 ng/L and 45 ng/L for deionized water, drinking and reclaimed waters (1:10 diluted with deionized water), respectively. Sucralose was detected in 82% of the reclaimed water samples at concentrations reaching up to 18 μg/L. The monthly average for a period of one year was 9.1 ± 2.9 μg/L. The calculated mass loads per capita of sucralose discharged through WWTP effluents based on the concentrations detected in wastewaters in the U. S. is 5.0 mg/day/person. As expected, the concentrations observed in drinking water were much lower but still relevant reaching as high as 465 ng/L. In order to evaluate the stability of sucralose, photodegradation experiments were performed in natural waters. Significant photodegradation of sucralose was observed only in freshwater at 254 nm. Minimal degradation (<20%) was observed for all matrices under more natural conditions (350 nm or solar simulator). The only photolysis product of sucralose identified by high resolution mass spectrometry was a de-chlorinated molecule at m/z 362.0535, with molecular formula C12H20Cl2O8. Conclusions Online SPE LC-APCI/MS/MS developed in the study was applied to more than 100 environmental samples. Sucralose was frequently detected (>80%) indicating that the conventional treatment process employed in the sewage treatment plants is not efficient for its removal. Detection of sucralose in drinking waters suggests potential contamination of surface and ground waters sources with anthropogenic wastewater streams. Its high resistance to photodegradation, minimal sorption and high solubility indicate that sucralose could be a good tracer of anthropogenic wastewater intrusion into the environment.

Exploring the Use of Everglades Agricultural Area Canal Water as Base Medium for the Mass Production of Algae for Biofuels

Freshwater use is a major concern in the mass production of algae for biofuels. This project examined the use of canal water obtained from the Everglades Agricultural Area as a base medium for the mass production of algae. This water is not suitable for human consumption, and it is currently used for crop irrigation. A variety of canals were found to be suitable for water collection. Comparison of two methods for algal production showed no significant difference in biomass accumulation. It was discovered that synthetic reticulated foam can be used for algal biomass collection and harvest, and there is potential for its application in large-scale operations. Finally, it was determined that high alkaline conditions may help limit contaminants and competing organisms in growing algae cultures.

Data compilation on the biological response to ocean acidification: environmental and experimental context of data sets and related literature

The exponential growth of studies on the biological response to ocean acidification over the last few decades has generated a large amount of data. To facilitate data comparison, a data compilation hosted at the data publisher PANGAEA was initiated in 2008 and is updated on a regular basis (doi:10.1594/PANGAEA.149999). By January 2015, a total of 581 data sets (over 4 000 000 data points) from 539 papers had been archived. Here we present the developments of this data compilation five years since its first description by Nisumaa et al. (2010). Most of study sites from which data archived are still in the Northern Hemisphere and the number of archived data from studies from the Southern Hemisphere and polar oceans are still relatively low. Data from 60 studies that investigated the response of a mix of organisms or natural communities were all added after 2010, indicating a welcomed shift from the study of individual organisms to communities and ecosystems. The initial imbalance of considerably more data archived on calcification and primary production than on other processes has improved. There is also a clear tendency towards more data archived from multifactorial studies after 2010. For easier and more effective access to ocean acidification data, the ocean acidification community is strongly encouraged to contribute to the data archiving effort, and help develop standard vocabularies describing the variables and define best practices for archiving ocean acidification data.

Guimarães Rosa: travessia

This study develops a reading of five tales of Guimarães Rosa: “A hora e vez de Augusto Matraga”, “Campo Geral”, “A Benfazeja”, “Esses Lopes” and “Meu tio o Iauaretê”, included, respectively, in Sagarana, Corpo de Baile, Primeiras Estórias, Tutaméia and Estas Estórias. Then, undertakes a brief course in order to observe in the creative genesis of the author a manichean theme, whose view of the world, with poetic intensity, opposes the good and the evil. This research thus has drawn upon the Mythical Symbolism, in Literary Criticism and aspects of Metaphisics, in addition to analyzing classic literary essays in approaching of histories masterpiece structure. It has also demonstrate that in the rosiano hinterland the contradictory contributes to the speculation about the human man, in the continuous learning of his passing

(Tables, page 17-18), Chemical composition of ferromanganese concretions of the lakes of Northern Savolax in Eastern Finland

This paper discusses the occurrence of manganese-rich concretions in the lakes of Northern Savolax in Eastern Finland. The samples were collected in the summer of 1905 and left to dry for 2 months at room temperature. The quantity of H2O mentionned in the analysis was obtained by dessication at 155°C. The amount of Mn is calulated as MnO2 although other valences might be present in the samples. The contents in CO2 and organic substances was not determined. J. Aschan determined that as a general rule, in Finland the manganese rich lake deposits are rather associated with soft bottom sediments while the iron rich deposits are more assocaited with hard or sandy bottoms.

Internal deformation and kinematic indicators within a tripartite Mass Transport Deposit, NW Argentina

Acknowledgments This work was carried out with support from CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) – Brazil, BG-Brazil and the University of Aberdeen. We would like to thank the following geologists for their support, camaraderie and countless hours of fieldwork: Claus Fallgatter, Victoria Valdez, Carla Puigdomenech, Guilherme Bozetti, Roberto Noll Filho and Arthur Giovannini, and we thank Lorena Moscardelli and an anonymous reviewer, whose constructive comments helped to improve the manuscript.

Characterization of urban aerosol in Cork city (Ireland) using aerosol mass spectrometry

Ambient wintertime background urban aerosol in Cork city, Ireland, was characterized using aerosol mass spectrometry. During the three-week measurement study in 2009, 93% of the ca. 1 350 000 single particles characterized by an Aerosol Time-of-Flight Mass Spectrometer (TSI ATOFMS) were classified into five organic-rich particle types, internally mixed to different proportions with elemental carbon (EC), sulphate and nitrate, while the remaining 7% was predominantly inorganic in nature. Non-refractory PM1 aerosol was characterized using a High Resolution Time-of-Flight Aerosol Mass Spectrometer (Aerodyne HR-ToF-AMS) and was also found to comprise organic aerosol as the most abundant species (62 %), followed by nitrate (15 %), sulphate (9 %) and ammonium (9 %), and chloride (5 %). Positive matrix factorization (PMF) was applied to the HR-ToF-AMS organic matrix, and a five-factor solution was found to describe the variance in the data well. Specifically, "hydrocarbon-like" organic aerosol (HOA) comprised 20% of the mass, "low-volatility" oxygenated organic aerosol (LV-OOA) comprised 18 %, "biomass burning" organic aerosol (BBOA) comprised 23 %, non-wood solid-fuel combustion "peat and coal" organic aerosol (PCOA) comprised 21 %, and finally a species type characterized by primary m/z peaks at 41 and 55, similar to previously reported "cooking" organic aerosol (COA), but possessing different diurnal variations to what would be expected for cooking activities, contributed 18 %. Correlations between the different particle types obtained by the two aerosol mass spectrometers are also discussed. Despite wood, coal and peat being minor fuel types used for domestic space heating in urban areas, their relatively low combustion efficiencies result in a significant contribution to PM1 aerosol mass (44% and 28% of the total organic aerosol mass and non-refractory total PM1, respectively).Ambient wintertime background urban aerosol in Cork city, Ireland, was characterized using aerosol mass spectrometry. During the three-week measurement study in 2009, 93% of the ca. 1 350 000 single particles characterized by an Aerosol Time-of-Flight Mass Spectrometer (TSI ATOFMS) were classified into five organic-rich particle types, internally mixed to different proportions with elemental carbon (EC), sulphate and nitrate, while the remaining 7% was predominantly inorganic in nature. Non-refractory PM1 aerosol was characterized using a High Resolution Time-of-Flight Aerosol Mass Spectrometer (Aerodyne HR-ToF-AMS) and was also found to comprise organic aerosol as the most abundant species (62 %), followed by nitrate (15 %), sulphate (9 %) and ammonium (9 %), and chloride (5 %). Positive matrix factorization (PMF) was applied to the HR-ToF-AMS organic matrix, and a five-factor solution was found to describe the variance in the data well. Specifically, "hydrocarbon-like" organic aerosol (HOA) comprised 20% of the mass, "low-volatility" oxygenated organic aerosol (LV-OOA) comprised 18 %, "biomass burning" organic aerosol (BBOA) comprised 23 %, non-wood solid-fuel combustion "peat and coal" organic aerosol (PCOA) comprised 21 %, and finally a species type characterized by primary m/z peaks at 41 and 55, similar to previously reported "cooking" organic aerosol (COA), but possessing different diurnal variations to what would be expected for cooking activities, contributed 18 %. Correlations between the different particle types obtained by the two aerosol mass spectrometers are also discussed. Despite wood, coal and peat being minor fuel types used for domestic space heating in urban areas, their relatively low combustion efficiencies result in a significant contribution to PM1 aerosol mass (44% and 28% of the total organic aerosol mass and non-refractory total PM1, respectively).

Source apportionment of PM2.5 in Cork Harbour, Ireland using a combination of single particle mass spectrometry and quantitative semi-continuous measurements

An aerosol time-of-flight mass spectrometer (ATOFMS) was deployed for the measurement of the size resolved chemical composition of single particles at a site in Cork Harbour, Ireland for three weeks in August 2008. The ATOFMS was co-located with a suite of semi-continuous instrumentation for the measurement of particle number, elemental carbon (EC), organic carbon (OC), sulfate and particulate matter smaller than 2.5 μm in diameter (PM2.5). The temporality of the ambient ATOFMS particle classes was subsequently used in conjunction with the semi-continuous measurements to apportion PM2.5 mass using positive matrix factorisation. The synergy of the single particle classification procedure and positive matrix factorisation allowed for the identification of six factors, corresponding to vehicular traffic, marine, long-range transport, various combustion, domestic solid fuel combustion and shipping traffic with estimated contributions to the measured PM2.5 mass of 23%, 14%, 13%, 11%, 5% and 1.5% respectively. Shipping traffic was found to contribute 18% of the measured particle number (20–600 nm mobility diameter), and thus may have important implications for human health considering the size and composition of ship exhaust particles. The positive matrix factorisation procedure enabled a more refined interpretation of the single particle results by providing source contributions to PM2.5 mass, while the single particle data enabled the identification of additional factors not possible with typical semi-continuous measurements, including local shipping traffic.

The Relationship of Fear of Intimacy, Body Image Avoidance and Body Mass Index

Issues of body image and ability to achieve intimacy are connected to body weight, yet remain largely unexplored and have not been evaluated by gender. The underlying purpose of this research was to determine if avoidant attitudes and perceptions of one’s body may hold implications toward its use in intimate interactions, and if an above average body weight would tend to increase this avoidance. The National Health and Nutrition Examination Survey (NHANES, 1999-2002) finds that 64.5% of US adults are overweight, with 61.9% of women and 67.2% of men. The increasing prevalence of overweight and obesity in men and women shows no reverse trend, nor have prevention and treatment proven effective in the long term. The researcher gathered self-reported age, gender, height and weight data from 55 male and 58 female subjects (determined by a prospective power analysis with a desired medium effect size (r =.30) to determine body mass index (BMI), determining a mean age of 21.6 years and mean BMI of 25.6. Survey instruments consisted of two scales that are germane to the variables being examined. They were (1) Descutner and Thelen of the University of Missouri’s (1991) Fear-of-Intimacy scale and (2) Rosen, Srebnik, Saltzberg, and Wendt’s (1991) Body Image Avoidance Questionnaire. Results indicated that as body mass index increases, fear of intimacy increases (p<0.05) and that as body mass index increases, body image avoidance increases (p<0.05). The relationship that as body image avoidance increases, fear of intimacy increases was not supported, but approached significance at (p<0.07). No differences in these relationships were determined between gender groups. For age, the only observed relationship was that of a difference between scores for age groups [18 to 22 (group 1) and ages 23 to 34 (group 2)] for the relationship of body image avoidance and fear of intimacy (p<0.02). The results suggest that the relationship of body image avoidance and fear of intimacy, as well as age, bear consideration toward the escalating prevalence of overweight and obesity. An integrative approach to body weight that addresses issues of body image and intimacy may prove effective in prevention and treatment.