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.

Importance of water source in controlling leaf leaching losses in a dwarf red mangrove (Rhizophora mangle L.) wetland

The southern Everglades mangrove ecotone is characterized by extensive dwarf Rhizophora mangle L. shrub forests with a seasonally variable water source (Everglades – NE Florida Bay) and residence times ranging from short to long. We conducted a leaf leaching experiment to understand the influence that water source and its corresponding water quality have on (1) the early decay of R. mangle leaves and (2) the early exchange of total organic carbon (TOC) and total phosphorus (TP) between leaves and the water column. Newly senesced leaves collected from lower Taylor River (FL) were incubated in bottles containing water from one of three sources (Everglades, ambient mangrove, and Florida Bay) that spanned a range of salinity from 0 to 32‰, [TOC] from 710 to 1400 μM, and [TP] from 0.17 to 0.33 μM. We poisoned half the bottles in order to quantify abiotic processes (i.e., leaching) and assumed that non-poisoned bottles represented both biotic (i.e., microbial) and abiotic processes. We sacrificed bottles after 1,2, 5, 10, and 21 days of incubation and quantified changes in leaf mass and changes in water column [TOC] and [TP]. We saw 10–20% loss of leaf mass after 24 h—independent of water treatment—that leveled off by Day 21. After 3 weeks, non-poisoned leaves lost more mass than poisoned leaves, and there was only an effect of salinity on mass loss in poisoned incubations—with greatest leaching-associated losses in Everglades freshwater. Normalized concentrations of TOC in the water column increased by more than two orders of magnitude after 21 days with no effect of salinity and no difference between poisoned and non-poisoned treatments. However, normalized [TP] was lower in non-poisoned incubations as a result of immobilization by epiphytic microbes. This immobilization was greatest in Everglades freshwater and reflects the high P demand in this ecosystem. Immobilization of leached P in mangrove water and Florida Bay water was delayed by several days and may indicate an initial microbial limitation by labile C during the dry season.

Elemental analysis of biological matrices by laser ablation high resolution inductively coupled plasma mass spectrometry (LA-HR-ICP-MS) and high resolution inductively coupled plasma mass spectrometry (HR -ICP -MS)

The need for elemental analysis of biological matrices such as bone, teeth, and plant matter for sourcing purposes has emerged within the forensic and geochemical laboratories. Trace elemental analyses for the comparison of materials such as glass by inductively coupled plasma mass spectrometry (ICP-MS) and laser ablation ICP-MS has been shown to offer a high degree of discrimination between different manufacturing sources. Unit resolution ICP-MS instruments may suffer from some polyatomic interferences including 40Ar16O+, 40Ar 16O1H+, and 40Ca 16O+ that affect iron measurement at trace levels. Iron is an important element in the analysis of glass and also of interest for the analysis of several biological matrices. A comparison of the analytical performance of two different ICP-MS systems for iron analysis in glass for determining the method detection limits (MDLs), accuracy, and precision of the measurement is presented. Acid digestion and laser ablation methods are also compared. Iron polyatomic interferences were reduced or resolved by using dynamic reaction cell and high resolution ICP-MS. MDLs as low as 0.03 μg g-1 and 0.14 μg g-1 for laser ablation and solution based analyses respectively were achieved. The use of helium as a carrier gas demonstrated improvement in the detection limits of both iron isotopes (56Fe and 57Fe) in medium resolution for the HR-ICP-MS and with a dynamic reaction cell (DRC) coupled to a quadrupole ICP-MS system. ^ The development and application of robust analytical methods for the quantification of trace elements in biological matrices has lead to a better understanding of the potential utility of these measurements in forensic chemical analyses. Standard reference materials (SRMs) were used in the development of an analytical method using HR-ICP-MS and LA-HR-ICP-MS that was subsequently applied on the analysis of real samples. Bone, teeth and ashed marijuana samples were analyzed with the developed method. ^ Elemental analysis of bone samples from 12 different individuals provided discrimination between individuals, when femur and humerus bones were considered separately. Discrimination of 14 teeth samples based on elemental composition was achieved with the exception of one case where samples from the same individual were not associated with each other. The discrimination of 49 different ashed plant (cannabis) samples was achieved using the developed method. ^

Temperature, salinity, methane concentration, oxidation rates and methanotrophic cell number at Svalbard seeps in summer 2011 and 2012 (cruises POS419 and MSM21/4)

Large amounts of the greenhouse gas methane are released from the seabed to the water column where it may be consumed by aerobic methanotrophic bacteria. This microbial filter is consequently the last marine sink for methane before its liberation to the atmosphere. The size and activity of methanotrophic communities, which determine the capacity of the water column methane filter, are thought to be mainly controlled by nutrient and redox dynamics, but little is known about the effects of ocean currents. Here, we report measurements of methanotrophic activity and biomass (CARD-FISH) at methane seeps west of Svalbard, and related them to physical water mass properties (CTD) and modelled current dynamics. We show that cold bottom water containing a large number of aerobic methanotrophs was rapidly displaced by warmer water with a considerably smaller methanotrophic community. This water mass exchange, caused by short-term variations of the West Spitsbergen Current, constitutes a rapid oceanographic switch severely reducing methanotrophic activity in the water column. Strong and fluctuating currents are widespread oceanographic features common at many methane seep systems and are thus likely to globally affect methane oxidation in the ocean water column.

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.

Data on the biogeochemical cycle of methane in the ocean

Geological, mineralogical and microbiological aspects of the methane cycle in water and sediments of different areas in the oceans are under consideration in the monograph. Original and published estimations of formation- and oxidation rates of methane with use of radioisotope and isotopic methods are given. The role of aerobic and anaerobic microbial oxidation of methane in production of organic matter and in formation of authigenic carbonates is considered. Particular attention is paid to processes of methane transformation in areas of its intensive input to the water column from deep-sea hydrothermal sources, mud volcanoes, and cold methane seeps.

Stable isotope ratios of a oyster Hyotissa hyotis from the northern Red Sea

This study explores the giant oyster Hyotissa hyotis as a novel environmental archive in tropical reef environments of the Indo-Pacific. The species is a typical accessory component in coral reefs, can reach sizes of tens of centimetres, and dates back to the Late Pleistocene. Here, a 70.2-mm-long oxygen and carbon isotope transect through the shell of a specimen collected at Safaga Bay, northern Red Sea, in May 1996, is presented. The transect runs perpendicularly to the foliate and vesicular layers of the inner ostracum near the ligament area of the oyster. The measured d18O and d13C records show sinusoidal fluctuations, which are independent of shell microstructure. The d13C fluctuations exhibit the same wavelength as the d18O fluctuations but are phase shifted. The d18O record reflects the sea surface temperature variations from 1957 until 1996, possibly additionally influenced by the local evaporation. Due to locally enhanced evaporation in the semi-enclosed Safaga Bay, the d18Oseawater value is estimated at 2.17 per mil, i.e., 0.3-0.8 per mil higher than published open surface water d18O values (1.36-1.85 per mil) from the region. The mean water temperature deviates by only 0.4°C from the expected value, and the minimum and maximum values are 0.5°C lower and 2.9°C higher, respectively. When comparing the mean monthly values, however, the sea surface temperature discrepancy between reconstructed and global grid datasets is always <1.0°C. The d13C signal is weakly negatively correlated with regional chlorophyll a concentration and with the sunshine duration, which may reflect changes in the bivalve's respiration. The study emphasises the palaeogeographic context in isotope studies based on fossils, because coastal embayments might not reflect open-water oceanographic conditions.

Star formation in a stellar mass-selected sample of galaxies to z=3 from the GOODS-NICMOS Survey

We present a study of the star-forming properties of a stellar mass-selected sample of galaxies in the GOODS (Great Observatories Origins Deep Survey) NICMOS Survey (GNS), based on deep Hubble Space Telescope (HST) imaging of the GOODS North and South fields. Using a stellar mass-selected sample, combined with HST/ACS and Spitzer data to measure both ultraviolet (UV) and infrared-derived star formation rates (SFRs), we investigate the star forming properties of a complete sample of ∼1300 galaxies down to log M_*= 9.5 at redshifts 1.5 < z < 3. Eight per cent of the sample is made up of massive galaxies with M_*≥ 10^11 M_⊙. We derive optical colours, dust extinctions and UV and infrared SFR to determine how the SFR changes as a function of both stellar mass and time. Our results show that SFR increases at higher stellar mass such that massive galaxies nearly double their stellar mass from star formation alone over the redshift range studied, but the average value of SFR for a given stellar mass remains constant over this ∼2 Gyr period. Furthermore, we find no strong evolution in the SFR for our sample as a function of mass over our redshift range of interest; in particular we do not find a decline in the SFR among massive galaxies, as is seen at z < 1. The most massive galaxies in our sample (log M_*≥ 11) have high average SFRs with values SFR_UV, corr= 103 ± 75 M_⊙ yr^−1, and yet exhibit red rest-frame (U−B) colours at all redshifts. We conclude that the majority of these red high-redshift massive galaxies are red due to dust extinction. We find that A_2800 increases with stellar mass, and show that between 45 and 85 per cent of massive galaxies harbour dusty star formation. These results show that even just a few Gyr after the first galaxies appear, there are strong relations between the global physical properties of galaxies, driven by stellar mass or another underlying feature of galaxies strongly related to the stellar mass.

Stochastic descriptors to study the fate and potential of naive T cell clonotypes in the periphery

The population of naive T cells in the periphery is best described by determining both its T cell receptor diversity, or number of clonotypes, and the sizes of its clonal subsets. In this paper, we make use of a previously introduced mathematical model of naive T cell homeostasis, to study the fate and potential of naive T cell clonotypes in the periphery. This is achieved by the introduction of several new stochastic descriptors for a given naive T cell clonotype, such as its maximum clonal size, the time to reach this maximum, the number of proliferation events required to reach this maximum, the rate of contraction of the clonotype during its way to extinction, as well as the time to a given number of proliferation events. Our results show that two fates can be identified for the dynamics of the clonotype: extinction in the short-term if the clonotype experiences too hostile a peripheral environment, or establishment in the periphery in the long-term. In this second case the probability mass function for the maximum clonal size is bimodal, with one mode near one and the other mode far away from it. Our model also indicates that the fate of a recent thymic emigrant (RTE) during its journey in the periphery has a clear stochastic component, where the probability of extinction cannot be neglected, even in a friendly but competitive environment. On the other hand, a greater deterministic behaviour can be expected in the potential size of the clonotype seeded by the RTE in the long-term, once it escapes extinction.

Regulatory factors controlling muscle mass : competition between innate immune function and anabolic signals in regulation of atrogin-1 in Atlantic salmon

The research was supported by an industrial PhD studentship between University of Aberdeen and by BioMar Ltd., for Z. Heidari.

Regulatory factors controlling muscle mass : competition between innate immune function and anabolic signals in regulation of atrogin-1 in Atlantic salmon

The research was supported by an industrial PhD studentship between University of Aberdeen and by BioMar Ltd., for Z. Heidari.

Fast and efficient : Postnatal growth and energy expenditure in an Arctic-breeding waterbird, the Red-throated Loon (Gavia stellata)

Peer reviewed

Regulatory factors controlling muscle mass : competition between innate immune function and anabolic signals in regulation of atrogin-1 in Atlantic salmon

The research was supported by an industrial PhD studentship between University of Aberdeen and by BioMar Ltd., for Z. Heidari.

Investigating the Structure of FtsZ to Understand its Functional Role in Bacterial Cell Division

FtsZ, a bacterial tubulin homologue, is a cytoskeleton protein that plays key roles in cytokinesis of almost all prokaryotes. FtsZ assembles into protofilaments (pfs), one subunit thick, and these pfs assemble further to form a “Z ring” at the center of prokaryotic cells. The Z ring generates a constriction force on the inner membrane, and also serves as a scaffold to recruit cell-wall remodeling proteins for complete cell division in vivo. FtsZ can be subdivided into 3 main functional regions: globular domain, C terminal (Ct) linker, and Ct peptide. The globular domain binds GTP to assembles the pfs. The extreme Ct peptide binds membrane proteins to allow cytoplasmic FtsZ to function at the inner membrane. The Ct linker connects the globular domain and Ct peptide. In the present studies, we used genetic and structural approaches to investigate the function of Escherichia coli (E. coli) FtsZ. We sought to examine three questions: (1) Are lateral bonds between pfs essential for the Z ring? (2) Can we improve direct visualization of FtsZ in vivo by engineering an FtsZ-FP fusion that can function as the sole source of FtsZ for cell division? (3) Is the divergent Ct linker of FtsZ an intrinsically disordered peptide (IDP)?

One model of the Z ring proposes that pfs associate via lateral bonds to form ribbons; however, lateral bonds are still only hypothetical. To explore potential lateral bonding sites, we probed the surface of E. coli FtsZ by inserting either small peptides or whole FPs. Of the four lateral surfaces on FtsZ pfs, we obtained inserts on the front and back surfaces that were functional for cell division. We concluded that these faces are not sites of essential interactions. Inserts at two sites, G124 and R174 located on the left and right surfaces, completely blocked function, and were identified as possible sites for essential lateral interactions. Another goal was to find a location within FtsZ that supported fusion of FP reporter proteins, while allowing the FtsZ-FP to function as the sole source of FtsZ. We discovered one internal site, G55-Q56, where several different FPs could be inserted without impairing function. These FtsZ-FPs may provide advances for imaging Z-ring structure by super-resolution techniques.

The Ct linker is the most divergent region of FtsZ in both sequence and length. In E. coli FtsZ the Ct linker is 50 amino acids (aa), but for other FtsZ it can be as short as 37 aa or as long as 250 aa. The Ct linker has been hypothesized to be an IDP. In the present study, circular dichroism confirmed that isolated Ct linkers of E. coli (50 aa) and C. crescentus (175 aa) are IDPs. Limited trypsin proteolysis followed by mass spectrometry (LC-MS/MS) confirmed Ct linkers of E. coli (50 aa) and B. subtilis (47 aa) as IDPs even when still attached to the globular domain. In addition, we made chimeras, swapping the E. coli Ct linker for other peptides and proteins. Most chimeras allowed for normal cell division in E. coli, suggesting that IDPs with a length of 43 to 95 aa are tolerated, sequence has little importance, and electrostatic charge is unimportant. Several chimeras were purified to confirm the effect they had on pf assembly. We concluded that the Ct linker functions as a flexible tether allowing for force to be transferred from the FtsZ pf to the membrane to constrict the septum for division.

Elemental Analysis of Biological Matrices by Laser Ablation High Resolution Inductively Coupled Plasma Mass Spectrometry (LA-HR-ICP-MS) and High Resolution Inductively Coupled Plasma Mass Spectrometry (HR-ICP-MS)

The need for elemental analysis of biological matrices such as bone, teeth, and plant matter for sourcing purposes has emerged within the forensic and geochemical laboratories. Trace elemental analyses for the comparison of aterials such as glass by inductively coupled plasma mass spectrometry (ICP-MS) and laser ablation ICP-MS has been shown to offer a high degree of discrimination between different manufacturing sources. Unit resolution ICP-MS instruments may suffer from some polyatomic interferences including 40Ar16O+, 40Ar16O1H+, and 40Ca16O+ that affect iron measurement at trace levels. Iron is an important element in the analysis of glass and also of interest for the analysis of several biological matrices. A comparison of the nalytical performance of two different ICP-MS systems for iron analysis in glass for determining the method detection limits (MDLs), accuracy, and precision of the measurement is presented. Acid digestion and laser ablation methods are also compared. Iron polyatomic interferences were reduced or resolved by using dynamic reaction cell and high resolution ICP-MS. MDLs as low as 0.03 ìg g-1 and 0.14 ìg g-1 for laser ablation and solution based analyses respectively were achieved. The use of helium as a carrier gas demonstrated improvement in the detection limits of both iron isotopes (56Fe and 57Fe) in medium resolution for the HR-ICP-MS and with a dynamic reaction cell (DRC) coupled to a quadrupole ICP-MS system. The development and application of robust analytical methods for the quantification of trace elements in biological matrices has lead to a better understanding of the potential utility of these measurements in forensic chemical analyses. Standard reference materials (SRMs) were used in the development of an analytical method using HR-ICP-MS and LA-HR-ICP-MS that was subsequently applied on the analysis of real samples. Bone, teeth and ashed marijuana samples were analyzed with the developed method. Elemental analysis of bone samples from 12 different individuals provided discrimination between individuals, when femur and humerus bones were considered separately. Discrimination of 14 teeth samples based on elemental composition was achieved with the exception of one case where samples from the same individual were not associated with each other. The discrimination of 49 different ashed plant (cannabis)samples was achieved using the developed method.