Role of lipid-mobilising factor (LMF) in protecting tumour cells from oxidative damage

Lipid-mobilising factor (LMF) is produced by cachexia-inducing tumours and is involved in the degradation of adipose tissue, with increased oxidation of the released fatty acids through an induction of uncoupling protein (UCP) expression. Since UCP-2 is thought to be involved in the detoxification of free radicals if LMF induced UCP-2 expression in tumour cells, it might attenuate free radical toxicity. As a model system we have used MAC13 tumour cells, which do not produce LMF. Addition of LMF caused a concentration-dependent increase in UCP-2 expression, as determined by immunoblotting. This effect was attenuated by the β3 antagonist SR59230A, suggesting that it was mediated through a β3 adrenoreceptor. Co-incubation of LMF with MAC13 cells reduced the growth-inhibitory effects of bleomycin, paraquat and hydrogen peroxide, known to be free radical generators, but not chlorambucil, an alkylating agent. There was no effect of LMF alone on cellular proliferation. These results indicate that LMF antagonises the antiproliferative effect of agents working through a free radical mechanism, and may partly explain the unresponsiveness to the chemotherapy of cachexia-inducing tumours. © 2004 Cancer Research UK.

Hypothesis: is Alzheimer's disease a metal-induced immune disorder?

A hypothesis that a metal-induced immune disorder may be involved in the pathogenesis of some forms of Alzheimer's disease (AD) is presented. The classical complement pathway is activated in AD and T cells and reactive microglia appear in the brain. Studies of metal induced autoimmunity and the use of compounds containing aluminium as vaccine adjuvants suggest that metals can activate complement and can be taken up by antigen presenting cells. The consequent immune response could contribute to neuronal damage, beta-amyloid deposition and cell death. The strengths and weaknesses of this hypothesis are discussed and tests of some aspects are proposed.

An aqueous extract of Fagonia cretica induces DNA damage, cell cycle arrest and apoptosis in breast cancer cells via FOXO3a and p53 expression

Background - Plants have proved to be an important source of anti-cancer drugs. Here we have investigated the cytotoxic action of an aqueous extract of Fagonia cretica, used widely as a herbal tea-based treatment for breast cancer. Methodology/Principal Findings - Using flow cytometric analysis of cells labeled with cyclin A, annexin V and propidium iodide, we describe a time and dose-dependent arrest of the cell cycle in G0/G1 phase of the cell cycle and apoptosis following extract treatment in MCF-7 (WT-p53) and MDA-MB-231 (mutant-p53) human breast cancer cell lines with a markedly reduced effect on primary human mammary epithelial cells. Analysis of p53 protein expression and of its downstream transcription targets, p21 and BAX, revealed a p53 associated growth arrest within 5 hours of extract treatment and apoptosis within 24 hours. DNA double strand breaks measured as ?-H2AX were detected early in both MCF-7 and MDA-MB-231 cells. However, loss of cell viability was only partly due to a p53-driven response; as MDA-MB-231 and p53-knockdown MCF-7 cells both underwent cell cycle arrest and death following extract treatment. p53-independent growth arrest and cytotoxicity following DNA damage has been previously ascribed to FOXO3a expression. Here, in MCF-7 and MDA-MB-231 cells, FOXO3a expression was increased significantly within 3 hours of extract treatment and FOXO3 siRNA reduced the extract-induced loss of cell viability in both cell lines. Conclusions/Significance - Our results demonstrate for the first time that an aqueous extract of Fagonia cretica can induce cell cycle arrest and apoptosis via p53-dependent and independent mechanisms, with activation of the DNA damage response. We also show that FOXO3a is required for activity in the absence of p53. Our findings indicate that Fagonia cretica aqueous extract contains potential anti-cancer agents acting either singly or in combination against breast cancer cell proliferation via DNA damage-induced FOXO3a and p53 expression.

The predictive ability of clinical tests for contact lens induced dry eye

Approximately half of current contact lens wearers suffer from dryness and discomfort, particularly towards the end of the day. Contact lens practitioners have a number of dry eye tests available to help them to predict which of their patients may be at risk of contact lens drop out and advise them accordingly. This thesis set out to rationalize them to see if any are of more diagnostic significance than others. This doctorate has found: (1) The Keratograph, a device which permits an automated, examiner independent technique for measuring non invasive tear break up time (NITBUT) measured NITBUT consistently shorter than measurements recorded with the Tearscope. When measuring central corneal curvature the spherical equivalent power of the cornea was measured as being significantly flatter than with a validated automated keratometer. (2) Non-invasive and invasive tear break-up times significantly correlated to each other, but not the other tear metrics. Symptomology, assessed using the OSDI questionnaire, correlated more with those tests indicating possible damage to the ocular surface (including LWE, LIPCOF and conjunctival staining) than with tests of either tear volume or stability. Cluster analysis showed some statistically significant groups of patients with different sign and symptom profiles. The largest cluster demonstrated poor tear quality with both non-invasive and invasive tests, low tear volume and more symptoms. (3) Care should be taken in fitting patients new to contact lenses if they have a NITBUT less than 10s or an OSDI comfort rating greater than 4.2 as they are more likely to drop-out within the first 6 months. Cluster analysis was not found to be beneficial in predicting which patients will succeed with lenses and which will not. A combination of the OSDI questionnaire and a NITBUT measurement was most useful both in diagnosing dry eye and in predicting contact lens drop out.

Shear induced toughening in bonded joints:experiments and analysis

Bonded joint specimens were fabricated from composite adherends and either an epoxy or a urethane adhesive. In mixed-mode fracture experiments, the epoxy bonded specimens generally failed by subinterfacial fracture in the composite, while specimens bonded with urethane failed very close to the adhesive/substrate interface. For the epoxy bonded specimens, fracture toughness did not change significantly with mode-mix, but for urethane bonded joints, fracture toughness increased with increasing shear load. Finite element analysis, which modeled specimens bonded with the two adhesives, showed similar trends. The different toughening behaviors for the two bonded joints can be attributed to dissipation of energy through inelastic deformation, which was insignificant in the epoxy-bonded joints but substantial when the urethane was used as the bonding agent.

Prediction of welding distortion in butt joint of thin plates

This paper investigates distortions and residual stresses induced in butt joint of thin plates using Metal Inert Gas welding. A moving distributed heat source model based on Goldak's double-ellipsoid heat flux distribution is implemented in Finite Element (FE) simulation of the welding process. Thermo-elastic-plastic FE methods are applied to modelling thermal and mechanical behaviour of the welded plate during the welding process. Prediction of temperature variations, fusion zone and heat affected zone as well as longitudinal and transverse shrinkage, angular distortion, and residual stress is obtained. FE analysis results of welding distortions are compared with existing experimental and empirical predictions. The welding speed and plate thickness are shown to have considerable effects on welding distortions and residual stresses. © 2009 Elsevier Ltd. All rights reserved.

In vitro protective effect and antioxidant mechanism of Resveratrol induced by Dapsone Hydroxylamine in human cells

Dapsone (DDS) hydroxylamine metabolites cause oxidative stress- linked adverse effects in patients, such as methemoglobin formation and DNA damage. This study evaluated the ameliorating effect of the antioxidant resveratrol (RSV) on DDS hydroxylamine (DDSNHOH) mediated toxicity in vitro using human erythrocytes and lymphocytes. The antioxidant mechanism was also studied using in-silico methods. In addition, RSV provided intracellular protection by inhibiting DNA damage in human lymphocytes induced by DDS-NHOH. However, whilst pretreatment with RSV (10-1000 μM significantly attenuated DDS-NHOH-induced methemoglobinemia, but it was not only significantly less effective than methylene blue (MET), but also post-treatment with RSV did not reverse methemoglobin formation, contrarily to that observed with MET. DDS-NHOH inhibited catalase (CAT) activity and reactive oxygen species (ROS) generation, but did not alter superoxide dismutase (SOD) activity in erythrocytes. Pretreatment with RSV did not alter these antioxidant enzymes activities in erythrocytes treated with DDS-NHOH. Theoretical calculations using density functional theory methods showed that DDS-NHOH has a pro-oxidant effect, whereas RSV and MET have antioxidant effect on ROS. The effect on methemoglobinemia reversion for MET was significantly higher than that of RSV. These data suggest that the pretreatment with resveratrol may decrease heme-iron oxidation and DNA damage through reduction of ROS generated in cells during DDS therapy.

Rapid tonicity induced re-localisation of endogenous aquaporin 4 in primary rat astrocytes - a therapeutic target for cytotoxic brain oedema?

It is estimated that 69-75 million people worldwide will suffer a traumatic brain injury (TBI) or stroke each year. Brain oedema caused by TBI or following a stroke, together with other disorders of the brain cost Europe €770 billion in 2014. Aquaporins (AQP) are transmembrane water channels involved in many physiologies and are responsible for the maintenance of water homeostasis. They react rapidly to changes in osmolarity by transporting water through their highly selective central pore to maintain tonicity and aid in cell volume regulation. We have previously shown that recombinant AQP1-GFP trafficking occurs in a proteinkinase C-microtubule dependant manner in HEK-293 cells in response to hypotonicity. This trafficking mechanism is also reliant on the presence of calcium and its messenger-binding protein calmodulin and results in increased cell surface expression of AQP1 in a time-scale of ~30 seconds. There is currently very little research into the trafficking mechanisms of endogenous AQPs in primary cells. AQP4 is the most abundantly expressed AQP within the brain, it is localised to the astrocytic end-feet, in contact with the blood vessels at the blood-brain-barrier. In situations where the exquisitely-tuned osmotic balance is disturbed, high water permeability can become detrimental. AQP4-mediated water influx causes rapid brain swelling, resulting in death or long term brain damage. Previous research has shown that AQP4 knock-out mice were protected from the formation of cytotoxic brain oedema in a stroke model, highlighting AQP4 as a key drug target for this pathology. As there are currently no treatments available to restrict the flow of water through AQP4 as all known inhibitors are either cytotoxic or non-specific, controlling the mechanisms involved in the regulation of AQP4 in the brain could provide a therapeutic solution to such diseases. Using cell surface biontinylation of endogenous AQP4 in primary rat astrocytes followed by neutraavidin based ELISA we have shown that AQP4 cell surface localisation increases by 2.7 fold after 5 minutes hypotonic treatment at around 85 mOsm/kg H2O. We have also shown that this rapid relocalisation of AQP4 is regulated by PKA, calmodulin, extra-cellular calcium and actin. In summary we have shown that rapid translocation of endogenous AQP4 occurs in primary rat astrocytes in response to hypotonic stimuli; this mechanism is PKA, calcium, actin and calmodulin dependant. AQP4 has the potential to provide a treatment for the development of brain oedema.

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 discrimination and association of float glass and the quantitative analysis of liquids from aerosols and microdrops using laser induced breakdown spectroscopy

Glass is a common form of trace evidence found at many scenes of crimes in the form of small fragments. These glass fragments can transfer to surrounding objects and/or persons and may provide forensic investigators valuable information to link a suspect to the scene of a crime. Since the elemental composition of different glass sources can be very similar, a highly discriminating technique is required to distinguish between fragments that have originated from different sources. ^ The research presented here demonstrates that Laser Induced Breakdown Spectroscopy (LIBS) is a viable analytical technique for the association and discrimination of glass fragments. The first part of this research describes the optimization of the LIBS experiments including the use of different laser wavelengths to investigate laser-material interaction. The use of a 266 nm excitation laser provided the best analytical figures of merit with minimal damage to the sample. The resulting analytical figures of merit are presented. The second part of this research evaluated the sensitivity of LIBS to associate or discriminate float glass samples originating from the same manufacturing plants and produced at approximately the same time period. Two different sample sets were analyzed ranging in manufacturing dates from days to years apart. Eighteen (18) atomic emission lines corresponding to the elements Sr, K, Fe, Ca, Al, Ba, Na, Mg and Ti, were chosen because of their detection above the method detection limits and for presenting differences between the samples. Ten elemental ratios producing the most discrimination were selected for each set. When all the ratios are combined in a comparison, 99% of the possible pairs were discriminated using the optimized LIBS method generating typical analytical precisions of ∼5% RSD. ^ The final study consisted of the development of a new approach for the use of LIBS as a quantitative analysis of ultra-low volume solution analysis using aerosols and microdrops. Laser induced breakdown spectroscopy demonstrated to be an effective technique for the analysis of as low as 90 pL for microdrop LIBS with 1 pg absolute LOD and 20 µL for aerosol LIBS with an absolute LOD of ∼100 fg.^

Effects of architectural features of air-permeable roof cladding materials on wind-induced uplift loading

Widespread damage to roofing materials (such as tiles and shingles) for low-rise buildings, even for weaker hurricanes, has raised concerns regarding design load provisions and construction practices. Currently the building codes used for designing low-rise building roofs are mainly based on testing results from building models which generally do not simulate the architectural features of roofing materials that may significantly influence the wind-induced pressures. Full-scale experimentation was conducted under high winds to investigate the effects of architectural details of high profile roof tiles and asphalt shingles on net pressures that are often responsible for damage to these roofing materials. Effects on the vulnerability of roofing materials were also studied. Different roof models with bare, tiled, and shingled roof decks were tested. Pressures acting on both top and bottom surfaces of the roofing materials were measured to understand their effects on the net uplift loading. The area-averaged peak pressure coefficients obtained from bare, tiled, and shingled roof decks were compared. In addition, a set of wind tunnel tests on a tiled roof deck model were conducted to verify the effects of tiles' cavity internal pressure. Both the full-scale and the wind tunnel test results showed that underside pressure of a roof tile could either aggravate or alleviate wind uplift on the tile based on its orientation on the roof with respect to the wind angle of attack. For shingles, the underside pressure could aggravate wind uplift if the shingle is located near the center of the roof deck. Bare deck modeling to estimate design wind uplift on shingled decks may be acceptable for most locations but not for field locations; it could underestimate the uplift on shingles by 30-60%. In addition, some initial quantification of the effects of roofing materials on wind uplift was performed by studying the wind uplift load ratio for tiled versus bare deck and shingled versus bare deck. Vulnerability curves, with and without considering the effects of tiles' cavity internal pressure, showed significant differences. Aerodynamic load provisions for low-rise buildings' roofs and their vulnerability can thus be more accurately evaluated by considering the effects of the roofing materials.

Development of Innovative Load Transfer Mechanism to Reduce Hurricane-Induced Failures in New and Existing Residential Construction

Implicit in current design practice of minimum uplift capacity, is the assumption that the connection's capacity is proportional to the number of fasteners per connection joint. This assumption may overestimate the capacity of joints by a factor of two or more and maybe the cause of connection failures in extreme wind events. The current research serves to modify the current practice by proposing a realistic relationship between the number of fasteners and the capacity of the joint. The research is also aimed at further development of non-intrusive continuous load path (CLP) connection system using Glass Fiber Reinforced Polymer (GFRP) and epoxy. Suitable designs were developed for stud to top plate and gable end connections and tests were performed to evaluate the ultimate load, creep and fatigue behavior. The objective was to determine the performance of the connections under simulated sustained hurricane conditions. The performance of the new connections was satisfactory.

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.

Efeitos do Kinesio Taping® sobre os marcadores clínicos indiretos de dano muscular induzido pelo exercício excêntrico: ensaio clínico randomizado

Objective: analyze the effect of Kinesio Taping (KT) on the indirect clinical markers of muscle damage induced by eccentric exercises in the elbow flexors in healthy individuals. Materials and methods: It is a randomized controlled trial involving sixty volunteers at age group between 18 and 28 years randomly selected. The sample into three groups with twenty participants: control group (CG) – eccentric protocol without KT, KT group – eccentric with tensioned KT, placebo group – eccentric protocol KT with no tension. The evaluations took place at four moments; the first one was the basis line (AV1), after the second protocol (AV2) and the following two groups 24 (AV3) and 48 hours (AV4) after the intervention protocol. The muscle damage was induced by sixteen maximum eccentric contractions of the elbow flexors from the non-dominant limb, divided in two sets of eight repetitions, at 60º/s, with two minutes interval. The variables analyzed were: the joint amplitude in rest, the level of pain, the joint position sense (JPS) followed of isokinetic checking with electromyographic sign capitation. These data were analyzed in software SPSS 20.0. The normality was identified by Kolmogorov-Smimov examination and then, being used the ANOVA mixed model with significance of 5%. Outcomes: a decrease was observed at joint amplitude moreover, an immediate increase of pain wich increased after 24 and remained until 48 hours at all groups searched. There was not difference at the JPS. The variables peak torque, average peak torque, total work and mean power mean reduced until 48 hours after muscle lesion in all groups. Among the groups, there was no difference in EMG values and for any of the variables. Conclusion: The KT did not influence at the indirect clinical markers of muscle lesion induced by eccentric exercises in the elbow flexors in healthy people.

Efeitos do Kinesio Taping® sobre os marcadores clínicos indiretos de dano muscular induzido pelo exercício excêntrico: ensaio clínico randomizado

Objective: analyze the effect of Kinesio Taping (KT) on the indirect clinical markers of muscle damage induced by eccentric exercises in the elbow flexors in healthy individuals. Materials and methods: It is a randomized controlled trial involving sixty volunteers at age group between 18 and 28 years randomly selected. The sample into three groups with twenty participants: control group (CG) – eccentric protocol without KT, KT group – eccentric with tensioned KT, placebo group – eccentric protocol KT with no tension. The evaluations took place at four moments; the first one was the basis line (AV1), after the second protocol (AV2) and the following two groups 24 (AV3) and 48 hours (AV4) after the intervention protocol. The muscle damage was induced by sixteen maximum eccentric contractions of the elbow flexors from the non-dominant limb, divided in two sets of eight repetitions, at 60º/s, with two minutes interval. The variables analyzed were: the joint amplitude in rest, the level of pain, the joint position sense (JPS) followed of isokinetic checking with electromyographic sign capitation. These data were analyzed in software SPSS 20.0. The normality was identified by Kolmogorov-Smimov examination and then, being used the ANOVA mixed model with significance of 5%. Outcomes: a decrease was observed at joint amplitude moreover, an immediate increase of pain wich increased after 24 and remained until 48 hours at all groups searched. There was not difference at the JPS. The variables peak torque, average peak torque, total work and mean power mean reduced until 48 hours after muscle lesion in all groups. Among the groups, there was no difference in EMG values and for any of the variables. Conclusion: The KT did not influence at the indirect clinical markers of muscle lesion induced by eccentric exercises in the elbow flexors in healthy people.