Mechanism of muscle protein degradation in Cancer Cachexia

A protein-mobilising factor of estimated molecular weight 24 KDa (p24) was purified both from the cachexia-inducing MAC 16 tumour and the urine of cachectic cancer patients by a combination of ammonium sulphate precipitation and affinity chromatography using a monoclonal antibody developed against the murine material. Administration of p24 to non tumour-bearing mice caused a decrease in body weight 24 h after the first injection, which was attenuated by prior treatment with the monoclonal antibody. Loss of body weight was accompanied by an accelerated loss of skeletal muscle protein, as determined by the release of tyrosine from this tissue. This was associated with an increased release of PGE2 and both protein degradation and PGE2 release were attenuated by the monoclonal antibody. Loss of protein mass arose from both a decrease in the rate of protein synthesis and an elevation of protein breakdown; the latter due to an activation of the ubiquitin-proteasome proteolytic system. In isolated muscle, p24 was capable of promoting protein breakdown and this was also associated with increased PGE2 levels. Both tyrosine and PGE2 release, were inhibited by PGE2 inhibitors and a specific inhibitor of cPLA2. When added to muscle cells in culture, p24 caused an elevation in the rates of total and myofibrillar protein breakdown and a depression in the rate of protein synthesis which was inhabitable by short-term incubation in insulin, suggesting that p24 may inhibit protein synthesis by causing an arrest in the translational process.

Intermediate pyrolysis studies of aquatic biomass and potential applications in the BtVB-process

Aquatic biomass is seen as one of the major feedstocks to overcome difficulties associated with 1st generation biofuels, such as competition with food production, change of land use and further environmental issues. Although, this finding is widely accepted only little work has been carried out to investigate thermo-chemical conversion of algal specimen to produce biofuels, power and heat. This work aims at contributing fundamental knowledge for thermo-chemical processing of aquatic biomass via intermediate pyrolysis. Therefore, it was necessary to install and commission an analytical pyrolysis apparatus which facilitates intermediate pyrolysis process conditions as well as subsequent separation and detection of pyrolysates (Py- GC/MS). In addition, a methodology was established to analyse aquatic biomass under intermediate conditions by Thermo-Gravimetric Analysis (TGA). Several microalgae (e.g. Chlamydomonas reinhardtii, Chlorella vulgaris) and macroalgae specimen (e.g. Fucus vesiculosus) from main algal divisions and various natural habitats (fresh and saline water, temperate and polar climates) were chosen and their thermal degradation under intermediate pyrolysis conditions was studied. In addition, it was of interest to examine the contribution of biochemical constituents of algal biomass onto the chemical compounds contained in pyrolysates. Therefore, lipid and protein fractions were extracted from microalgae biomass and analysed separately. Furthermore, investigations of residual algal materials obtained by extraction of high valuable compounds (e.g. lipids, proteins, enzymes) were included to evaluate their potential for intermediate pyrolysis processing. On basis of these thermal degradation studies, possible applications of algal biomass and from there derived materials in the Bio-thermal Valorisation of Biomass-process (BtVB-process) are presented. It was of interest to evaluate the combination of the production of high valuable products and bioenergy generation derived by micro- and macro algal biomass.

Role of reactive oxygen species in protein degradation in murine myotubes induced by proteolysis-inducing factor and angiotensin II

The antioxidants butylated hydroxytoluene (BHT, 1 mM) and d-α-tocopherol (10 μM) completely attenuated protein degradation in murine myotubes in response to both proteolysis-inducing factor (PIF) and angiotensin II (Ang II), suggesting that the formation of reactive oxygen species (ROS) plays an important role in this process. Both PIF and Ang II induced a rapid and transient increase in ROS formation in myotubes, which followed a parabolic dose-response curve, similar to that for total protein degradation. Antioxidant treatment attenuated the increase in expression and activity of the ubiquitin-proteasome proteolytic pathway by PIF and Ang II, by preventing the activation of the transcription factor nuclear factor-κB (NF-κB), through inhibition of phosphorylation of the NF-κB inhibitor protein (I-κB) and its subsequent degradation. ROS formation by both PIF and Ang II was attenuated by diphenyleneiodonium (10 μM), suggesting that it was mediated through the NADPH oxidase system. ROS formation was also attenuated by trifluoroacetyl arachidonic acid (10 μM), a specific inhibitor of cytosolic phospholipase A2, U-73122 (5 μM) and D609 (200 μM), inhibitors of phospholipase C and calphostin C (300 nM), a highly specific inhibitor of protein kinase C (PKC), all known activators of NADPH oxidase. Myotubes containing a dominant-negative mutant of PKC did not show an increase in ROS formation in response to either PIF or Ang II. The two Rac1 inhibitors W56 (200 μM) and NSC23766 (10 μM) also attenuated both ROS formation and protein degradation induced by both PIF and Ang II. Rac1 is known to mediate signalling between the phosphatidylinositol-3 kinase (PI-3K) product and NADPH oxidase, and treatment with LY24002 (10 μM), a highly selective inhibitor of PI-3K, completely attenuated ROS production in response to both PIF and Ang II, and inhibited total protein degradation, while the inactive analogue LY303511 (100 μM) had no effect. ROS formation appears to be important in muscle atrophy in cancer cachexia, since treatment of weight losing mice bearing the MAC16 tumour with d-α-tocopherol (1 mg kg- 1) attenuated protein degradation and increased protein synthesis in skeletal muscle. © 2007 Elsevier Inc. All rights reserved.

Mechanism of induction of muscle protein degradation by angiotensin II

Angiotensin I and II have been shown to directly induce protein degradation in skeletal muscle through an increased activity and expression of the ubiquitin-proteasome proteolytic pathway. This investigation determines the role of the nuclear transcription factor nuclear factor-κB (NF-κB) in this process. Using murine myotubes as a surrogate model system both angiotensin I and II were found to induce activation of protein kinase C (PKC), with a parabolic dose-response curve similar to the induction of total protein degradation. Activation of PKC was required for the induction of proteasome expression, since calphostin C, a highly specific inhibitor of PKC, attenuated both the increase in total protein degradation and in proteasome expression and functional activity increased by angiotensin II. PKC is known to activate I-κB kinase (IKK), which is responsible for the phosphorylation and subsequent degradation of I-κB. Both angiotensin I and II induced an early decrease in cytoplasmic I-κB levels followed by nuclear accumulation of NF-κB. Using an NF-κB luciferase construct this was shown to increase transcriptional activation of NF-κB regulated genes. Maximal luciferase expression was seen at the same concentrations of angiotensin I/II as those inducing protein degradation. Total protein degradation induced by both angiotensin I and II was attenuated by resveratrol, which prevented nuclear accumulation of NF-κB, confirming that activation of NF-κB was responsible for the increased protein degradation. These results suggest that induction of proteasome expression by angiotensin I/II involves a signalling pathway involving PKC and NF-κB. © 2005 Elsevier Inc. All rights reserved.

Downregulation of ubiquitin-dependent protein degradation in murine myotubes during hyperthermia by eicosapentaenoic acid

Muscle atrophy in a number of acute wasting conditions is associated with an increased activity and expression of the ubiquitin-proteasome proteolytic pathway. Although different initiators are involved, it is possible that the intracellular signalling events leading to upregulation of this pathway are the same in all catabolic conditions. This study investigates hyperthermia in murine myotubes as a model for increased protein degradation through the ubiquitin-proteasome pathway. The effect of eicosapentaenoic acid (EPA) on this process should identify common elements, since EPA has been shown to attenuate induction of the ubiquitin-proteasome pathway in cancer cachexia. Increasing the temperature of myotubes caused a progressive increase in protein degradation. This was associated with an increased proteasome 'chymotrypsin-like' enzyme activity, as well as increased expression of both mRNA and protein for 20S proteasome subunits and the ubiquitin-conjugating enzyme (E214k). This upregulation was not seen in cultures treated with EPA (50 μM), suggesting that it acts to prevent transcriptional activation of the ubiquitin-proteasome pathway in hyperthermia. These results suggest that protein catabolism in hyperthermia and cancer cachexia is mediated through a common pathway. © 2005 Elsevier Inc. All rights reserved.

Pharmaceutical process optimisation of bulk lyophilisates:implications of powder handling

Lyophilisation or freeze drying is the preferred dehydrating method for pharmaceuticals liable to thermal degradation. Most biologics are unstable in aqueous solution and may use freeze drying to prolong their shelf life. Lyophilisation is however expensive and has seen lots of work aimed at reducing cost. This thesis is motivated by the potential cost savings foreseen with the adoption of a cost efficient bulk drying approach for large and small molecules. Initial studies identified ideal formulations that adapted well to bulk drying and further powder handling requirements downstream in production. Low cost techniques were used to disrupt large dried cakes into powder while the effects of carrier agent concentration were investigated for powder flowability using standard pharmacopoeia methods. This revealed superiority of crystalline mannitol over amorphous sucrose matrices and established that the cohesive and very poor flow nature of freeze dried powders were potential barriers to success. Studies from powder characterisation showed increased powder densification was mainly responsible for significant improvements in flow behaviour and an initial bulking agent concentration of 10-15 %w/v was recommended. Further optimisation studies evaluated the effects of freezing rates and thermal treatment on powder flow behaviour. Slow cooling (0.2 °C/min) with a -25°C annealing hold (2hrs) provided adequate mechanical strength and densification at 0.5-1 M mannitol concentrations. Stable bulk powders require powder transfer into either final vials or intermediate storage closures. The targeted dosing of powder formulations using volumetric and gravimetric powder dispensing systems where evaluated using Immunoglobulin G (IgG), Lactate Dehydrogenase (LDH) and Beta Galactosidase models. Final protein content uniformity in dosed vials was assessed using activity and protein recovery assays to draw conclusions from deviations and pharmacopeia acceptance values. A correlation between very poor flowability (p<0.05), solute concentration, dosing time and accuracy was revealed. LDH and IgG lyophilised in 0.5 M and 1 M mannitol passed Pharmacopeia acceptance values criteria with 0.1-4 while formulations with micro collapse showed the best dose accuracy (0.32-0.4% deviation). Bulk mannitol content above 0.5 M provided no additional benefits to dosing accuracy or content uniformity of dosed units. This study identified considerations which included the type of protein, annealing, cake disruption process, physical form of the phases present, humidity control and recommended gravimetric transfer as optimal for dispensing powder. Dosing lyophilised powders from bulk was demonstrated as practical, time efficient, economical and met regulatory requirements in cases. Finally the use of a new non-destructive technique, X-ray microcomputer tomography (MCT), was explored for cake and particle characterisation. Studies demonstrated good correlation with traditional gas porosimetry (R2 = 0.93) and morphology studies using microscopy. Flow characterisation from sample sizes of less than 1 mL was demonstrated using three dimensional X-ray quantitative image analyses. A platinum-mannitol dispersion model used revealed a relationship between freezing rate, ice nucleation sites and variations in homogeneity within the top to bottom segments of a formulation.

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.

Role of Hydrological Process Representation on Erosion, Deposition, and Sediment Yield Estimate

Soil erosion by water is a major driven force causing land degradation. Laboratory experiments, on-site field study, and suspended sediments measurements were major fundamental approaches to study the mechanisms of soil water erosion and to quantify the erosive losses during rain events. The experimental research faces the challenge to extent the result to a wider spatial scale. Soil water erosion modeling provides possible solutions for scaling problems in erosion research, and is of principal importance to better understanding the governing processes of water erosion. However, soil water erosion models were considered to have limited value in practice. Uncertainties in hydrological simulations are among the reasons that hindering the development of water erosion model. Hydrological models gained substantial improvement recently and several water erosion models took advantages of the improvement of hydrological models. It is crucial to know the impact of changes in hydrological processes modeling on soil erosion simulation.

This dissertation work first created an erosion modeling tool (GEOtopSed) that takes advantage of the comprehensive hydrological model (GEOtop). The newly created tool was then tested and evaluated at an experimental watershed. The GEOtopSed model showed its ability to estimate multi-year soil erosion rate with varied hydrological conditions. To investigate the impact of different hydrological representations on soil erosion simulation, a 11-year simulation experiment was conducted for six models with varied configurations. The results were compared at varied temporal and spatial scales to highlight the roles of hydrological feedbacks on erosion. Models with simplified hydrological representations showed agreement with GEOtopSed model on long temporal scale (longer than annual). This result led to an investigation for erosion simulation at different rainfall regimes to check whether models with different hydrological representations have agreement on the soil water erosion responses to the changing climate. Multi-year ensemble simulations with different extreme precipitation scenarios were conducted at seven climate regions. The differences in erosion simulation results showed the influences of hydrological feedbacks which cannot be seen by purely rainfall erosivity method.

Degradation of Biofumigant Isothiocyanates and Allyl Glucosinolate in Soil and Their Effects on the Microbial Community Composition

Brassicales species rich in glucosinolates are used for biofumigation, a process based on releasing enzymatically toxic isothiocyanates into the soil. These hydrolysis products are volatile and often reactive compounds. Moreover, glucosinolates can be degraded also without the presence of the hydrolytic enzyme myrosinase which might contribute to bioactive effects. Thus, in the present study the stability of Brassicaceae plant-derived and pure glucosinolates hydrolysis products was studied using three different soils ( model biofumigation). In addition, the degradation of pure 2-propenyl glucosinolate was investigated with special regard to the formation of volatile breakdown products. Finally, the influence of pure glucosinolate degradation on the bacterial community composition was evaluated using denaturing gradient gel electrophoresis of 16S rRNA gene amplified from total community DNA. The model biofumigation study revealed that the structure of the hydrolysis products had a significant impact on their stability in the soil but not the soil type. Following the degradation of pure 2-propenyl glucosinolate in the soils, the nitrile as well as the isothiocyanate can be the main degradation products, depending on the soil type. Furthermore, the degradation was shown to be both chemically as well as biologically mediated as autoclaving reduced degradation. The nitrile was the major product of the chemical degradation and its formation increased with iron content of the soil. Additionally, the bacterial community composition was significantly affected by adding pure 2-propenyl glucosinolate, the effect being more pronounced than in treatments with myrosinase added to the glucosinolate. Therefore, glucosinolates can have a greater effect on soil bacterial community composition than their hydrolysis products.

MSW: from pollution/degradation source to resource

Municipal Solid Waste is one of the biggest challenges that cities are facing: MSW is considered of the main sources of energy consumption, urban degradation and pollution. This paper defines the major negative effects of MSW on cities and proposes new solutions to guide waste policies. Most contemporary waste management efforts are focused at regional government level and based on high tech waste disposal by methods such as landfill and incineration. However, these methods are becoming increasingly expensive, energy inefficient and pollutant: waste disposal is not sustainable and will have negative implications for future generations. In this paper are proposed all the principle solutions that could be undertaken. New policy instruments are presented updating and adapting policies and encouraging innovation for less wasteful systems. Waste management plans are fundamental to increase the ability of urban areas effectively to adapt to waste challenges. These plans have to give an outline of waste streams and treatment options and provide a scenario for the following years that significantly reduce landfills and incinerators in favor of prevention, reuse and recycling. The key aim of an urban waste management plan is to set out the work towards a zero waste economy as part of the transition to a sustainable economy. Other questions remain still opened: How to change people’s behavior? What is the role of environmental education and risk perception? It is sure that the involvement of the various stakeholders and the wider public in the planning process should aim at ensuring acceptance of the waste policy.

A III-V channel field effect transistor for non-classical CMOS: process optimisation for improved gate stack function

This thesis describes a collection of studies into the electrical response of a III-V MOS stack comprising metal/GaGdO/GaAs layers as a function of fabrication process variables and the findings of those studies. As a result of this work, areas of improvement in the gate process module of a III-V heterostructure MOSFET were identified. Compared to traditional bulk silicon MOSFET design, one featuring a III-V channel heterostructure with a high-dielectric-constant oxide as the gate insulator provides numerous benefits, for example: the insulator can be made thicker for the same capacitance, the operating voltage can be made lower for the same current output, and improved output characteristics can be achieved without reducing the channel length further. It is known that transistors composed of III-V materials are most susceptible to damage induced by radiation and plasma processing. These devices utilise sub-10 nm gate dielectric films, which are prone to contamination, degradation and damage. Therefore, throughout the course of this work, process damage and contamination issues, as well as various techniques to mitigate or prevent those have been investigated through comparative studies of III-V MOS capacitors and transistors comprising various forms of metal gates, various thicknesses of GaGdO dielectric, and a number of GaAs-based semiconductor layer structures. Transistors which were fabricated before this work commenced, showed problems with threshold voltage control. Specifically, MOSFETs designed for normally-off (VTH > 0) operation exhibited below-zero threshold voltages. With the results obtained during this work, it was possible to gain an understanding of why the transistor threshold voltage shifts as the gate length decreases and of what pulls the threshold voltage downwards preventing normally-off device operation. Two main culprits for the negative VTH shift were found. The first was radiation damage induced by the gate metal deposition process, which can be prevented by slowing down the deposition rate. The second was the layer of gold added on top of platinum in the gate metal stack which reduces the effective work function of the whole gate due to its electronegativity properties. Since the device was designed for a platinum-only gate, this could explain the below zero VTH. This could be prevented either by using a platinum-only gate, or by matching the layer structure design and the actual gate metal used for the future devices. Post-metallisation thermal anneal was shown to mitigate both these effects. However, if post-metallisation annealing is used, care should be taken to ensure it is performed before the ohmic contacts are formed as the thermal treatment was shown to degrade the source/drain contacts. In addition, the programme of studies this thesis describes, also found that if the gate contact is deposited before the source/drain contacts, it causes a shift in threshold voltage towards negative values as the gate length decreases, because the ohmic contact anneal process affects the properties of the underlying material differently depending on whether it is covered with the gate metal or not. In terms of surface contamination; this work found that it causes device-to-device parameter variation, and a plasma clean is therefore essential. This work also demonstrated that the parasitic capacitances in the system, namely the contact periphery dependent gate-ohmic capacitance, plays a significant role in the total gate capacitance. This is true to such an extent that reducing the distance between the gate and the source/drain ohmic contacts in the device would help with shifting the threshold voltages closely towards the designed values. The findings made available by the collection of experiments performed for this work have two major applications. Firstly, these findings provide useful data in the study of the possible phenomena taking place inside the metal/GaGdO/GaAs layers and interfaces as the result of chemical processes applied to it. In addition, these findings allow recommendations as to how to best approach fabrication of devices utilising these layers.

Evaluation of isoprene degradation in the detailed tropospheric chemical mechanism, MCM v3, using environmental chamber data

The isoprene degradation mechanism included in version 3 of the Master Chemical Mechanism (MCM v3) has been evaluated and refined, using the Statewide Air Pollution Research Center (SAPRC) environmental chamber datasets on the photo-oxidation of isoprene and its degradation products, methacrolein (MACR) and methylvinyl ketone (MVK). Prior to this, the MCM v3 butane degradation chemistry was also evaluated using chamber data on the photo-oxidation of butane, and its degradation products, methylethyl ketone (MEK), acetaldehyde (CH3CHO) and formaldehyde (HCHO), in conjunction with an initial evaluation of the chamber-dependent auxiliary mechanisms for the series of relevant chambers. The MCM v3 mechanisms for both isoprene and butane generally performed well and were found to provide an acceptable reaction framework for describing the NOx-photo-oxidation experiments on the above systems, although a number of parameter modifications and refinements were identified which resulted in an improved performance. All these relate to the magnitude of sources of free radicals from organic chemical process, such as carbonyl photolysis rates and the yields of radicals from the reactions of O3 with unsaturated oxygenates, and specific recommendations are made for refinements. In addition to this, it was necessary to include a representation of the reactions of O(3P) with isoprene, MACR and MVK (which were not previously treated in MCM v3), and conclusions are drawn concerning the required extent of free radical formation from these reactions. Throughout the study, the performance of MCM v3 was also compared with that of the SAPRC-99 mechanism, which was developed and optimized in conjunction with the chamber datasets.

Exploring the potential of novel biomixtures and Lentinula edodes fungus for the degradation of selected pesticides. Evaluation for use in biobed systems

An approach to reduce the contamination of water sourceswith pesticides is the use of biopurificaction systems. The active core of these systems is the biomixture. The composition of biomixtures depends on the availability of local agro-industrial wastes and design should be adapted to every region. In Portugal, cork processing is generally regarded as environmentally friendly and would be interesting to find applications for its industry residues. In this work the potential use of different substrates in biomixtures, as cork (CBX); cork and straw, coat pine and LECA (Light Expanded Clay Aggregates), was tested on the degradation of terbuthylazine, difenoconazole, diflufenican and pendimethalin pesticides. Bioaugmentation strategies using the white-rot fungus Lentinula edodes inoculated into the CBX, was also assessed. The results obtained from this study clearly demonstrated the relevance of using natural biosorbents as cork residues to increase the capacity of pesticide dissipation in biomixtures for establishing biobeds. Furthermore, higher degradation of all the pesticides was achieved by use of bioaugmented biomixtures. Indeed, the biomixtures inoculated with L. edodes EL1were able to mineralize the selected xenobiotics, revelling that these white-rot fungi might be a suitable fungus for being used as inoculum sources in on-farm sustainable biopurification system, in order to increase its degradation efficiency. After 120 days, maximum degradation of terbuthylazine, difenoconazole, diflufenican and pendimethalin, of bioaugmented CBX, was 89.9%, 75.0%, 65.0% and 99.4%, respectively. The dominant metabolic route of terbuthylazine in biomixtures inoculated with L. edodes EL1 proceeded mainly via hydroxylation, towards production of terbuthylazine-hydroxy-2 metabolite. Finally, sorption process to cork by pesticides proved to be a reversible process,working cork as a mitigating factor reducing the toxicity to microorganisms in the biomixture, especially in the early stages.