Targeting protein–protein interactions within the cyclic AMP signaling system as a therapeutic strategy for cardiovascular disease

The cAMP signaling system can trigger precise physiological cellular responses that depend on the fidelity of many protein–protein interactions, which act to bring together signaling intermediates at defined locations within cells. In the heart, cAMP participates in the fine control of excitation–contraction coupling, hence, any disregulation of this signaling cascade can lead to cardiac disease. Due to the ubiquitous nature of the cAMP pathway, general inhibitors of cAMP signaling proteins such as PKA, EPAC and PDEs would act non-specifically and universally, increasing the likelihood of serious ‘off target’ effects. Recent advances in the discovery of peptides and small molecules that disrupt the protein–protein interactions that underpin cellular targeting of cAMP signaling proteins are described and discussed

Development of molecular tools to enhance understanding of antiviral RNAi in mosquitoes

Mosquito-borne arboviruses are a considerable threat to human and animal health across the world. Many of them are classed as emerging or remerging pathogens and the incidence of disease for a number of serious viral infections has increased as they expand their geographical and host ranges. As with other invertebrates, mosquitoes lack the adaptive immune response present in vertebrates and instead rely on their innate immune defences to modulate viral infections. Nevertheless, in contrast to vertebrates, arboviral infections in their arthropod vector are non-pathogenic and have no cytopathic effect or detrimental impact on their survival. The response considered to be the most important for antiviral defence in mosquitoes is RNA interference (RNAi) which is a sequence-specific, RNA silencing mechanism. Most of what is known about antiviral RNAi in arthropods has been established in Drosophila as the model insect organism. These studies have benefited from an extensive range of genetic mutants, molecular tools, reporter assays and genetic profiling. The absence of these tools for use in mosquito research is a substantial deficit for arboviral studies in their natural vector system and must be rectified in order to fully understand the influence vector immunity has on virus transmission. This thesis discusses the development of a ‘molecular tool-box’ for advancing the acquisition of knowledge in this area. Efficient RNAi gene silencing and its effect on the antiviral RNAi response was established in vitro using Semliki Forest virus (SFV) as model arbovirus. This assay determined that knock-down of Argonaute-2 had the most substantial impact on virus replication compared to the knockdown of other RNAi proteins. In addition, the limited detection of virus-derived small RNAs, key molecules of the antiviral RNAi response by Northern blot analysis provides further support to previous evidence that SFV may circumvent the antiviral response by sequestering its genomic RNA, resulting in restricted access by the RNAi machinery and preventing the generation of large quantities of virus-derived small RNAs. However, some SFV-derived small RNAs are known to be produced and these have been shown to generate a pattern of ‘hot’ and ‘cold’ spots along the full-length coding sequences. This thesis has determined that this pattern is not exclusive to viral-derived dsRNA trigger molecules but is also exhibited following the treatment of mosquito cells in culture with non-viral dsRNA. This implies that all exogenous dsRNA is processed by RNAi in a similar manner. This study has also characterised the presence of an RNA-dependent RNA polymerase (RdRP) encoded by Aedes aegypti mosquitoes. RdRPs are important for the amplification and spread of the RNAi signal in other organisms such as plants and worms; however, only one study suggested the existence of one in Drosophila. Although, this project proposed the presence and transcription of a homologue of the Drosophila RdRP in the Aedes aegypti-derived Aag2 cell line, protein knockdown assays revealed that it has no effect on virus replication in vitro; suggesting that it does not function as an RdRP. Due to the lack of antibodies against the major RNAi proteins Dicer-1, Dicer-2, Argonaute-1 and Argonaute-2 in mosquitoes, these were designed and screened which allowed the identification of several candidates for the detection of the proteins in mosquito cells in culture. Further to this, recombinant forms of the RNAi initiator protein Dicer-2 and the slicer protein Argonaute-2 were successfully generated and tested in vitro using different promoters to establish their use for future temporal and spatial kinetic studies. It was concluded that of the promoters tested the most successful for the expression of these reporter constructs was the subgenomic promoter of SFV. On the other hand a second promoter, the PUb promoter, may prove more suitable in the future. Finally, this project studied the antiviral capabilities of a non-haematophagous mosquito cell line which would not come across an arboviral infection by traditional blood- feeding routes. Instead the mosquito larvae sustain their adult life stages by feeding on the larvae of other species which may be vertically infected. A cell line derived from Toxorhynchites amboinensis was characterised and was shown to carry out RNAi if induced by dsRNA suggesting that they are able to mount an antiviral response to acquired infections. This study also determined that the cell line contains an endogenous insect specific virus and, although the source of this is unknown, it adds an interesting new dimension to mosquito antiviral immunity. This thesis enhances RNAi research in Aedes mosquitoes by presenting novel molecular tools and reporter assays which will be highly valuable for facilitating future investigations. The studies performed also add to what is already understood regarding the interaction between SFV and mosquito antiviral immunity through the RNAi response

Characterization of carbon monoxide, methane and nonmethane hydrocarbons in emerging cities of Saudi Arabia and Pakistan and in Singapore

We investigate the composition of 63 C2-C10 nonmethane hydrocarbons (NMHCs), methane (CH4) and carbon monoxide (CO), in Jeddah, Mecca, and Madina (Saudi Arabia), in Lahore, (Pakistan), and in Singapore. We established a database with which to compare and contrast NMHCs in regions where ambient levels and emissions are poorly characterized, but where conditions are favorable to the formation of tropospheric ozone, and where measurements are essential for improving emission inventories and modeling. This dataset will also serve as a base for further analysis of air pollution in Western Saudi Arabia including, but not limited to, the estimation of urban emissions and long range pollution transport from these regions. The measured species showed enhanced levels in all Saudi Arabian cities compared to the local background but were generally much lower than in Lahore. In Madina, vehicle exhaust was the dominant NMHC source, as indicated by enhanced levels of combustion products and by the good correlation between NMHCs and CO, while in Jeddah and Mecca a combination of sources needs to be considered. Very high NMHC levels were measured in Lahore, and elevated levels of CH4 in Lahore were attributed to natural gas. When we compared our results with 2010 emissions from the MACCity global inventory, we found discrepancies in the relative contribution of NMHCs between the measurements and the inventory. In all cities, alkenes (especially ethene and propene) dominated the hydroxyl radical (OH) reactivity (kOH) because of their great abundance and their relatively fast reaction rates with OH

Improving regional ozone modeling through systematic evaluation of errors using the aircraft observations during the International Consortium for Atmospheric Research on Transport and Transformation

During the operational phase of the ICARTT field experiment in 2004, the regional air quality model STEM showed a strong positive surface bias and a negative upper troposphere bias (compared to observed DC-8 and WP-3 observations) with respect to ozone. After updating emissions from NEI 1999 to NEI 2001 (with a 2004 large point sources inventory update), and modifying boundary conditions, low-level model bias decreases from 11.21 to 1.45 ppbv for the NASA DC-8 observations and from 8.26 to −0.34 for the NOAA WP-3. Improvements in boundary conditions provided by global models decrease the upper troposphere negative ozone bias, while accounting for biomass burning emissions improved model performance for CO. The covariances of ozone bias were highly correlated to NOz, NOy, and HNO3 biases. Interpolation of bias information through kriging showed that decreasing emissions in SE United States would reduce regional ozone model bias and improve model correlation coefficients. The spatial distribution of forecast errors was analyzed using kriging, which identified distinct features, which when compared to errors in postanalysis simulations, helped document improvements. Changes in dry deposition to crops were shown to reduce substantially high bias in the forecasts in the Midwest, while updated emissions were shown to account for decreases in bias in the eastern United States. Observed and modeled ozone production efficiencies for the DC-8 were calculated and shown to be very similar (7.8) suggesting that recurring ozone bias is due to overestimation of NOx emissions. Sensitivity studies showed that ozone formation in the United States is most sensitive to NOx emissions, followed by VOCs and CO. PAN as a reservoir of NOx can contribute to a significant amount of surface ozone through thermal decomposition

Material concepts for top of rail friction management – Classification, characterisation and application

The concept of managing and adjustment of friction between the wheel and rail has a long history within the operation of railways systems. In the past, adjustment/management has been limited to gauge face lubrication and the use of sanding equipment. The introduction of the top of rail (TOR) friction modifier (FM) over the last 20 years now allows for the modification of the friction at the top of rail–wheel tread interface. This paper focusses on the concept of TOR friction adjustment. Recent developments have led to a new generation of products, defined here as, TOR lubricants (oil and/or grease-based) and hybrid materials (oil/water mixtures), which are non-drying or slow drying. Definitions and functional difference are detailed and contrasted with that of the water-based drying FM. The water-based TOR-FM once applied rapidly dries, mixes with the existing third-body layer, and allows for the accommodation of shear displacement. TOR lubricants and hybrid materials rely on mixed boundary layer lubrication, contrary to application of the water-based TOR-FM. It has been shown that the adhesion level is highly influenced by the lubricant application rates. The risks and benefits (lateral force reduction, corrugation mitigation, and impact on energy consumption and influence on rolling contact fatigue) are discussed for all product classifications. However, a lack of data exists for the TOR lubricants especially in the area of rolling contact fatigue where laboratory studies have identified the possibility of crack interaction. Whilst it can be seen that TOR lubricants have the ability to provide similar benefits to that of a water-based FM, they exhibit a strong dependency on the application rate which may lend itself to adhesion and RCF issues. Further work is recommended in this area

Supporting adult unpaid carers via an online dancing intervention: A feasibility/acceptability study

Unpaid carers often experience poor mental and physical health linked to their caring role. Engagement in physical activity has been shown to alleviate these negative health outcomes, but it is harder for carers to find the time, energy and space to exercise. This qualitative study, based in the UK, explored the feasibility of an online, dance-based physical activity intervention with six female unpaid carers. Five themes resulted from the thematic analysis of the pre- and post-intervention interviews: Perceived physical health benefits of the intervention; Perceived mental health benefits of the intervention; Satisfactoriness of the dance classes; Impact of caring responsibilities on participation; and Suggestions for future classes. Further research is required to measure the effectiveness of the dance intervention in improving mental and physical wellbeing with larger samples including a wider mix of carers in terms of gender, age and health conditions of the care recipients, as well as international samples. Future research should also consider the barriers that some carers may face when accessing an online intervention, and alternative forms of exercise that may appeal to other groups of carers (e.g., male carers, older adult carers)