An Introduction: Quantification of the Hippocampal BDNF Content of Maternally Separated Rats Using a Western Blot Protocol

Among industrialized nations, the United States has the worst incidence of child maltreatment with 3 million cases per year (U.S. Department of Health and Human Services, 2012). Studies have shown that individuals who were maltreated when they were young are more susceptible to drug abuse such as alcohol, cocaine, and nicotine (Maddahian, Newcomb, & Bentle, 1988). Early life stress (ELS) causes hyperactivation of the Hypothalamic-Pituitary- Adrenal (HPA) Axis (Heim et al., 2000; Plotsky et al., 2005). The dysregulation of the HPA axis causes the secretion of glucocorticoid stress hormones by large amounts, which in return attenuates hippocampal Brain-derived neurotrophic factor (BDNF) (Smith, Makino, Kvetnansky, & Post, 1995). BDNF is a neurotrophin that helps the growth, maintenance, and survival of neurons and is also involved in neuronal plasticity. To mimic ELS, maternal separation is used as an animal model. Studies have shown that adult maternally separated rats have decreased hippocampal mature BDNF (Lippman et al., 2007). Exercise alters some of the effects of ELS by protecting hippocampal BDNF from the down-regulation caused by the hyperactivation of HPA axis (Maniam & Morris, 2010; Neeper et al., 1996). The purpose of this research is to investigate the effects of ELS and exercise on behaviors related to substance abuse and hippocampal BDNF content. When we attempted to measure hippocampal BDNF by the enzyme-linked immunosorbent assay (ELISA), we did not observe the previously-published down-regulation of BDNF in the hippocampi of maternally separated animals (Dold, 2013). Thus, a western blot protocol was developed for the quantification of BDNF in the same protein samples that were previously quantified using ELISA. Western blot is more specific in targeting protein due to SDS-page capability to separate protein components in accordance to their size. BDNF undergoes post-translational processing, such that both pro- BDNF (the 32 kD precursor) and mature BDNF (14 kD) are present and biologically active in rat brain tissue. Thus, western blot will allow us to distinguish between these BDNF proteins, which is important in our study since we are primarily interested in mature BDNF. We expect maternal separation (MS) to attenuate the hippocampal mature BDNF content. Furthermore, we expect that the mature BDNF content will positively correlate with the total running activity

Polymeric Microsensors for Intraoperative Contact Pressure Measurement

Biocompatible sensors have been demonstrated using traditional microfabrication techniques modified for polymer substrates and utilize only materials suitable for implantation or bodily contact. Sensor arrays for the measurement of the load condition of polyethylene spacers in the total knee arthroplasty (TKA) prosthesis have been developed. Arrays of capacitive sensors are used to determine the three-dimensional strain within the polyethylene prosthesis component. Data from these sensors can be used to give researchers a better understanding of component motion, loading, and wear phenomena for a large range of activities. This dissertation demonstrates both analytically and experimentally the fabrication of these sensor arrays using biocompatible polymer substrates and dielectrics while preserving industry-standard microfabrication processing for micron-level resolution. An array of sensors for real-time measurement of pressure profiles is the long-term goal of this research. A custom design using capacitive-based sensors is an excellent selection for such measurement, giving high spatial resolution across the sensing surface and high load resolution for pressures applied normal to that surface while operating at low power

The Salmonella specific, σE-regulated, STM1250 and AgsA, function with the sHsps IbpA and IbpB, to counter oxidative stress and survive macrophage killing

The host presents an array of environments which induce bacterial stress including changes in pH, antimicrobial compounds and reactive oxygen species. The bacterial envelope sits at the interface between the intracellular and extracellular environment and its maintenance is essential for Salmonella cell viability under a range of conditions, including during infection. In this study, we aimed to understand the contribution of the σH- and σE-regulated small heat shock proteins IbpA, IbpB, and AgsA and the putative σE-regulated stress response protein STM1250 to the Salmonella envelope stress response. Due to shared sequence identity, regulatory overlap, and the specificity of STM1250 and AgsA to Salmonella sp., we hypothesized that functional overlap exists between these four stress response proteins, which might afford a selective advantage during Salmonella exposure to stress. We present here new roles for three small heat shock proteins and a putative stress response protein in Salmonella that are not limited to heat shock. We have shown that, compared to WT, a quadruple mutant is significantly more sensitive to hydrogen peroxide, has a lower minimum bactericidal concentration to the cationic antimicrobial peptide polymyxin B, and is attenuated in macrophages

Metallo-Supramolecular Assemblies as Functional Architectures

This thesis concerns the rational design of supramolecular metal-complexes and assemblies through combination of tripodal ligands and geometrically directing metallotectons. The aim of this work is to impart functionality into the resultant complexes, with a view towards applications exploiting the inherent luminescent emission of the systems. The metallotectons discussed in this thesis all derive from low-spin d6 metal centres, with incorporation of ancillary ligands designed to retain and modulate the resultant luminescent emission leading to high-fidelity control over the emissive outcome. Three families of complexes were synthesised; incorporating Re(I), Ru(II) and Ir(III) in combination with supporting ancillary ligands and a pair of bi-dentate bipyridine-appended cavitand ligands. The photophysical properties of these systems were investigated and rationalised with respect to both the structural motif of the cavitand ligands and the structure of the metallotecton. The Ir(III) family of complexes was expanded upon to give rise to both tri-metallic and mono-metallic systems, retaining vacant coordination sites. These vacant sites were subsequently exploited in the formation of heterometallic and heteronuclear assemblies employing secondary Ir(III) metallotectons and Ru(II) metallotectons respectively. This novel, modular approach allows for high-fidelity control over the emission properties and gives rise to a ‘function-driven’ route towards metallo-supramolecular design. The self-assembly of the aforementioned d6-metallotectons in combination with mono-dentate cavitand ligands was also investigated, leading to the formation of the first example of an ambi-dentate heteroleptic Re(I) metallocryptophane and a Ru(II)-cornered metallo-cube. A family of novel luminescent Ir(III) metallocryptophanes were also formed, one of which was crystallographically elucidated. The emission colour was modulated between blue-green and intense yellow luminescence depending on the nature of the ligand, and with large internal cavities these cages possess potential in host-guest chemistry

Chromatin compaction in Cornelia de Lange syndrome

Cornelia de Lange Syndrome (CdLS) is a multisystem genetic disorder caused by mutations in the cohesin complex. It is believed that cohesin is able to regulate gene expression with CTCF by holding chromatin in topological complexes, such as active chromatin hubs, and that CdLS is caused by loss of these complexes causing aberrant gene expression. In order to determine if loss of these complexes in CdLS resulted in a general change in the compaction of chromatin, I undertook a series of analyses of the nucleus in CdLS patient lymphoblastoid cell lines (LCLs), compared to wildtype, and later in RNAi knockdown models of CdLS. By fluorescent in situ hybridisation (FISH) I studied the chromatin compaction of different regions of the genome, and found that in some, but not all, CdLS cell lines, gene-rich regions have less compact chromatin compared to wildtype. RNAi knockdown of two proteins that are mutated in CdLS, NIPBL and SMC1, also resulted in decompaction of regions of the genome, however these were different regions than in the patient LCLs, perhaps due to variation between cell lines. This change was not due to the interaction between cohesin and CTCF, as I found that knockdown of CTCF did not result in changes in chromatin compaction. I have also looked at the published data for gene expression in CdLS, and in mouse and Drosophila models of CdLS, and have found no correlation between the genes misexpressed in CdLS in the three species, nor between three cell lines of the same species. These data suggest that the variation in chromatin compaction observed in CdLS may not be due to an interaction between cohesin and CTCF, and that cohesin can act independently of CTCF to regulate gene expression

Reduced Cardiovascular Disease Deaths in 21 Western Countries 1989-91 V 2013-154: What is the UK doing Right or What is the USA doing Wrong?

Objectives: To compare UK Cardiovascular Disease Deaths (CDD) with twenty Other Western Countries (OWC). Design: Population-controlled-based study using WHO data on CDD people aged 55-74 and Age-Standardised-Death-Rates (ASDR) rates per million (pm) contrasts UK and OWC outcomes between 1989-2015 and World Bank % GDP Expenditure-on-Health (%GDPEH) data. Setting: Twenty-one Western Countries. Participants: National populations. Outcome measures: Reduced CDD for people aged 55-74 and ASDR confidence intervals determines statistical differences between UK and OWC over the period. Result: All countries substantially reduced CDD 55-74, highest current rates America at 3440pm, Finland 3197pm, Greece 3173 to lowest, France 1522pm, Australia 1634pm and Japan 1866pm. Previously UK was 4th highest but fell to 8th at 2524pm, significantly reducing CDD more than 15 OWC, though three had greater falls than Britain. ASDR fell substantially everywhere but the UK had second biggest reduction, significantly reducing total CDD deaths more than 15 OWC. Highest 1980-2015 average %GDPEH was USA at 12.7%, UK’s 7.6% was lowest suggesting British cardiac services achieved more with proportionately less. Conclusion: Improvements in primary and secondary prevention of cardiovascular disease occurred in every country, we speculate whether the UK success might be attributable to the Pan UK public health innovations, the National Service Framework (2000) to reduce myocardial infarction, and, National Framework for long term conditions (2005) but further research is required to identify the effective mechanisms. These results should be a morale boost for patients and their families and or all in the cardiac services, especially in the UK

Mortality in the USA, the UK and Other Western Countries, 1989-2015: What Is Wrong With the US?

This population-based study compares U.S. effectiveness with 20 Other Western Countries (OWC) in reducing mortality 1989-1991 and 2013-2015 and, responding to criticisms of Britain's National Health Service, directly compares U.S. with U.K. child (0-4), adult (55-74), and 24 global mortality categories. World Health Organization Age-Standardized Death Rates (ASDR) data are used to compare American and OWC mortality over the period, juxtaposed against national average percentages of Gross Domestic Product (GDP) Expenditure on Health (%GDPEH) drawn from World Bank data. America's average %GDPEH was highest at 13.53% and Britain's the lowest at 7.68%. Every OWC had significantly greater ASDR reductions than America. Current U.S. child and adult mortality rates are 46% and 19% higher than Britain's. Of 24 global diagnostic mortalities, America had 16 higher rates than Britain, notably for Circulatory Disease (24%), Endocrine Disorders (70%), External Deaths (53%), Genitourinary (44%), Infectious Disease (65%), and Perinatal Deaths (34%). Conversely, U.S. rates were lower than Britain's for Neoplasms (11%), Respiratory (12%), and Digestive Disorder Deaths (11%). However, had America matched the United Kingdom's ASDR, there would have been 488,453 fewer U.S. deaths. In view of American %GDPHE and their mortality rates, which were significantly higher than those of the OWC, these results suggests that the U.S. health care system is the least efficient in the Western world

Population-Based Study of Child Mortality (0-4) and Income Inequality in Japan and the Developed world 1989-91 v 2012-14: Any Excess Deaths Between the Most Unequal Countries?

Introduction: Parental child `neglect’ is usually linked to parents but can apply to nations using the criteria explicit in UNICEF statement “in the last analysis Child-Mortality-Rates (CMR) indicates how well a nation meets the needs of its children”. Hence under-five (0-4) CMR rates of Japan and twenty Other Developed Countries (ODC) are compared within the context of relative poverty. Method: WHO data yields CMR rates per million (pm), analysed between 1989-91 and 2012-14 to compare Japan against ODC. World Bank Income Inequality data used as a measure of relative poverty. Excess deaths calculated by matching the most unequal Income Inequality country’s CMR with the most equal nation. Results: All countries reduced CMR substantially. The highest CMR was in USA 1383pm, followed by three English-speaking countries. Japan at 597pm was 19th of 21. USA and New Zealand were double Japan’s CMR, whilst twelve ODC had rates 25% higher than Japan. Most unequal Income Inequality USA at 15.9 times, Japan the most equal at 4.5 times. Income Inequality and CMR were strongly correlated (+0.6188 p<0.005). The countries with the lowest Income Inequality, had lowest CMR namely Finland Japan, Norway and Sweden. America not matching Japan’s CMR, meant an average excess of 16,838 US children’s deaths annually. Discussion: The strong statistical association between higher CMR and Income Inequality, suggests that one factor in Japan’s results is the lower social inequality, unlike Canada, New Zealand, the UK and USA. Does Japan’s results indicate cultural factors suggesting Japan is more child orientated than English-speaking countries