Electrochemical detection of biomolecules at liquid-liquid interfaces

This thesis is concerned with the electrochemistry of proteins at liquid-liquid interfaces. Understanding the behaviour of proteins at the liquid-liquid interface is of fundamental importance to the development of liquid-liquid electrochemistry as a viable method for bioanalysis. This thesis explores new avenues of fundamental protein behaviour, including protein surfactant interactions, selective detection of proteins and protein accumulation at the liquid-liquid interface

DISC1, differential expression, and deconvolution; human and mouse RNA-Seq investigation of a t(1;11) translocation that predisposes to major mental illness

The t(1;11) translocation is a mutation unique to a Scottish pedigree, members of which have been diagnosed with schizophrenia, bipolar disorder, recurrent major depressive disorder and other related disorders. The translocation is significantly linked to increased risk of these diagnoses. It disrupts three genes, only one of which, DISC1, encodes a protein. A number of experiments have explored the function of DISC1 as a molecular scaffold and developmental regulator. DISC1 and its interactors have roles in processes of relevance to psychiatric disease. These include neuronal precursor proliferation, migration and integration in the developing and adult brain, neurite outgrowth, mitochondrial activity, which is particularly important in neurons due to their high energy demands, and intracellular trafficking, especially critical in neurons due to their highly elongated morphology. Although various DISC1 mutations have been investigated in the past, it is only with advances in technology that neural cells derived directly from translocation carriers, and therefore carrying the translocation plus their genetic background, have been generated and analysed. In addition a recently described mouse model mimics the effects of the translocation upon DISC1 expression. It does so by removing endogenous Disc1 exons corresponding to those distal to the breakpoint in translocation carriers, and fusing the remaining endogenous 5’ Disc1 genomic sequence to human chromosome 11 genomic sequence distal to the translocation breakpoint. The result is a chimeric gene with 5’ mouse Disc1 joined to a segment of human DISC1FP1, the non-coding fusion partner of DISC1 located on chromosome 11. This leads to loss of wild-type Disc1 and prediction of chimeric transcripts encoding aberrant C-terminally truncated forms of Disc1. This thesis builds on the work of previous researchers to characterise the RNAsequenced transcriptome of ‘cortical’ neurons derived from induced pluripotent stem cells from various members of the pedigree. Both heterozygous and homozygous mutant mice have also been utilised to generate RNA-sequencing data from the hippocampus and cortex. The thesis not only describes the differential expression of genes and exons, but also carries out a series of analyses to examine whether proportions of certain cell types are altered, as well as whether differentially expressed genes are highly associated with specific cell types. RNA-Seq data have been analysed for differential expression at the gene and individual exon level using DESeq2 and DEXSeq, respectively. This has revealed over 1,200 differentially expressed genes in human neurons carrying the translocation, which predict changes to functions relating to intracellular transport and synaptic activity. In addition, a number of genes have been verified by RT-qPCR as being differentially expressed in these neurons. These include genes of known relevance to schizophrenia such as DRD2, which encodes the D2 dopamine receptor, NTRK2, which encodes the BDNF receptor NTRK2, and BBS1 which encodes the DISC1 interactor and centrosomal protein BBS1. The human neurons also show significant overlap with previously published dysregulated genes in human neurons carrying other DISC1 mutations, as well as with genes associated with schizophrenia by large-scale genome wide association and copy number variation studies. Human neuron RNA-Seq data have also been examined for evidence of local effects of the translocation upon gene expression, and no obvious strong effect was found. The pattern of gene dysregulation in heterozygous mutant mouse cortex overlaps with that of the mutant human neurons. Gene expression changes in the mutant mouse cortex have also been verified by RT-qPCR in the genes Arc and Avp, and the list of implicated genes also shows overlap with genes associated with schizophrenia by large-scale genome wide association and copy number variation studies. An RNA-Seq deconvolution analysis was carried out to look for evidence of altered proportions of cell types at both the broad and more specific cell type level. This compared the observed expression of hundreds of genes in in the RNA-Seq samples against their expression in publically available RNA-Seq data of specific cell types. There does not appear to be any strong and consistent effect of the t(1;11) or mouse mutation on cell proportions. However, the data indicate greater than expected dysregulation of genes that are highly enriched in specific cell types. This includes certain subtypes of astrocyte. Mutant mouse cortex also shows dysregulation of genes associated with several subtypes of interneuron and pyramidal neuron, including parvalbumin positive interneurons. This indicates that, while the proportions of cell types appears to be unaffected by the translocation or mouse mutation, specialised cellular functions may be perturbed. To conclude, this thesis highlights a number of processes which appear to be disturbed by the translocation and mouse mutation. In all models, RNA-Seq evidence suggests signalling pathways of known relevance to psychiatric disease have been affected without significant alteration of cell proportions. This concurs with histological analyses of the mouse model by previous researchers. This thesis also describes the overlap between genes implicated in the study of this unique mutation as well as those implicated by studies seeking common or rare mutations predisposing to schizophrenia, supporting the hypothesis that different genomic risk variants and mutations converge upon certain molecular pathways that are especially important in this illness. The implication that the t(1;11) may alter the activities of certain cell types is also notable and future work can elucidate the cell-specific effects of the translocation

Cardiac Substrate Utilization and Relationship to Invasive Exercise Hemodynamic Parameters in HFpEF

We conducted transcardiac blood sampling in healthy subjects and subjects with heart failure with preserved ejection fraction (HFpEF) to compare cardiac metabolite and lipid substrate use. We demonstrate that fatty acids are less used by HFpEF hearts and that lipid extraction is influenced by hemodynamic factors including pulmonary pressures and cardiac index. The release of many products of protein catabolism is apparent in HFpEF compared to healthy myocardium. In subgroup analyses, differences in energy substrate use between female and male hearts were identified

Terrorism studies: What we have forgotten and what we now know

In our current troubled times, terrorism and the threat of attacks on liberal states preoccupies both policymakers and much of the scholarly community. Four important books are reviewed here. These works represent the evolution of thinking on terrorism over the last three turbulent decades. Revisiting earlier thinking and bringing debates up to date about how to understand and respond to violent threats allows us to ponder what we ‘now know’ and may not know about terrorism and liberal states

Developments in Transduction, Connectivity and AI/Machine Learning for Point-of-Care Testing

We review some emerging trends in transduction, connectivity and data analytics for Point-of-Care Testing (POCT) of infectious and non-communicable diseases. The patient need for POCT is described along with developments in portable diagnostics, specifically in respect of Lab-on-chip and microfluidic systems. We describe some novel electrochemical and photonic systems and the use of mobile phones in terms of hardware components and device connectivity for POCT. Developments in data analytics that are applicable for POCT are described with an overview of data structures and recent AI/Machine learning trends. The most important methodologies of machine learning, including deep learning methods, are summarised. The potential value of trends within POCT systems for clinical diagnostics within Lower Middle Income Countries (LMICs) and the Least Developed Countries (LDCs) are highlighted

Tephrochronology

Tephrochronology is the use of primary, characterized tephras or cryptotephras as chronostratigraphic marker beds to connect and synchronize geological, paleoenvironmental, or archaeological sequences or events, or soils/paleosols, and, uniquely, to transfer relative or numerical ages or dates to them using stratigraphic and age information together with mineralogical and geochemical compositional data, especially from individual glass-shard analyses, obtained for the tephra/cryptotephra deposits. To function as an age-equivalent correlation and chronostratigraphic dating tool, tephrochronology may be undertaken in three steps: (i) mapping and describing tephras and determining their stratigraphic relationships, (ii) characterizing tephras or cryptotephras in the laboratory, and (iii) dating them using a wide range of geochronological methods. Tephrochronology is also an important tool in volcanology, informing studies on volcanic petrology, volcano eruption histories and hazards, and volcano-climate forcing. Although limitations and challenges remain, multidisciplinary applications of tephrochronology continue to grow markedly