Disc-embedded grinding mill towards process integtrated hydro-mechanical cell lysis on centrifugal microfluidic platforms

For the first time we utilize the spindle motor intrinsic to centrifugal microfluidic systems to drive a grinding mill for cell lysis in a stator-rotor concept. This mechanical concept particularly enables the lysis of armored cells and, as a further benefit, avoids the addition of chemicals that potentially inhibit subsequent reactions. As a proof of concept we demonstrate the lysis of the silica-shelled algae Phaeodactylum Tricornutum

Identification of sleep apnea events using discrete wavelet transform of respiration, ECG and accelerometer signals

Sleep apnea is a common sleep disorder in which patient sleep patterns are disrupted due to recurrent pauses in breathing or by instances of abnormally low breathing. Current gold standard tests for the detection of apnea events are costly and have the addition of long waiting times. This paper investigates the use of cheap and easy to use sensors for the identification of sleep apnea events. Combinations of respiration, electrocardiography (ECG) and acceleration signals were analysed. Results show that using features, formed using the discrete wavelet transform (DWT), from the ECG and acceleration signals provided the highest classification accuracy, with an F1 score of 0.914. However, the novel employment of just the accelerometer signal during classification provided a comparable F1 score of 0.879. By employing one or a combination of the analysed sensors a preliminary test for sleep apnea, prior to the requirement for gold standard testing, can be performed

Automated on-disc total RNA extraction from whole blood towards point-of-care for early-stage diagnostics

We present a novel integrated, centrifugo-pneumatic micro-homogenizer (“μHomogenizer”) for automated sample preparation and total RNA extraction from whole blood. Using a Trizol based protocol, this novel μHomogenizer efficiently lyses whole blood spiked with E. coli, retains the organic-mixed fraction and yields the aqueous phase with the total RNA content. By the interplay of microfluidic design and a protocol of rotational frequencies, we concatenate (and parallelize) a sequence of five subsequent liquid handling operations that complete in less than 10 minutes. A comparison of the total nucleotide content yields similar performance as conventional, essentially manual off-disc sample preparation methods

Optical sensing system based on wireless paired emitter detector diode device and ionogels for lab-on-a-disc water quality analysis

This work describes the first use of a wireless paired emitter detector diode device (PEDD) as an optical sensor for water quality monitoring in a lab-on-a-disc device. The microfluidic platform, based on an ionogel sensing area combined with a low-cost optical sensor is applied for pH (quantitative) and qualitative turbidity monitoring of water samples at the point-of-need. The autonomous capabilities of the PEDD system, combined with the portability and wireless communication of the full device, provide the flexibility needed for on-site water testing. Water samples from local fresh and brackish sources were successfully analysed using the device, showing very good correlation with standard bench-top systems

A structural and biochemical model of processive chitin synthesis

Chitin synthases (CHS) produce chitin, an essential component of the fungal cell wall. The molecular mechanism of processive chitin synthesis is not understood, limiting the discovery of new inhibitors of this enzyme class. We identified the bacterial glycosyltransferase NodC as an appropriate model system to study the general structure and reaction mechanism of CHS. A high throughput screening-compatible novel assay demonstrates that a known inhibitor of fungal CHS also inhibit NodC. A structural model of NodC, on the basis of the recently published BcsA cellulose synthase structure, enabled probing of the catalytic mechanism by mutagenesis, demonstrating the essential roles of the DD and QXXRW catalytic motifs. The NodC membrane topology was mapped, validating the structural model. Together, these approaches give insight into the CHS structure and mechanism and provide a platform for the discovery of inhibitors for this antifungal target

Complement system biomarkers in first episode psychosis

Several lines of evidence implicate immunological/inflammatory factors in development of schizophrenia. Complement is a key driver of inflammation, and complement dysregulation causes pathology in many diseases. Here we exploredwhether complement dysregulation occurred in first episode psychosis (FEP) andwhether this provides a source of biomarkers. Eleven complement analytes (C1q, C3, C4, C5, factor B [FB], terminal complement complex [TCC], factor H [FH], FH-related proteins [FHR125], Properdin, C1 inhibitor [C1inh], soluble complement receptor 1 [CR1]) plus C-reactive protein (CRP) were measured in serum from 136 first episode psychosis (FEP) cases and 42 mentally healthy controls using established in-house or commercial ELISA. The relationship between caseness and variables (analytes measured, sex, age, ethnicity, tobacco/cannabis smoking) was tested by multivariate logistic regression. Whenmeasured individually, only TCC was significantly different between FEP and controls (p=0.01). Stepwise selection demonstrated interdependence between some variables and revealed other variables that significantly and independently contributed to distinguishing cases and controls. The finalmodel included demographics (sex, ethnicity, age, tobacco smoking) and a subset of analytes (C3, C4, C5, TCC, C1inh, FHR125, CR1). A receiver operating curve analysis combining these variables yielded an area under the curve of 0.79 for differentiating FEP from controls. This model was confirmed by multiple replications using randomly selected sample subsets. The data suggest that complement dysregulation occurs in FEP, supporting an underlying immune/inflammatory component to the disorder. Classification of FEP cases according to biological variables rather than symptoms would help stratify cases to identify those that might most benefit from therapeuticmodification of the inflammatory response

Patterns of adaptive and purifying selection in the genomes of phocid seals

Modern genomic sequencing technologies provide the opportunity to address long-standing questions in molecular evolution with empirical data. In this dissertation, I combine this new technology with advances in statistical population genetics to describe how deleterious mutations and adaptive evolution have shaped the genomic evolution of phocid seals. In Chapter 1, I model historical demographic processes using whole genome sequences of eight seal taxa: the Hawaiian monk seal, the Mediterranean monk seal, the northern elephant seal, the southern elephant seal, the Weddell seal, the grey seal, the Baltic ringed seal, and the Saimaa ringed seal. Through this, I establish that the endangered monk seal species have long-term small population sizes, as do grey seals. On the other hand, the elephant seals, Weddell seal, and ringed seals had much larger populations in the distant past. Notably, the most recent glaciation (c. 12,000-120,000 years ago) appeared to have a dramatic effect on phocid populations throughout the world. With this knowledge of historical population sizes, I test a fundamental premise of molecular evolution: that the rate of mutation accumulation will be higher in smaller populations due to less efficient purifying selection. I show that there is not a higher substitution rate or overall rate of mutation accumulation in the long-term small populations of monk seals compared to other seal species. On the contrary, overall rates of mutation accumulation appear to be lower in monk seals and grey seals, both of which show smaller long-term population sizes compared to the other species. This suggests either that the distribution of fitness effects may differ across seal species in a way that depends on population size and history. In Chapter 2, I use population genomic data and a newly developed statistical model to detect positive selection in the protein coding genes of phocid seals (monk seals, elephant seals, Weddell seals, grey seals, and ringed seals). In addition, I use a phylogenetic framework to detect parallel evolution across multiple lineages of seals, relating to traits such as polar adaptations, hypoxia tolerance during long dives, and mating behavior. I develop a new bioinformatic tool to process raw BAM files and transform them into useable input for MASS-PRF, a tool to detect selection from polymorphism and divergence data. Through these analyses, I identify thousands of genes that show positive selection across multiple seal lineages. Genes associated with immune function, sperm competition, and blubber composition show positive selection in all lineages, highlighting how complex and important these traits are in seals. In the deep-diving elephant seals, the list of positively selected genes was enriched for genes relating to cardiac muscle development and function, providing important insight into how adaptive protein evolution has helped allow these seals to survive sustained bradycardia during dives that last over an hour. Weddell seals, on the other hand, showed enrichment for genes relating to neuronal development, which may relate to molecular adaptations that allow their neurons to survive hypoxic conditions during long dives. Because MASS-PRF allows for site-specific tests of selection, I am able to show how parallel evolution in the same genes across lineages sometimes may or may not involve positive selection at the same genic site. In Chapter 3, I use the population genomic data from Chapter 2 to model the distribution of fitness effects (DFE) of segregating alleles in each population. Due to sample size issues, only parameters for the Hawaiian monk seal were confidently estimated. Using the site frequency spectrum of synonymous sites, I show that the Hawaiian monk seal has had a long-term effective population size below 5000, in agreement with the results from Chapter 1. In addition, I should that after the arrival of humans in Hawaii, the monk seal experienced a 95% decline in effective population size, in line with the current census size of fewer than 1500 individuals. Conditioning the model on the Hawaiian monk seal demographic parameters, I am able to estimate the shape of DFE in Hawaiian monk seals using the site frequency spectrum of nonsynonymous sites. I estimate a DFE for the Hawaiian monk seal that is nearly identical to the one estimated in humans. This DFE, however, is different than the one estimated for mouse, with the seal and human DFEs having a higher proportion of more strongly deleterious alleles. This pattern cannot be explained by phylogenetic relatedness or differences in phenotypic complexity, but instead is likely related to differences in effective population size. I discuss how the geometric model of evolution predicts such a shift in DFE in response to the epistatic effect of fixed deleterious mutations in smaller populations

The ergonomics and design of an inclusive best-fit solution to workbenches

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University.In a time when the developed world, is trying to reduce the human and economic costs of musculo-skeletal disorders (MSDs), any contribution to such an endeavour would be welcome. These economic costs are estimated to be in the tens of billions of Euro in the EU countries and similarly in the USA, the cost in human pain has not been measured. It may surprise many that in spite of all the advancements in science and technology, that two generations of people, who are very significantly taller than the people of a century ago, are still working in industry and in education at benches, which have not changed, either in height or design in centuries. Some, like wheelchair users do not have the opportunity to work at a bench at all. At the outset this research project, had the primary objective of determining an ergonomic best-fit, for a broad range of users of workbenches. These included the young school going population (12-13 year olds), the senior students (16 plus years old), adults, and a cohort of surrogate wheelchair users. The research also endeavoured to determine if adolescents, who were of the same stature as adults, had the same workbench ergonomics requirements. The secondary objective, which was completely dependant on the first, was to design a bench, which would suit the ergonomic requirements of this diverse group. The research has identified the best-fit workbench heights for the total cohort, while recognising the individual differences in relation to bench height ergonomics, for each of the sub-groups tested. The findings of the research have shown, that using surrogate wheelchair users to determine ergonomic data for this type of activity is fully justified. In combining the raw data for a similar number of wheelchair users, a best-fit bench height has been confirmed at 100 mm above knee height. There are no significant differences between the ergonomic requirements for males and females at workbenches. Body part discomfort has been reduced significantly, for the wheelchair users, at the identified height and endurance has been extended. Importantly the career options for wheelchair users have been extended, empowering them to make broader career choices. The outcomes of the research relating to three groups making up the able-bodied cohort have shown that an ergonomic best-fits possible, which suits the needs of this diverse group. A height of 150 mm under elbow height has been identified as best-fit, and this reduces the discomfort considerably while extending endurance. Robust working heights have been identified, but the female working heights at workbenches, are not as robust as for the males. For all groups it has been shown that bench height has a significant effect on body part discomfort and endurance, and while there were differences in efficiency, which were not quite significant, it is suggested that working in an ergonomically compromising position must, in the long term, in addition to increasing the risk of MSDs, likely also influence productivity, and quality of work. An inclusive test-workbench has been designed and built which satisfies the ergonomic needs of the diverse user group described above