Tear analysis and lens-tear interactions:Part I. Protein fingerprinting with microfluidic technology

The purpose of this work is to establish the application of a fully automated microfluidic chip based protein separation assay in tear analysis. It is rapid, requires small sample volumes and is vastly superior to, and more convenient than, comparable conventional gel electrophoresis assays. The protein sizing chip technology was applied to three specific fields of analysis. Firstly tear samples were collected regularly from subjects establishing the baseline effects of tear stimulation, tear state and patient health. Secondly tear samples were taken from lens wearing eyes and thirdly the use of microfluidic technology was assessed as a means to investigate a novel area of tear analysis, which we have termed the 'tear envelope'. Utilising the Agilent 2100 Bioanalyzer in combination with the Protein 200 Plus LabChip kit, these studies investigated tear proteins in the range of 14-200 kDa. Particular attention was paid to the relative concentrations of lysozyme, tear lipocalin, secretory IgA (sIgA), IgG and lactoferrin, together with the overall tear electropherogram 'fingerprint'. Furthermore, whilst lens-tear interaction studies are generally thought of as an investigation into the effects of tears components on the contact lens material, i.e. deposition studies, this report addresses the reverse phenomenon-the effect of the lens, and particularly the newly inserted lens, on the tear fluid composition and dynamics. The use of microfluidic technology provides a significant advance in tear studies and should prove invaluable in tear diagnostics and contact lens performance analysis.

Determination of recombination coefficients for nanocrystalline silicon embedded in hydrogenated amorphous silicon

The spectroscopic pump-probe reflectance method was used to investigate recombination dynamics in samples of nanocrystalline silicon embedded in a matrix of hydrogenated amorphous silicon. We found that the dynamics can be described by a rate equation including linear and quadratic terms corresponding to recombination processes associated with impurities and impurity-assisted Auger ionization, respectively. We determined the values of the recombination coefficients using the initial concentrations method. We report the coefficients of 1.5 × 1011 s-1 and 1.1 × 10-10 cm3 s-1 for the impurity-assisted recombination and Auger ionization, respectively.

Experiments in the dissociative recombination of xenon and krypton

Experiments were conducted using the Time of Flight (TOF) method to identify the final product states of the dissociative recombination reaction of krypton and xenon. In the dissociative recombination (DR) reaction the molecular ion breaks up into product atoms whose velocities can be measured. These velocities can then be used to identify the final product states. The DR of krypton had been studied by Shiu and Biondi using spectrometric techniques. They observed the 5p states. Hardy et al. using TOF techniques had observed the 5s states. Mitchell et al. studied the DR of xenon. They observed the 6p and 5d states of xenon. In this laboratory using the TOF method I have recently identified the 5s, 6p and the 4d final states of the DR of krypton. Then I was able to identify the 5d, 7s, 6d, and 6p′ final product states of the DR of xenon. The study of the DR of these heavy inert gases can shed light on the theory of the DR of heavy polyatomic gases, which is not well developed. ^

Investigation of generation -recombination noise and related processes in aluminum gallium arsenide TEGFETs and Hall structures with quantum wells

Experimental and theoretical studies regarding noise processes in various kinds of AlGaAs/GaAs heterostructures with a quantum well are reported. The measurement processes, involving a Fast Fourier Transform and analog wave analyzer in the frequency range from 10 Hz to 1 MHz, a computerized data storage and processing system, and cryostat in the temperature range from 78 K to 300 K are described in detail. The current noise spectra are obtained with the “three-point method”, using a Quan-Tech and avalanche noise source for calibration. ^ The properties of both GaAs and AlGaAs materials and field effect transistors, based on the two-dimensional electron gas in the interface quantum well, are discussed. Extensive measurements are performed in three types of heterostructures, viz., Hall structures with a large spacer layer, modulation-doped non-gated FETs, and more standard gated FETs; all structures are grown by MBE techniques. ^ The Hall structures show Lorentzian generation-recombination noise spectra with near temperature independent relaxation times. This noise is attributed to g-r processes in the 2D electron gas. For the TEGFET structures, we observe several Lorentzian g-r noise components which have strongly temperature dependent relaxation times. This noise is attributed to trapping processes in the doped AlGaAs layer. The trap level energies are determined from an Arrhenius plot of log (τT2) versus 1/T as well as from the plateau values. The theory to interpret these measurements and to extract the defect level data is reviewed and further developed. Good agreement with the data is found for all reported devices. ^

Development of microsatellite markers in Cannabis Sativa for fingerprinting and genetic relatedness analyses

Microsatellite markers were developed for Cannabis sativa L. (marijuana) to estimate the level of polymorphism, usefulness for DNA typing (genotype identification), and to measure the genetic relationships between the different plants. Twelve different oligonucleotide probes were used to screen an enriched microsatellite library of Cannabis sativa in which 49% of the clones contained microsatellite sequences. Characterization of microsatellite loci in Cannabis revealed that GA/CT was the most abundant class of isolated microsatellites representing 50% overall. Eleven polymorphic SSR markers were developed, derived from dinucleotide motifs and eight from trinucleotide motifs. A total of 52 alleles were detected averaging 4.7 alleles/locus. The expected heterozygosity of the eleven loci ranged between 0.368 and 0.710 and the common probability of identical genotypes was 1.8 x 107. The loci identified 27 unique profiles of the 41 Cannabis samples. The eleven microsatellite markers developed in this study were found to be useful for DNA fingerprinting and for assessing genetic relationships in Cannabis.

Genetic Analysis of Mitotic Recombination in Saccharomyces cerevisiae

Mitotic genome instability can occur during the repair of double-strand breaks (DSBs) in DNA, which arise from endogenous and exogenous sources. Studying the mechanisms of DNA repair in the budding yeast, Saccharomyces cerevisiae has shown that Homologous Recombination (HR) is a vital repair mechanism for DSBs. HR can result in a crossover event, in which the broken molecule reciprocally exchanges information with a homologous repair template. The current model of double-strand break repair (DSBR) also allows for a tract of information to non-reciprocally transfer from the template molecule to the broken molecule. These “gene conversion” events can vary in size and can occur in conjunction with a crossover event or in isolation. The frequency and size of gene conversions in isolation and gene conversions associated with crossing over has been a source of debate due to the variation in systems used to detect gene conversions and the context in which the gene conversions are measured.

In Chapter 2, I use an unbiased system that measures the frequency and size of gene conversion events, as well as the association of gene conversion events with crossing over between homologs in diploid yeast. We show mitotic gene conversions occur at a rate of 1.3x10-6 per cell division, are either large (median 54.0kb) or small (median 6.4kb), and are associated with crossing over 43% of the time.

DSBs can arise from endogenous cellular processes such as replication and transcription. Two important RNA/DNA hybrids are involved in replication and transcription: R-loops, which form when an RNA transcript base pairs with the DNA template and displaces the non-template DNA strand, and ribonucleotides embedded into DNA (rNMPs), which arise when replicative polymerase errors insert ribonucleotide instead of deoxyribonucleotide triphosphates. RNaseH1 (encoded by RNH1) and RNaseH2 (whose catalytic subunit is encoded by RNH201) both recognize and degrade the RNA in within R-loops while RNaseH2 alone recognizes, nicks, and initiates removal of rNMPs embedded into DNA. Due to their redundant abilities to act on RNA:DNA hybrids, aberrant removal of rNMPs from DNA has been thought to lead to genome instability in an rnh201Δ background.

In Chapter 3, I characterize (1) non-selective genome-wide homologous recombination events and (2) crossing over on chromosome IV in mutants defective in RNaseH1, RNaseH2, or RNaseH1 and RNaseH2. Using a mutant DNA polymerase that incorporates 4-fold fewer rNMPs than wild type, I demonstrate that the primary recombinogenic lesion in the RNaseH2-defective genome is not rNMPs, but rather R-loops. This work suggests different in-vivo roles for RNaseH1 and RNaseH2 in resolving R-loops in yeast and is consistent with R-loops, not rNMPs, being the the likely source of pathology in Aicardi-Goutières Syndrome patients defective in RNaseH2.

Combining histology, stable isotope analysis and ZooMS collagen fingerprinting to investigate the taphonomic history and dietary behaviour of extinct giant tortoises from the Mare aux Songes deposit on Mauritius

Taphonomic research of bones can provide additional insight into a site's formation and development, the burial environment and ongoing post-mortem processes. A total of 30 tortoise (Cylindraspis) femur bone samples from the Mare aux Songes site (Mauritius)were studied histologically, assessing parameters such as presence and type of microbial alteration, inclusions, staining/infiltrations, the degree of microcracking and birefringence. The absence of microbial attack in the 4200 year old Mare aux Songes bones suggests the animals rapidly entered the soil whole-bodied and were sealed anoxically, although they suffered frombiological and chemical degradation (i.e. pyrite formation/oxidation, mineral dissolution and staining) related to changes in the site's hydrology. Additionally, carbon and nitrogen stable isotopeswere analysed to obtain information on the animals' feeding behaviour. The results show narrowly distributed δ13C ratios, indicating a terrestrial C3 plant-based diet, combined with a wide range in δ15N ratios. This is most likely related to the tortoises' drought-adaptive ability to change their metabolic processes, which can affect the δ15N ratios. Furthermore, ZooMS collagen fingerprinting analysis successfully identified two tortoise species (C. triserrata and C. inepta) in the bone assemblage,which,when combined with stable isotope data, revealed significantly different δ15N ratios between the two tortoise species. As climatic changes around this period resulted in increased aridity in the Mascarene Islands, this could explain the extremely elevated δ15N ratio in our dataset. The endemic fauna was able to endure the climatic changes 4200 years ago, although human arrival in the 17th century changed the original habitat to such an extent that it resulted in the extinction of several species. Fortunately we are still able to study these extinct tortoises due to the beneficial conditions of their burial environment, resulting in excellent bone preservation.

Fingerprinting of Mangrove Ecosystems along Northern Kerala Coast using Biomarker Approach

Mangrove forests are the most productive and bio-diverse wetlands on earth. It generate a large amount of litter in the form of leaves, branches, twigs, inflorescence and other debris and provides habitat for diverse flora and fauna of marine and terrestrial origin such as bacteria, fungi, algae, lichens, zooplankton, benthos, birds, reptiles and mammals. These systems act as nursery for many fishes and shellfishes. The other sources may also provide important organic carbon inputs; including allochthonous riverine or marine material, autochthonous production by benthic or epiphytic micro- or macroalgae, and local water column production by phytoplankton. Since mangrove sediments are very complex which receives autochthonous and allochthonous organic matter inputs, the information extracted from the analysis of mangrove sediments is the fingerprint of both natural and human-induced changes.

RECQ5 promotes recombination and mutagenesis at targeted nicks through disruption of RAD51 filaments

Thesis (Ph.D.)--University of Washington, 2016-08

'On the Application of Hierarchical Coevolutionary Genetic Algorithms: Recombination and Evaluation Partners'

This paper examines the use of a hierarchical coevolutionary genetic algorithm under different partnering strategies. Cascading clusters of sub-populations are built from the bottom up, with higher-level sub-populations optimising larger parts of the problem. Hence higher-level sub-populations potentially search a larger search space with a lower resolution whilst lower-level sub-populations search a smaller search space with a higher resolution. The effects of different partner selection schemes amongst the sub-populations on solution quality are examined for two constrained optimisation problems. We examine a number of recombination partnering strategies in the construction of higher-level individuals and a number of related schemes for evaluating sub-solutions. It is shown that partnering strategies that exploit problem-specific knowledge are superior and can counter inappropriate (sub-) fitness measurements.

Resonant five-body recombination in an ultracold gas of bosonic atoms

We combine theory and experiment to investigate five-body recombination in an ultracold gas of atomic cesium at negative scattering length. A refined theoretical model, in combination with extensive laboratory tunability of the interatomic interactions, enables the five-body resonant recombination rate to be calculated and measured. The position of the new observed recombination feature agrees with a recent theoretical prediction and supports the prediction of a family of universal cluster states at negative a that are tied to an Efimov trimer.

Genetic diversity analysis of isolates belonging to the Photobacterium phosphoreum species group collected from salmon products using AFLP fingerprinting

An accurate amplified fragment length polymorphism (AFLP) method, including three primer sets for the selective amplification step, was developed to display the phylogenetic position of Photobacterium isolates collected from salmon products. This method was efficient for discriminating the three species Photobacterium phosphoreum, Photobacterium iliopiscarium and Photobacterium kishitanii, until now indistinctly gathered in the Photobacterium phosphoreum species group known to be strongly responsible for seafood spoilage. The AFLP fingerprints enabled the isolates to be separated into two main clusters that, according to the type strains, were assigned to the two species P. phosphoreum and P. iliopiscarium. P. kishitanii was not found in the collection. The accuracy of the method was validated by using gyrB-gene sequencing and luxA-gene PCR amplification, which confirmed the species delineation. Most of the isolates of each species were clonally distinct and even those that were isolated from the same source showed some diversity. Moreover, this AFLP method may be an excellent tool for genotyping isolates in bacterial communities and for clarifying our knowledge of the role of the different members of the Photobacterium species group in seafood spoilage.