Investigating the Effect of Histone H4 Sumoylation on Chromatin Structure and Function

Thesis (Ph.D.)--University of Washington, 2016-06

The Relationship between Adolescent Social Network Structure and Perceptions of School Climate

Thesis (Ph.D.)--University of Washington, 2016-06

An investigation into effects of long-distance seed dispersal on organelle population genetic structure and colonization rate: a model analysis

A simulation-based modelling approach is used to examine the effects of stratified seed dispersal (representing the distribution of the majority of dispersal around the maternal parent and also rare long-distance dispersal) on the genetic structure of maternally inherited genomes and the colonization rate of expanding plant populations. The model is parameterized to approximate postglacial oak colonization in the UK, but is relevant to plant populations that exhibit stratified seed dispersal. The modelling approach considers the colonization of individual plants over a large area (three 500 km x 10 km rolled transects are used to approximate a 500 km x 300 km area). Our approach shows how the interaction of plant population dynamics with stratified dispersal can result in a spatially patchy haplotype structure. We show that while both colonization speeds and the resulting genetic structure are influenced by the characteristics of the dispersal kernel, they are robust to changes in the periodicity of long-distance events, provided the average number of long-distance dispersal events remains constant. We also consider the effects of additional physical and environmental mechanisms on plant colonization. Results show significant changes in genetic structure when the initial colonization of different haplotypes is staggered over time and when a barrier to colonization is introduced. Environmental influences on survivorship and fecundity affect both the genetic structure and the speed of colonization. The importance of these mechanisms in relation to the postglacial spread and genetic structure of oak in the UK is discussed.

Molecular structure and function of the glycine receptor chloride channel

The glycine receptor chloride channel (GlyR) is a member of the nicotinic acetylcholine receptor family of ligand-gated ion channels. Functional receptors of this family comprise five subunits and are important targets for neuroactive drugs. The GlyR is best known for mediating inhibitory neurotransmission in the spinal cord and brain stem, although recent evidence suggests it may also have other physiological roles, including excitatory neurotransmission in embryonic neurons. To date, four alpha-subunits (alpha1 to alpha4) and one beta-subunit have been identified. The differential expression of subunits underlies a diversity in GlyR pharmacology. A developmental switch from alpha2 to alpha1beta is completed by around postnatal day 20 in the rat. The beta-subunit is responsible for anchoring GlyRs to the subsynaptic cytoskeleton via the cytoplasmic protein gephyrin. The last few years have seen a surge in interest in these receptors. Consequently, a wealth of information has recently emerged concerning Glyl? molecular structure and function. Most of the information has been obtained from homomeric alpha1 GlyRs, with the roles of the other subunits receiving relatively little attention. Heritable mutations to human GlyR genes give rise to a rare neurological disorder, hyperekplexia (or startle disease). Similar syndromes also occur in other species. A rapidly growing list of compounds has been shown to exert potent modulatory effects on this receptor. Since GlyRs are involved in motor reflex circuits of the spinal cord and provide inhibitory synapses onto pain sensory neurons, these agents may provide lead compounds for the development of muscle relaxant and peripheral analgesic drugs.

Novel mitochondrial gene content and gene arrangement indicate illegitimate inter-mtDNA recombination in the chigger mite, Leptotrombidium pallidum

To better understand the evolution of mitochondrial (mt) genomes in the Acari (mites and ticks), we sequenced the mt genome of the chigger mite, Leptotrombidium pallidum (Arthropoda: Acari: Acariformes). This genome is highly rearranged relative to that of the hypothetical ancestor of the arthropods and the other species of Acari studied. The mt genome of L. pallidum has two genes for large subunit rRNA, a pseudogene for small subunit rRNA, and four nearly identical large noncoding regions. Nineteen of the 22 tRNAs encoded by this genome apparently lack either a T-arm or a D-arm. Further, the mt genome of L. pallidum has two distantly separated sections with identical sequences but opposite orientations of transcription. This arrangement cannot be accounted for by homologous recombination or by previously known mechanisms of mt gene rearrangement. The most plausible explanation for the origin of this arrangement is illegitimate inter-mtDNA recombination, which has not been reported previously in animals. In light of the evidence from previous experiments on recombination in nuclear and mt genomes of animals, we propose a model of illegitimate inter-mtDNA recombination to account for the novel gene content and gene arrangement in the mt genome of L. pallidum.

Variation in mangrove forest structure and sediment characteristics in Bocas del Toro, Panama

Mangrove forest structure and sediment characteristics were examined in the extensive mangroves of Bocas del Toro, Republic of Panama. Forest structure was characterized to determine if spatial vegetation patterns were repeated over the Bocas del Toro landscape. Using a series of permanent plots and transects we found that the forests of Bocas del Toro were dominated by Rhizophora maugle with very few individuals of Avicennia germinans and Laguncularia racemosa. Despite this low species diversity, there was large variation in forest structure and in edaphic conditions (salinity, concentration of available phosphorus, Eh and sulphide concentration). Aboveground biomass varied 20-fold, from 6.8 Mg ha(-1) in dwarf forests to 194.3 Mg ha(-1) in the forests fringing the land. But variation in forest structure was predictable across the intertidal zone. There was a strong tree height gradient from seaward fringe (mean tree height 3.9 m), decreasing in stature in the interior dwarf forests (mean tree height 0.7 m), and increasing in stature in forests adjacent to the terrestrial forest (mean tree height 4.1 m). The predictable variation in forest structure emerges due to the complex interactions among edaphic and plant factors. Identifying predictable patterns in forest structure will aid in scaling up the ecosystem services provided by mangrove forests in coastal landscapes.

Genetic population structure and call variation in a passerine bird, the satin bowerbird, Ptilonorhynchus violaceus

Geographic variation in vocalizations is widespread in passerine birds, but its origins and maintenance remain unclear. One hypothesis to explain this variation is that it is associated with geographic isolation among populations and therefore should follow a vicariant pattern similar to that typically found in neutral genetic markers. Alternatively, if environmental selection strongly influences vocalizations, then genetic divergence and vocal divergence may be disassociated. This study compared genetic divergence derived from 11 microsatellite markers with a metric of phenotypic divergence derived from male bower advertisement calls. Data were obtained from 16 populations throughout the entire distribution of the satin bowerbird, an Australian wet-forest-restricted passerine. There was no relationship between call divergence and genetic divergence, similar to most other studies on birds with learned vocalizations. Genetic divergence followed a vicariant model of evolution, with the differentiation of isolated populations and isolation-by-distance among continuous populations. Previous work on Ptilonorhynchus violaceus has shown that advertisement call structure is strongly influenced by the acoustic environment of different habitats. Divergence in vocalizations among genetically related populations in different habitats indicates that satin bowerbirds match their vocalizations to the environment in which they live, despite the homogenizing influence of gene flow. In combination with convergence of vocalizations among genetically divergent populations occurring in the same habitat, this shows the overriding importance that habitat-related selection can have on the establishment and maintenance of variation in vocalizations.

Molecular mechanisms for the variation of mitochondrial gene content and gene arrangement among chigger mites of the genus Leptotrombidium (Acari : Acariformes)

The gene content of a mitochondrial (mt) genome, i.e., 37 genes and a large noncoding region (LNR), is usually conserved in Metazoa. The arrangement of these genes and the LNR is generally conserved at low taxonomic levels but varies substantially at high levels. We report here a variation in mt gene content and gene arrangement among chigger mites of the genus Leptotrombidium. We found previously that the mt genome of Leptotrombidium pallidum has an extra gene for large-subunit rRNA (rrnL), a pseudo-gene for small-subunit rRNA (PrrnS), and three extra LNRs, additional to the 37 genes and an LNR typical of Metazoa. Further, the arrangement of mt genes of L. pallidum differs drastically from that of the hypothetical ancestor of the arthropods. To find to what extent the novel gene content and gene arrangement occurred in Leptotrombidium, we sequenced the entire or partial mt genomes of three other species, L. akamushi, L. deliense, and L. fletcheri. These three species share the arrangement of all genes with L. pallidum, except trnQ (for tRNA-glutamine). Unlike L. pallidum, however, these three species do not have extra rrnL or PrrnS and have only one extra LNR. By comparison between Leptotrombidium species and the ancestor of the arthropods, we propose that (1) the type of mt genome present in L. pallidum evolved from the type present in the other three Leptotrombidium species, and (2) three molecular mechanisms were involved in the evolution of mt gene content and gene arrangement in Leptotrombidium species.

Solution structure and characterization of the LGR8 receptor binding surface of insulin-like peptide 3

Insulin-like peptide 3 (INSL3), a member of the relaxin peptide family, is produced in testicular Leydig cells and ovarian thecal cells. Gene knock-out experiments have identified a key biological role in initiating testes descent during fetal development. Additionally, INSL3 has an important function in mediating male and female germ cell function. These actions are elicited via its recently identified receptor, LGR8, a member of the leucine-rich repeat-containing G-protein- coupled receptor family. To identify the structural features that are responsible for the interaction of INSL3 with its receptor, its solution structure was determined by NMR spectroscopy together with in vitro assays of a series of B-chain alanine-substituted analogs. Synthetic human INSL3 was found to adopt a characteristic relaxin/ insulin-like fold in solution but is a highly dynamic molecule. The four termini of this two-chain peptide are disordered, and additional conformational exchange is evident in the molecular core. Alanine-substituted analogs were used to identify the key residues of INSL3 that are responsible for the interaction with the ectodomain of LGR8. These include Arg(B16) and Val(B19), with His(B12) and Arg(B20) playing a secondary role, as evident from the synergistic effect on the activity in double and triple mutants involving these residues. Together, these amino acids combine with the previously identified critical residue, Trp(B27), to form the receptor binding surface. The current results provide clear direction for the design of novel specific agonists and antagonists of this receptor.

Effects of fire on the structure and composition of open eucalypt forests

Fires are integral to the healthy functioning of most ecosystems and are often poorly understood in policy and management, however, the relationship between floristic composition and habitat structure is intrinsically linked, particularly after fire. The aim of this study was to test whether the variability of habitat structure or floristic composition and abundance in forests at a regional scale can be explained in terms of fire frequency using historical data and experimental prescribed burns. We tested this hypothesis in open eucalypt forests of Fraser Island off the east coast of Australia. Fraser Island dunes show progressive stages in plant succession as access to nutrients decreases across the Island. We found that fire frequency was not a good predictor of floristic composition or abundance across dune systems; rather, its affects were dune specific. In contrast, habitat structure was strongly influenced by fire frequency, independent of dune system. A dense understorey occurred in frequently burnt areas, whereas infrequently burnt areas had a more even distribution of plant heights. Plant communities returned to pre-burn levels of composition and abundances within 6 months of a fire and frequently burnt areas were dominated by early successional species of plant. These ecosystems were characterized by low diversity and frequently burnt areas on the east coast were dominated by Pteridium. Greater midstorey canopy cover in low frequency areas reduces light penetration and allows other species to compete more effectively with Pteridium. Our results strongly indicate that frequent fires on the Island have resulted in a decrease in relative diversity through dominance of several species. Prescribed fire represents a powerful management tool to shape habitat structure and complexity of Fraser Island forests.

Structure and dynamics of multiple cationic vectors-siRNA complexation by all-atomic molecular dynamics simulations

Understanding the molecular mechanism of gene condensation is a key component to rationalizing gene delivery phenomena, including functional properties such as the stability of the gene-vector complex and the intracellular release of the gene. In this work, we adopt an atomistic molecular dynamics simulation approach to study the complexation of short strand duplex RNA with four cationic carrier systems of varying charge and surface topology at different charge ratios. At lower charge ratios, polymers bind quite effectively to siRNA, while at high charge ratios, the complexes are saturated and there are free polymers that are unable to associate with RNA. We also observed reduced fluctuations in RNA structures when complexed with multiple polymers in solution as compared to both free siRNA in water and the single polymer complexes. These novel simulations provide a much better understanding of key mechanistic aspects of gene-polycation complexation and thereby advance progress toward rational design of nonviral gene delivery systems.

Gene network and proteomic analyses of cardiac responses to pathological and physiological stress

Background—The molecular mechanisms underlying similarities and differences between physiological and pathological left ventricular hypertrophy (LVH) are of intense interest. Most previous work involved targeted analysis of individual signaling pathways or screening of transcriptomic profiles. We developed a network biology approach using genomic and proteomic data to study the molecular patterns that distinguish pathological and physiological LVH. Methods and Results—A network-based analysis using graph theory methods was undertaken on 127 genome-wide expression arrays of in vivo murine LVH. This revealed phenotype-specific pathological and physiological gene coexpression networks. Despite >1650 common genes in the 2 networks, network structure is significantly different. This is largely because of rewiring of genes that are differentially coexpressed in the 2 networks; this novel concept of differential wiring was further validated experimentally. Functional analysis of the rewired network revealed several distinct cellular pathways and gene sets. Deeper exploration was undertaken by targeted proteomic analysis of mitochondrial, myofilament, and extracellular subproteomes in pathological LVH. A notable finding was that mRNA–protein correlation was greater at the cellular pathway level than for individual loci. Conclusions—This first combined gene network and proteomic analysis of LVH reveals novel insights into the integrated pathomechanisms that distinguish pathological versus physiological phenotypes. In particular, we identify differential gene wiring as a major distinguishing feature of these phenotypes. This approach provides a platform for the investigation of potentially novel pathways in LVH and offers a freely accessible protocol (http://sites.google.com/site/cardionetworks) for similar analyses in other cardiovascular diseases.

The structure and dynamics of saxicolous lichen communities

The saxicolous lichen vegetation on Ordovician slate rock at the mouth of the River Dovey, South Merionethshire, Wales was described in relation to several environmental variables which include aspect, slope angle, light intensity, rock porosity, rock microtopography and rock stability. Each of the measured environmental variables was shown to influence the lichen vegetation. A number of groups of species which were characteristic of certain environments were described. The data from the saxicolous lichen communities were analysed using multivariate analysis. Qualitative and quantitative data were ordinated, the qualitative data being easier to interpret ecologically, and site number (which reflects distance from the sea and altitude), rock porosity and light intensity were shown to be important environmental variables. A classification of the data was also carried out. The results of the ordination and classification were combined together and a model constructed which describes saxicolous lichen vegetation. A method which uses the model as an aid to the design and interpretation of field experiments is described. The model is applied to an experiment which investigates the effect on growth of transplanting four saxicolous lichens to different aspects. Growth was inhibited in Physcia orbicularis and Parmelia conspersa on rock surfaces of northwest aspect compared with growth on rock surfaces of southeast aspect. Growth was inhibited in Parmelia glabratula ssp. fuliginosa on rock surfaces of southeast aspect compared with rock surfaces of northwesr aspect. The growth of Parmelia saxatilis was similar at both southeast and northwesr aspects. Growth inhibition or stimulation in thalli of Physcia orbicularis, Parmelia conspersa and Parmelia glabratula ssp. fuliginosa after transplantation was consistent with the predictions of the model while the results for Parmelia saxatilis were not as expected. There was evidence that the frequency of Parmelia conspersa and Parmelia glabratula at a site is related to an effect of the environment on the growth of the thalli. There was also evidence that the frequency of Physcia orbicularis at a site is related to an effect of the environment on the establishment phase of the thalli and for the competitive exclusion of Parmelia saxatilis thalli from southeast facing rock surfaces. The distribution of lichens in relation to height on nine rock surfaces was investigated. It was suggested that the distribution of the lichens was influenced by microclimatic factors which are related to height on the rock, environmental variables which are associated with the rock substratum (e.g. rock porosity and rock microtopography) and by historical factors. The pattern of one crustose and one foliose lichen on four rock surfaces of different aspect and slope was investigated. On the vertically inclined surface the density of small thalli of Buellia aethalea and Parmelia glabratula ssp fuliginosa was correlated with the microtopography of the surface in transects horizontally across the rock surface but not in transects vertically down the rock surface. there were consitent differences in the scale and intensity of pattern horizontally and vertically and also a decrease in the intensity of pattern vertically as the slope of the rock surface decreased. These results were consistent with the suggestion of a gradient of microclimatic factors up the rock. The differences in the scale and intensity of pattern in different size classes in the population were consistent with the changes in pattern with time which have been shown to occur during succession in sand dune and salt marsh vegetation. The relationship between thallus size and height on a rock surface and between the radial growth rate and location of a thallus on a rock surface were investigated. Thalli of Parmelia glabratula ssp. fuliginosa were larger at the top of the rock surface than at the bottom and the data were consistent with the suggestion that the colonisation of the rock surface began at the top and, in time, spread downwards. The radial growth rate of the thalli could not be related to variation in slope, porosity, microtopography or directly to height on the rock but could be related to the horizontal location of the thalli on the rock. These results were consistent with the suggestion that here is a gradient of microclimatic factors across the rock surface which is also modified by height on the rock surface. The succession of lichen communities was described by relating the vegetation to rock porosity, rock microtopography, species diversity and rock stability. An initial stage dominated by crustose lichens leads to communities dominated by crustose, foliose and fruticose species. In the late stages of the succession on some rock surfaces crustose species again become dominant. The occurrence of the climax state and cyclic vegetation change in lichen communities are discussed. A mthod of estimating the age structure of a lichen population by relating thallus size to growth rate is described. The sources of error in the method are discussed in some detail and several refinements suggested to increase the accuracy of the method. The population dynamics of Parmelia glabratula ssp. fuliginosa was investigated by applying life tables to the age structures of eight different populations. The data were consistent with a period of relatively constant recruitment of thalli into the populations. Mortality in lichen populations was divided into deaths which occur after fragmentation of the thallus and deaths which occur after catastrophic environmental events. THe data suggest that the rate of fragmenting death is dependent on the age of the thallus while the rate of catastrophic death is dependent on the number of thalli established in an age class. A comparison of the numbers of thalli in each age class in the eight populations suggested that population density is controlled firstly, by climate and secondly, by variables related to the local rock surface environment. The rate of fragmenting death is related to the diversity of the community and the influence of diversity together with environmental variables in fluctuating or cyclic changes in population number.