published_or_final_version / Surgery / Master / Master of Philosophy

One of the important contributions genetic studies have made to conservation is the ability to resolve taxonomy and define relationships among populations. However, this can be complicated when species exhibit hybridization. Hybridization can be an important part of the evolutionary process and a critical component in a species ability to adapt to a changing environment. Most hybrid zones are observed at ecotones between two distinct habitats and this may be important in defining the role of hybrid zones in the evolutionary process. I examined hybridization among the three distinct lineages of desert tortoises in the genus Gopherus. An important aspect of this study system is the presence of areas of overlap between divergent lineages of desert tortoise which allowed me to test hypotheses about which forces influence these taxonomic boundaries. Specifically, I tested hypotheses about the contribution of physical vs. ecological segregation and the relative importance of isolation and gene flow in the formation of these disparate desert tortoise lineages. I used mtDNA sequence data and 25 microsatellite loci to perform Bayesian clustering, clinal analyses and habitat suitability modeling to infer population structure and influence of landscape features at each contact zone. In both instances, I observed ecological niche partitioning and limited hybridization at ecotones. I then used mtDNA and four nDNA loci to perform a multi-locus phylogenetic analysis to estimate the species tree among desert tortoise lineages and tested for ancestral admixture with RNA-seq data using demographic inference employed in the software package ∂a∂i. My results validate taxonomic distinction among all three lineages without evidence of ancestral introgression. These data suggest that despite the presence of contemporary hybridization and incomplete reproductive isolation, divergence among these lineages is consistent with species-level differentiation. By clarifying the evolutionary processes that influence the distribution of desert tortoise lineages, this study will directly inform efforts to preserve the evolutionary potential of these threatened species. Ultimately, understanding the evolutionary history of desert tortoises not only clarifies the forces that have driven speciation in this group, but it also contributes to our knowledge of the biogeographic history of the southwestern deserts and how diversity is maintained within them.

In this dissertation, I present the results of my research on a prototypical interface of the metal oxide ZnO and the organic semiconductor C₆₀. I establish that the physics at such oxide / organic interfaces is complex and very different from the extensively investigated case of organic semiconductor / metal interfaces. The studies presented in this dissertation were designed to address and improve the understanding of the fundamental physics at such hybrid organic / inorganic interfaces. Using photoemission spectroscopies, I show that metal oxide defect states play an important role in determining the interfacial electronic properties, such as energy level alignment and charge carrier dynamics. In particular, I show that for hybrid interfaces, electronic phenomena are sensitive to the surface electronic structure of the inorganic semiconductor. I also demonstrate applications of photoemission spectroscopies which are unique in that they allow for a direct comparison of ultrafast charge carrier dynamics at the interface and the electronic structure of defect levels. The research presented here focuses on a achieving a significant understanding of the realistic and device relevant C₆₀ / ZnO hybrid interface. I show how the complex surface structure of ZnO can be modified by simple experimental protocols, with direct and dramatic consequences on the interfacial energy level alignment, carrier dynamics and carrier collection and injection efficiencies. As a result of this careful study of the electronic structure and dynamics at the C₆₀ / ZnO interface, a greater understanding of the role of gap states in interface hybridization and charge carrier localization is obtained. This dissertation constitutes a first step in achieving a fundamental understanding of hybrid interfacial electronic properties.

This dissertation will examine the phenotype of pain facilitatory neurons in the rostral ventromedial medulla (RVM) and its role in neuropathic pain states. Activation of the descending facilitation pathways might be the result of plasticity in the RVM that is driven, at least in part, by the presence and activity of cholecystokinin type-2 receptors (CCK2R) mRNA expressing neurons. The expression of either opioid mu receptors (MOR) or CCK2R mRNA in the RVM was confirmed by in situ hybridization (ISH). Pretreatment with CCK8(s)-saporin resulted in a significant loss of CCK2R mRNA positive cells in the RVM, concomitant with a blockade of CCK8(s) induced hyperalgesia. The same treatment also significantly reduced the number of neurons labeled for MOR mRNA, hinting that MOR and CCK2R mRNA signals may be co-localized in some RVM cells. Consistent with these data, pretreatment with dermorphin-saporin significantly reduced the number of MOR mRNA labeled cells in the RVM, blocked RVM CCK8(s) induced hyperalgesia and reduced the number of CCK2R mRNA positive cells in the RVM. The co-localization was further confirmed by a dual label ISH approach using 35S-labeled CCK2R and Digoxigenin-labeled MOR riboprobes. Data showed that over 80% of labeled RVM neurons co-expressed both MOR and CCK2R mRNA, ~15% expressed only CCK2R mRNA, and very few cells expressed only MOR mRNA. The above findings may suggest that elimination of CCK2R mRNA expressing neurons result in removal of the driving force for descending facilitation from RVM, hereby block the development of neuropathic pain. Rats pretreated with CCK8(s)-saporin conjugates had a full reversal of thermal sensory threshold and partial reversal of tactile threshold starting at day 5 after SNL. The lesion effects of RVM CCK-SAP were evaluated by ISH. Comparing to saporin pretreated groups, CCK8(s)-saporin pretreatment significantly reduced the numbers of CCK2R mRNA labeled neurons within RVM. The data suggest that selective ablation of CCK2R mRNA expressing cells in RVM is sufficient to block the development of neuropathic pain, and thus confirm the hypothesis that CCK2R mRNA expressing cells may be an important player in descending facilitation from RVM as a mechanism of neuropathic pain.

Nicotinic acetylcholine receptors (nAChR) are neurotransmitter-gated ion channels that exist as a family of subtypes defined by unique subunit compositions. nAChR containing α6 subunits (α6*-nAChR) have attracted interest because α6 subunits are thought to be localized in brain regions implicated in reward, mood and drug dependence. To provide new information necessary toward a more complete understanding of roles of α6*-nAChR in neuropsychiatric health and disease, three lines of investigation were pursued. A set of stably transfected, human, immortalized cell lines were generated that heterologously express nAChR α6 subunits in combination with other nAChR subunits found in reward brain regions (nAChR subunit combinations α6β2, α6β4, α6β2β3, α6β4β3, α6β2β3α5, α6β4β3α5, α6α4β2β3 and α6α4β4β3). The α6α4β2β3 combination may have a functional response to epibatidine that differs from that of the α4β2 nAChR. A unique binding site was identified in cells transfected with the α6β4β3α5 nAChR subunit combination. Messenger RNA fluorescence in situ hybridization (mRNA FISH) studies established regional and celluar distribution of nAChR α6 subunit mRNA in the mouse brain. The third line of study extended this work to examine potential co-expression of nAChR α6 subunits and glutamic acid decarboxylase (GAD) or tyrosine hydroxylase (TH) as labels of GABAergic and dopaminergic/catecholaminergic neurons respectively, using tandem mRNA FISH and fluorescence immunohistochemistry. nAChR α6 subunit signal in the substantia nigra (SN) and ventral tegmental area (VTA) was congruent with previous studies. Message was also detected in the amydala, dentate gyrus, striatum, zona incerta, and cingulate, entorhinal, perirhinal, piriform, and prelimbic cortices. nAChR α6 mRNA was coexpressed with GAD in the amygdala, dentate gyrus, striatum, SN, VTA and cingulate, entorhinal, prelimbic and prelimbic cortices. TH was exclusively co-localized with nAChR α6 mRNA in the SN and VTA. Findings suggest extended roles for α6*-nAChR in the brain, particularly in the control of GABAergic neuronal activity and/or GABA release. These studies provide new insights into the composition of α6*-nAChR, the localization and cellular origins of nAChR α6 subunit expression. Data collected suggest roles for α6*-nAChR in many brain regions, including those involved in higher order processes involved in drug dependence and reward, and in modulation of inhibitory neurotransmission.

Asexual reproduction and polyploidy are relatively rare in animals with chromosomal sex determination and always represent a derived condition. To accomplish asexual reproduction several changes in gene expression are required in the mechanism of oogenesis. Polyploidy increases the cell volume and also gives rise to alterations in general physiology. Nevertheless, there are asexual animals that not only survive but seem to be doing better than their sexual progenitors. This is expressed in the distribution pattern called geographical parthenogenesis. Using molecular phylogeny, I here examine the evolution of Otiorynchid weevils, mainly Otiorhynchus scaber and sulcatus in an attempt to trace the evolutionary history and find out what causes the variation in success of different parthenogens. I also evaluate the contribution of asexuality, hybridity and polyploidy as explanations behind geographical parthenogenesis in insects. I conclude that what is called O. scaber is, in fact, a set of geographical polyploids as polyploidy and not asexuality explains the difference in clonal success. I also argue that O. sulcatus is a recently formed clonal species of non-hybrid origin that may well be a good example of a true general purpose genotype. I find little support for asexuality or a hybrid origin as explanations behind geographical parthenogenesis in insects. Finally, I argue that polyploidy in all eukaryotes should be seen as an opportunity for the species evolution, not as a limitation that ensures the demise of the taxa.

No description available.

Magistro darbe tiriami tuopų genties medžių tarprūšiniai hibridai, jų morfologiniai ir genotipiniai skirtumai. Darbo objektas. Tuopų genties hibridinių medžių: Populus alba x Populus tremula (Nr. 21), P. tremuloides x P. tremula (Nr.3) bendri palikuonys. Darbo tikslas. Atlikti tuopų genties tarprūšinių hibridų palikuonių pirminį įvertinimą. Darbo metodai: hibridizacijos, audinių kultūrų, DNR žymenų, statistiniai, tyrimo duomenų analizės, testavimo, skaičiavimo. Darbo metodika. Hibridizacija atlikta laboratorijos sąlygomis – mot. P. alba × P. tremula × tėv. P. tremuloides × P. tremula. Mezginės su nesubrendusiais gemalais subrandinamos ir sudaiginamos naudojant 3 skirtingus maitinamųjų terpių variantus. Užauginami hibridų mikroūgliai ir adaptuojami aplinkos sąlygoms specialioje patalpoje (pirminė adaptacija). Po 6 mėnesių įvertinami adaptuotų hibridų kiekybiniai požymiai. Hibridiniai medeliai perkeliami auginti į medelyną lauko sąlygomis (antrinė adaptacija) ir po metų atliekamas fenotipinių požymių įvertinimas. Darbo rezultatai ir išvados. Gauti rezultatai rodo, kad didesnė koncentracija citokininų klasės fitohormono slopino gemalų organogenezę, kai tuo tarpu maitinamoji terpė be fitohormonų sudarė palankiausias sąlygas augalų pilnam išsivystymui pirmame kultivavimo etape. Tolimesni tyrimai parodė, kad 0,5 mg/l BA( Benzyladenino) teigiamai veikė gemalų ( nesudygusių pirmame etape) organogenezę tolimesniuose kultivavimo etapuose. Atlikti regenerantų aukščio matavimai, kurių... [toliau žr. visą tekstą] / In this research work the morphologic and genotypic differences of s were investigated. Research object. The common offsprings of hybrid poplar trees: Populus alba x Populus tremula (No. 21) and P. tremuloides x P. tremula (No. 3). Research aim. Primary assessment of the offsprings obtained after crossing of interspecific Populus hybrids. Research methods: hybridization, tissue culture, DNA markers, statistics, analysis of research data, testing, computation. Research proceedings. Hybridization of P. alba x P. tremula (maternal tree) x P. tremuloides x P. tremula (paternal tree) was done under laboratory conditions. The ovaries with immature embryos were maturated and germinated using 3 different variants of nutrient medium. The hybrid microshoots were grown and later adapted under environmental conditions in the special room (primary adaptation). After 6 months the quantitive features of adopted hybrids were estimated. Young hybrid trees were transferred to the nursery for cultivation under nature conditions. (secondary adaptation). A year later the estimation of phenotypic features was done. Research results and conclusions. The obtained results indicate that higher concentration of cytokinin class phytohormone repressed organogenesis of the embryos, whereas nutrient medium without phytohormones provided the most favourable conditions for the full plant development during the first cultivation stage. The invetigations during following cultivation stages revealed that 0,5... [to full text]

Successful preservation of Earth’s biodiversity requires an understanding of the processes that generate new species. The generation of species without gene exchange is considered predominant; however, a growing body of evidence indicates that populations can diverge while exchanging genes, and that this may be common. Previous research hypothesized that blue-footed (Sula nebouxii) and Peruvian (S. variegata) boobies diverged from their common ancestor while exchanging genes. Here, I combine ecological and genetic perspectives to thoroughly evaluate this hypothesis. Using a panel of eight molecular markers, I estimate population differentiation for each species. I find evidence of weak population differentiation for both species, an uncommon pattern in seabirds, and argue that specialization to an unpredictable food resource has shaped contemporary population differentiation. Next, I use molecular markers and morphology to evaluate the hybrid status of five morphologically aberrant individuals. I report that all are likely F1 (first generation) hybrids, and are the product of crosses between female Peruvian boobies and male blue-footed boobies. Sex biases in pairing may occur because of an underlying preference for elaborate courtship displays. I then expand the dataset to 19 loci and use cline theory and Bayesian assignment tests to characterize the hybrid zone, to examine introgression, and to evaluate the hybrid status of the aberrant individuals. The hybrid zone is most likely maintained by strong endogenous and exogenous selection against hybrids and dispersal of parentals into the hybrid zone (a tension zone), and introgression is low for nuclear loci and absent for mitochondrial loci. Finally, I test the hypothesis that this species pair diverged from their common ancestor with gene flow using recently developed analyses and multiple loci. Divergence without gene flow is rejected and unidirectional introgression of sex-linked loci during divergence is reported. The results of this study support the hypotheses that: 1) populations can diverge while exchanging genes; 2) the Z chromosome may play a role in avian speciation; and 3) organisms specialized to variable foraging environments should exhibit low population differentiation. This study adds to our understanding of both population differentiation and speciation in seabirds, and the generation of new species more generally. / Thesis (Ph.D, Biology) -- Queen's University, 2011-04-06 13:55:32.151

I investigated how orchid biology, floral morphology, and diversity of surrounding floral and pollinator communities affected reproductive success and hybridization of Cypripedium candidum and C. parviflorum. Floral dimensions, including pollinator exit routes were smallest in C. candidum, largest in C. parviflorum, with hybrids intermediate and overlapping with both. This pattern was mirrored in the number of insect visitors, fruit set, and seed set. Exit route size seemed to restrict potential pollinators to a subset of visiting insects, which is consistent with reports from other rewardless orchids. Overlap among orchid taxa in morphology, pollinators, flowering phenology, and spatial distribution, may affect the frequency and direction of pollen transfer and hybridization. The composition and abundance of co-flowering rewarding plants seems to be important for maintaining pollinators in orchid populations. Comparisons with orchid fruit set indicated that individual co-flowering species may be facilitators or competitors for pollinator attention, affecting orchid reproductive success.