Indiana University-Purdue University Indianapolis (IUPUI) / Blow flies are often utilized in the field of forensic science due to their ability to aid in the estimation of time since death. Currently, estimations of postmortem interval require assumptions to be made and are prone to a margin of error, prompting research that may contribute to more accurate postmortem interval estimations and help to fill in the gaps of unknown information. Blow flies are necrophagous, feeding on feces and carrion, and therefore, are constantly sampling the environment. This behavior can be exploited in order to monitor the biodiversity in an environment. Through analysis of DNA isolated from the guts of blow flies, information can be obtained regarding what animals have died in an environment, what animals are still living in that environment, and the abundance and diversity of the animals present in a specific environment. Using fly-derived ingested DNA is a viable method for vertebrate resource identification and biodiversity monitoring. Over the course of a two-summer sampling period, in and around two national parks, a total of 162 blow fly (Phormia regina) samples returned a positive vertebrate DNA identification, with 33 species identified from five animal orders. Of the total number of flies collected and analyzed, 23.58% returned a positive vertebrate species identification. The method detected both abundant and common species based on National Park surveys, as well as some uncommon or unknown to the park species. In the SE region, 9 individuals belonging to the Rodentia order, 12 individuals belonging to the Artiodactyla order, 21 individuals belonging to the Carnivora order, 1 individual belonging to the Cingulata order, and 3 individuals belonging to the Lagomorph order were detected. In the SE region, 63% of the individuals detected belonged to the common category, 14% of the individuals detected belonged to the uncommon category, and 23% of the individuals detected belonged to the not in park/unknown category. In the NW region, 42 individuals belonging to the Rodentia order, 46 individuals belonging to the Artiodactyla order, and 28 individuals belonging to the Carnivora order were detected. In the NW region, 52% of the individuals detected belonged to the abundant category, 36% of the individuals detected belonged to the common category, and 12% of the individuals detected belonged to the uncommon category. The relative biodiversity of the sampled environment can be inferred. In the SE region, the Shannon Biodiversity Index was calculated to be 2.28 with an evenness of 0.844, while in the NW region, the Shannon Biodiversity Index was calculated to be 2.79 with an evenness of 0.855. Unsurprisingly, there was greater biodiversity in the Northwest Park samples than in the Southeast Park samples. Additionally, the ideal weather conditions for blow fly collection were determined be at a temperature of between 60- and 80-degrees Fahrenheit, a relative humidity between 50% and 60%, no precipitation, and a wind speed between 2 and 8 miles per hour. This information has further implications in the field of forensic science, specifically dealing with wildlife forensics, pathogen distributions, and can help to improve accuracy in regards to postmortem interval (PMI) estimations.

Changes in the structure, content and morphology of chromosomes accumulate over evolutionary time and contribute to cell, developmental and organismal biology. The axolotl (Ambystoma mexicanum) is an important model for studying these changes because: 1) it provides important phylogenetic perspective for reconstructing the evolution of vertebrate genomes and amphibian karyotypes, 2) its genome has evolved to a large size (~10X larger than human) but has maintained gene orders, and 3) it possesses potentially young sex chromosomes that have not undergone extensive differentiation in the structure that is typical of many other vertebrate sex chromosomes (e.g. mammalian XY chromosomes and avian ZW chromosomes). Early chromosomal studies were performed through cytogenetics, but more recent methods involving next generation sequencing and comparative genomics can reveal new information. Due to the large size and inherent complexity of the axolotl genome, multiple approaches are needed to cultivate the genomic and molecular resources essential for expanding its utility in modern scientific inquiries. This dissertation describes our efforts to improve the genomic and molecular resources for the axolotl and other salamanders, with the aim of better understanding the events that have driven the evolution of vertebrate (and amphibian) chromosomes. First, I review our current state of knowledge with respect to genome and karyotype evolution in the amphibians, present a case for studying sex chromosome evolution in the axolotl, and discuss solutions for performing analyses of large vertebrate genomes. In the second chapter, I present a study that resulted in the optimization of methods for the capture and sequencing of individual chromosomes and demonstrate the utility of the approach in improving the existing Ambystoma linkage map and generating targeted assemblies of individual chromosomes. In the third chapter, I present a published work that focuses on using this approach to characterize the two smallest chromosomes and provides an initial characterization of the huge axolotl genome. In the fourth chapter, I present another study that details the development of a dense linkage map for a newt, Notophthalmus viridescens, and its use in comparative analyses, including the discovery of a specific chromosomal fusion event in Ambystoma at the site of a major effect quantitative trait locus for metamorphic timing. I then describe the characterization of the relatively undifferentiated axolotl sex chromosomes, identification of a tiny sex-specific (W-linked) region, and a strong candidate for the axolotl sex-determining gene. Finally, I provide a brief discussion that recapitulates the main findings of each study, their utility in current studies, and future research directions. The research in this dissertation has enriched this important model with genomic and molecular resources that enhance its use in modern scientific research. The information provided from evolutionary studies in axolotl chromosomes shed critical light on vertebrate genome and chromosome evolution, specifically among amphibians, an underrepresented vertebrate clade in genomics, and in homomorphic sex chromosomes, which have been largely unstudied in amphibians.

The objective of this thesis was to investigate the effects of mammalian grazers, such as microtine rodents and reindeer, (top-down effects) and nutrient availability (bottom- up effects) on the plant community of a tundra heath. I conducted a large-scale fertilization experiment and studied the impact of grazers using exclosures. I measured the effects of fertilization and grazing on soil microbial activity and nutrient cycling. I investigated the responses to fertilization of the invertebrate community, I studied the effects on the quality of bilberry as food for mammalian herbivores, and I looked at how concentrations of nutrients and carbon-based secondary defences against herbivory fluctuated between seasons in unfertilized and fertilized treatments. The results of my thesis show that the plant community investigated is exposed to a strong top-down control by mammalian herbivores. On the fertilized and grazed areas the aboveground biomass of the vascular plant community did not increase compared to unfertilized areas. However, the productivity of the plant community was clearly nutrient- limited. During the eight years of the experiment, on the fertilized areas plant biomass was significantly increased inside the herbivore exclosures In my study mammalian herbivores at comparatively low densities and grazing outside the growing season were sufficient to control the biomass of a heterogeneous plant community. Microtine rodents (Norwegian lemmings and grey-sided voles) preferred the fertilized areas for overwintering. The food plant quality of bilberry for grey-sided voles was improved on the fertilized areas throughout the year. Grazing decreased the nitrogen storage in the aboveground plant biomass. Reindeer and rodents had also important indirect effects on the plant community by decelerating soil nutrient cycling and soil microbial activity. This effect may be accelerated by the impact of herbivore on plant species composition. Graminoids, which contained the highest nitrogen concentrations in their tissues, increased rapidly on the fertilized areas, but their abundance was significantly lower on grazed fertilized areas. The invertebrate community was detritus-based and received their energy indirectly from the litter via soil microbes and detritivores. Fertilization increased the biomass of invertebrate carnivores, but had no effect on the biomass of invertebrate herbivores. Apparent competition between detritivores and invertebrate herbivores, mediated by carnivorous invertebrates predating on both of them, is supposed to keep the densities and grazing pressure of invertebrate herbivores low. Grazing damage by invertebrates was very low and only 0.021 % of the total vascular plant biomass was removed. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 2001, härtill 6 uppsstser.</p> / digitalisering@umu

Recurrent processes are a general feature of living systems, from the cell cycle to circadian day-night rhythms to hibernation and flowering cycles. During development and life, numerous recurrent processes are controlled by genetic oscillators, a specific class of genetic regulatory networks that generates oscillations in the level of gene products. A vital mechanism controlled by genetic oscillators is the rhythmic and sequential segmentation of the elongating body axis of vertebrate embryos. During this process, a large collection of coupled genetic oscillators gives rise to spatio-temporal wave patterns of oscillating gene expression at tissue level, forming a dynamic prepattern for the precursors of the vertebrae. While such systems of genetic oscillators have been studied extensively over the past years, many fundamental questions about their collective behavior remain unanswered. In this thesis, we study the behavior and the properties of genetic oscillators from the single oscillator scale to the complex pattern forming system involved in vertebrate segmentation. Genetic oscillators are subject to fluctuations because of the stochastic nature of gene expression. To study the effects of noisy biochemical coupling on genetic oscillators, we propose a theory in which both the internal dynamics of the oscillators as well as the coupling process are inherently stochastic. We find that stochastic coupling of oscillators profoundly affects their precision and synchronization properties, key features for their viability as biological pacemakers. Moreover, stochasticity introduces phenomena not known from deterministic systems, such as stochastic switching between different modes of synchrony. During vertebrate segmentation, genetic oscillators play a key role in establishing a segmental prepattern on tissue scale. We study the spatio-temporal patterns of oscillating gene expression using a continuum theory of coupled phase oscillators. We investigate the effects of different biologically relevant factors such as delayed coupling due to complex signaling processes, local tissue growth, and tissue shortening on pattern formation and segmentation. We find that the decreasing tissue length induces a Doppler effect that contributes to the rate of segment formation in a hitherto unanticipated way. Comparison of our theoretical findings with experimental data reveals the occurrence of such a Doppler effect in vivo. To this end, we develop quantification methods for the spatio-temporal patterns of gene expression in developing zebrafish embryos. On a cellular level, tissues have a discrete structure. To study the interplay of cellular processes like cell division and random cell movement with pattern formation, we go beyond the coarse-grained continuum theories and develop a three-dimensional cell-based model of vertebrate segmentation, in which the dynamics of the segmenting tissue emerges from the collective behavior of individual cells. We show that this model is able to describe tissue formation and segmentation in a self-organized way. It provides the first step of theoretically describing pattern formation and tissue dynamics during vertebrate segmentation in a unified framework involving a three-dimensional tissue with cells as distinct mechanical entities. Finally, we study the synchronization dynamics of generic oscillator systems whose coupling is subject to phase shifts and time delays. Such phase shifts and time delays are induced by complex signaling processes as found, e.g., between genetic oscillators. We show how phase shifts and coupling delays can alter the synchronization dynamics while leaving the collective frequency of the synchronized oscillators invariant. We find that in globally coupled systems, fastest synchronization occurs for non-vanishing coupling delays while in spatially extended systems, fastest synchronization can occur on length scales larger than the coupling range, giving rise to novel synchronization scenarios. Beyond their potential relevance for biological systems, these results have implications for general oscillator systems, e.g., in physics and engineering. In summary, we use discrete and continuous theories of genetic oscillators to study their dynamic behavior, comparing our theoretical results to experimental data where available. We cover a wide range of different topics, contributing to the general understanding of genetic oscillators and synchronization and revealing a hitherto unknown mechanism regulating the timing of embryonic pattern formation.

Species are vital entities in biology. Species are generally considered to be discrete entities, consisting of a group of (usually interbreeding) individuals that are similar in phenotype and genetic composition, yet differ in significant ways from other species. The study of speciation has focussed on understanding general evolutionary mechanisms involved in the accumulation of differences both at the genetic and phenotypic level. In this thesis, I investigate incipient speciation, an early stage of divergence towards evolutionary independence in closely related natural populations. I make ample use of recent advances in sequencing technology that allow 1) characterizing phenotypic divergence at the level of the transcriptome and 2) delineate patterns of genetic variation at genome-scale from which processes are inferred by using principles of population genetic theory. In the first paper, we assembled a draft genome of the hooded crow and investigated population differentiation across a famous European hybrid zone. Comparing sequence differentiation peaks between and within the colour morphs, we could identify regions of the genome that show differentiation only between colour morphs and that could be related to gene expression profiles of the melanogenesis pathway coding for colour differences. The second paper expands on the first paper in that it includes crow population samples from across the entire Palaearctic distribution spanning two additional zones of contact between colour morphs. The results suggest that regions associated with selection against gene flow between colour morphs were largely idiosyncratic to each contact zone and emerged against a background of conserved 'islands of differentiation' due to shared linked selection. The third paper focusses on five killer whale ecotypes with distinct feeding and habitat specific adaptations. Differing levels of sequence differentiation between these ecotypes places them along a speciation continuum and provides a unique temporal cross-section of the speciation process. Using genome scans we identified regions of the genome that show ecotype specific differentiation patterns which might contain candidate genes involved in adaptation. In the fourth and final paper, I assumed a comparative genomic perspective to the problem of heterogeneous genomic differentiation during population divergence. The relatively high correlations in the diversity landscapes as well as differentiation patterns between crow, flycatcher and Darwin's Finch populations is best explained by conservation in broad-scale recombination rate and/or  association with telomeres and centromeres conducive to shared, linked selection.

Orientador: Lúcia Elvira Alvares / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T17:37:00Z (GMT). No. of bitstreams: 1 Pedrosa_AngelicaVasconcelos_M.pdf: 1992620 bytes, checksum: ed7d02568860e3ccf1e489cf2928818e (MD5) Previous issue date: 2013 / Resumo: A correta padronização do corpo do embrião requer a atividade de diferentes vias de sinalização. Dentre elas, uma que se destaca é via de sinalização Wnt de polaridade celular planar (Wnt/PCP), que é responsável pelo controle da polaridade celular e pela organização celular de diversos tecidos nos animais. Uma vez interrompida, a via Wnt/PCP pode causar falhas no fechamento do tubo neural, provocando defeitos congênitos. Em seres humanos, mutações em componentes-chave da via Wnt/PCP como as proteínas codificadas pelos genes Vangl1 e Vangl2 têm sido associadas à graves malformações geradas por falhas no fechamento do tubo neural. Estruturalmente, ambos os genes Vangl1 e Vangl2 codificam proteínas de superfície transmembranares, essenciais para o desenvolvimento apropriado do embrião. O presente trabalho teve como objetivo a caracterização do padrão de expressão dos genes Vangl1 e Vangl2 durante a embriogênese de Gallus gallus. Ensaios de hibridação in situ em embrião inteiro (whole mount) e cortes em vibratómo foram realizados com a finalidade de estabelecer temporal e espacialmente o padrão de expressão dos genes Vangl1 e Vangl2. Como resultado, observou-se que estes genes são expressos durante as etapas de gastrulação, neurulação e no início da organogênese do desenvolvimento embrionário de Gallus gallus. No início da gastrulação, os genes Vangl1 e Vangl2 possuem domínios de expressão comuns nos embriões de galinha, uma vez que ambos são expressos na linha primitiva, nódulo de Hensen e crescente cardiogênico. Contudo, nossos dados revelaram particularidades na expressão destes genes, uma vez que há uma predominância dos transcritos de Vangl1 na região posterior da linha primitiva, enquanto Vangl2 apresenta uma expressão uniforme ao longo desta estrutura. Em adição, enquanto Vangl1 é expresso na notocorda e em toda a extensão do nódulo de Hensen, Vangl2 é expresso no entorno desta estrutura. Ao longo da neurulação e na organogênese inicial, ambos os genes Vangl são expressos de maneira similar, em domínios que abrangem a placa, as pregas e o tubo neural. Outros importantes domínios de expressão dos Vangl correspondem às vesículas ópticas e óticas, às vesículas encefálicas particularmente na região das flexuras encefálicas, aos diferentes tipos de mesoderma (paraxial, intermediário e lateral) e ao assoalho da faringe. Ao comparar os resultados obtidos por hibridação in situ em galinha ao um levantamento bibliográfico sobre outros vertebrados, observou-se uma sobreposição dos domínios-chave de expressão nos diferentes organismos, demonstrando a conservação filogenética da atividade destes genes e sugerindo uma possível conservação funcional. Desta forma, nossos dados sugerem que os genes Vangl desempenham um importante papel no desenvolvimento embrionário de aves, possivelmente coordenando os movimentos morfogenéticos durante a gastrulação, bem como a formação da placa neural e posterior dobramento e fechamento do tubo neural, além de outros processos da embriogênese de aves / Abstract: The correct patterning of the embryo's body requires the activity of different signaling pathways. Among them, one that stands out is the Wnt Planar Cell Polarity Signaling Pathway (Wnt/PCP), which is responsible for controlling the cell polarity and cellular organization of many tissues in animals. Failures in the Wnt/PCP signaling can cause neural tube birth defects. In humans, mutations in key components of the Wnt/PCP as the Vangl1 and Vangl2 molecules were identified in patients with neural tube defects. Structurally, both Vangl1 and Vangl2 genes encode transmembrane surface proteins similar, which are essential to proper development. The present study aimed to characterize the expression pattern of Vangl1 and Vangl2 genes during embryogenesis in Gallus gallus. Whole-mount in situ hybridization assays and vibratome sectioning of embryos were conducted in order to establish the spatial and temporal expression pattern of Vangl1 and Vangl2 genes. Our results showed that these genes are expressed during gastrulation, neurulation and early organogenesis in Gallus gallus. At the onset of Gastrulation, Vangl1 and Vangl2 genes have common areas of expression in chicken embryos, since both are expressed in the primitive streak, Hensen's node and cardiogenic crescent. However, our data showed particularities in the expression of these genes, since there is a predominance of Vangl1 transcripts in the posterior region of the primitive streak while Vangl2 has a uniform expression throughout that structure. In addition, while Vangl1 is expressed in the notochord and in the full length of the Hensen's node, Vangl2 is expressed only around this structure. Throughout neurulation and early organogenesis, both Vangl genes are expressed in a similar manner on the neural plate, neural groove, neural folds and in the neural tube. Other important areas of Vangl expression correspond to optical and otic vesicles, the brain vesicles, the different types of mesoderm (paraxial, intermediate and lateral) and the floor of the pharynx. By comparing the chicken expression of Vangl genes with other vertebrates, we notice that there are overlapping expression patterns among key areas among different organisms, showing a phylogenetic conservation of expression domains and suggesting a possible functional conservation. Overall, our data suggests that Vangl genes play an important role in embryonic development of bird, possibly by coordinating the morphogenetic movements during gastrulation, as well as the formation of neural tube, among other processes during the birds embriogenesis / Mestrado / Biologia Celular / Mestra em Biologia Celular e Estrutural

Les connexions neuronales du système visuel forment des synapses spatialement distribuées en couches discrètes. Comprendre la base du ciblage spécifique axonale est critique pour déchiffrer la formation des réseaux neuronaux complexes. Dans une première étude, nous avons investigué le rôle de la protéine de la matrice extracellulaire Reelin dans la formation in vivo du circuit rétinotectal chez le poisson zèbre. Ce circuit se compose de cellules ganglionnaires de la rétine (CGRs) transmettant l’information visuelle au cerveau via la projection de leur axone dans les différentes couches du tectum optique. Nous avons démontré que la Reelin secrétée par de neurones inhibiteurs localisés dans les couches supérieures du tectum optique forme un gradient. L’induction de mutations délétères dans la voie de signalisation canonicale de la Reelin à l’aide d’outils génétiques a conduit à des défauts de ciblage des axones de CGRs. Nos résultats démontrent un nouveau rôle de la Reelin lors du développement du système visuel et la décrivent comme signature moléculaire nécessaire au ciblage et au positionnement précis des axones de CGRs.Dans une seconde étude, nous avons utilisé la technique CRISPR/Cas9 pour développer une nouvelle approche de mutagénèse conditionnelle chez le poisson zèbre. Nos résultats démontrent que la perturbation de gènes dans des tissues spécifiques peut être effectué par l’induction de l’expression de la protéine Cas9 via le système Gal4/UAS. Nous avons établis un outil pour induire l’apparition de mutations délétères dans des clones de cellules mais aussi dans des cellules individuelles, tous pouvant être suivit distinctement grâce à un marquage génétique. / Neuronal connections in the visual system are arranged in synaptic laminae. Understanding the basis of lamina-specific axonal targeting is critical to gain deeper insights on how complex neural networks form. In a first study we investigated the role of the ECM protein Reelin during zebrafish retinotectal circuit formation in vivo. Here retinal ganglion cells (RGCs) convey the visual information to the brain by projecting their axons to different layers of the optic tectum. We demonstrated that Reelin secreted by a specific class of tectal superficial inhibitory neurons is spatially distributed in a superficial-to-deep gradient within the tectal neuropil. Induced gene disruption for all the components of the canonical Reelin pathway expressed in the retinotectal system resulted in aberrant layering of RGC axons suggesting a role for Reelin pathway in axonal sublaminar segregation. Altogether our findings elucidate a new role for Reelin in vertebrate visual system development, during which it acts as molecular cue by imparting positional information for ingrowing RGCs.In a second study we took advantage of the CRISPR/Cas9 technology to develop a novel approach for conditional mutagenesis in zebrafish. Our results provide evidence that tissue-specific gene disruption can be achieved by driving Cas9 expression with the Gal4/UAS system. We established a tool to induce loss-of-function mutations in cell clones or single cells that can be followed by genetic labeling, enabling their phenotypic analysis. Our technique has the potential to be applied to a wide-range of model organisms, allowing systematic mutagenesis and labeling on a genome-wide scale.

Feral swine (Sus scrofa) are an invasive species in the Mississippi Alluvial Valley (MAV). They cause millions in damage annually to agriculture, and likely negatively affect native wildlife species. Using camera traps, I monitored 36 forest patches within the MAV to assess the effects of swine invasions on native wildlife species richness. I also modified the double-observer point count technique into a new method for estimating swine abundance with camera traps. Feral swine suppressed native vertebrate richness by 26% when compared to uninvaded patches. I validated the new double-observer technique by determining if it could detect an abundance-area relationship in wildlife populations and estimate a known decrease in abundance following swine removal. This technique was sensitive enough to detect the increase and decrease in abundance and estimated the number of individuals removed from the population relatively accurately. This technique may be useful in the future to manage feral swine populations.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Roman Reddy R. Ganta / Ehrlichia and Anaplasma species are rickettsial organisms which infect a variety of mammalian species. The organisms are transmitted from ticks and are maintained in reservoir hosts. Several pathogens have been identified in recent years as the causative agents for emerging infections in people. One of the primary reservoir hosts for the pathogens is the white tailed deer. In this study, 147 deer blood samples and 37 ticks were evaluated for the prevalence of Ehrlichia/Anaplasma species by TaqMan-based real time amplification assay and DNA sequence analysis. One hundred and thirteen (74%) samples tested positive with the Ehrlichia/Anaplasma genera-specific probe. Further analysis of the samples with the probes specific for human ehrlichiosis agents, E. chaffeensis and E. ewingii identified 4 (2.7%) and 7 (4.7%) positives, respectively. Test positives from 24 randomly selected samples were further evaluated by sequence analysis targeting to a 450 bp segment of 16S rRNA gene. All 24 samples were confirmed as positive for the Ehrlichia GA isolate # 4 (GenBank #U27104.1). DNAs from 37 pools of ticks collected from the white tailed deer were also evaluated. The TaqMan-based real time PCR assay with Anaplasma/Ehrlichia common probe identified 29 (78%) tick pools as positives whereas E. chaffeensis- and E. ewingii-specific probes identified three (8%) and one (3%) positives, respectively. The PCR and sequence analysis of tick samples identified Gram-negative bacteria species which included one endosymbiont of Rickettsia species (one tick pool), one Alcaligenes faecalis strain (three tick pools), five different Pseudomonas species (9 tick pools) and five different uncultured bacteria organisms (7 tick pools). Although the pathogenic potential of the white-tailed deer isolates of Anaplasma and Ehrlichia agents remains to be established, their high prevalence and the presence of human ehrlichiosis pathogens in white-tailed deer is similar to earlier findings. The high prevalence of the deer isolates of Anaplasma and Ehrlichia species demonstrates the need for further assessment of the pathogenic potential of these organisms to people and domestic animals.

To study the impact of environmental changes in a coastal marine ecosystem, it is necessary to use indicator species. It is crucial to understand the foraging performances that proceed from environmental changes. The aim of my thesis was to examine the influence of intrinsic and extrinsic factors on the foraging activity of the little penguins (Eudyptula minor). The thermocline allowed birds to approach optimal behaviour. However, the thermocline is an unstable element. I did not find any effect of individual characteristics on their foraging behaviour and success. My work suggests that environmental conditions are major factors that will influence the behaviour of little penguins, allowing me to conclude that little penguins are good ecological indicators.