The study of B and T cell receptor repertoires from high throughput sequencing is a recent development that allows for unprecedented resolution and quantification of the adaptive immune response. The immense diversity and long tailed distribution of these repertoires has up until now limited such studies to expanded clonal signatures or to analysis of imprecise signals with limited dynamic range collected by techniques such as radioactive and fluorescent labeling. This thesis presents a number of quantitative methods to characterize the repertoire and examine the questions of sequence diversity and inter-repertoire divergence of T cell repertoires. These approaches attempt to accurately parametrize the inherent distribution of T cell clones drawing from statistical tools derived from ecological literature and information theory. The methods presented are applied to T cell analyses of various tissue compartments of the human body, including peripheral blood mononucleocytes, thymic tissues, spleen, inguinal lymph nodes, lung lymph nodes and the brain. A number of applications are explored with strong implications for translational use in medicine. Novel insights are made into the mechanism of maintenance and compartmentalization of na{\"i}ve T cells from human donors of many different ages. Diversity and divergence of the tumor infiltrating sequence repertoire is measured in low grade gliomas and glioblastomas from cancer patients, and potential sequence based biomarkers are assessed for studying glioma phenotype progression. A careful investigation of the immune response to allogeneic stimulus reveals the effect of HLA on sequence sharing and diversity of the alloresponse, and quantifies for the first time using sequence data the fraction of T cells in a repertoire that are alloreactive. The use of repertoire sequencing and mathematical models within immunology is a new and emerging concept within the rapidly expanding field of systems immunology and will undoubtedly have a profound impact on the future of immunology research. It is hoped that the tools presented in this thesis will give insight into how to quantitatively explore the breadth and depth of the T cell receptor repertoire, and provide future directions for TCR repertoire analysis.

This dissertation is an exploration of phenomena on varying scales, built on the backbone of Cenozoic mammalian biochronologic units (Land Mammal ‘Ages’): the integration of fossil and geological data to constrain spatiotemporal patterns in evolution. I develop and test hypotheses about the origins and ordinal-level relationships of the extinct South American endemic placental order Notoungulata, as well as about some more specific macroevolutionary patterns at a familial level within notoungulates. Major novel outcomes include a new biochronologic timescale for the terrestrial Cenozoic of South America, numerically calibrated through synthesis of new and existing high-precision geochronological data (particularly U-Pb and 40Ar/39Ar dating), along with an explicit logical framework for Land Mammal “Age” calibration; description of two new interatheriid notoungulate taxa from the central Chilean Andes; a new phylogenetic hypothesis for the position of Notoungulata within Placentalia; and the first ever DNA sequences obtained from the extinct musk ox relative Bootherium bombifrons. The first study, chapter two, is a review of geochronologic (radiometric and magnetostratigraphic) constraints for the South American Land Mammal “Age” timescale. I present a revised, updated timescale, and a descriptive, logical framework for the synthesis of geochronologic and biochronologic data from a variety of sources and analytical methods. Significant changes to the calibration of individual SALMAs in this update are concentrated in the Paleogene, while Neogene calibrations have remained fairly stable, with small refinements to the core age ranges of the Huayquerian, Chasicoan, Colloncuran, and Friasian. This section also investigates the influence of latitudinal biotic provinciality on correlations and chronologic calibrations, particularly as far as provinciality reflects the climatic evolution of the continent. Marked provinciality is evident at least as early as the early Eocene, with faunas like Itaboraí differing from potential correlative faunas at higher latitudes, potentially representing unique periods in mammalian evolution, both faunally and chronologically. Instead of trying to recognize and correlate the classical high latitude SALMAs to highly distinctive tropical assemblages, the SALMA timescale should allow for the development of separate mammalian biochronologic zonations for low and high latitudes. Chapter three presents and describes two new notoungulate taxa, representing the first species formally described from the Los Queñes Fauna, a late Eocene mammal assemblage from the Andean Main Range of central Chile. These two taxa, Anabalcarcel ignimbritae and Jackconrad carreterensis, represent the earliest hypsodont interatheres known. Based on ancestral state reconstructions using parsimony, hypsodonty appeared no later than the latest Eocene (34.6 ± 0.8 Ma; likely correlative with the Mustersan South American Land Mammal Age) in interatheres, a time when this dental specialization was not yet pervasive among other mammalian herbivores. Tree-based comparative analyses revealed two significant taxonomic radiations of interatheres, the early radiation of basal interatheriids and a later radiation of hypselodont taxa after the interval of dramatic global climatic change associated with the Eocene/Oligocene boundary. In chapter four, I attempted to apply some of the geochronologic methods incorporated in the first two chapters, to date a newly discovered site in Abanico Formation, central Chilean Andes. This formation yields fossil mammals in numerous areas, including at several localities in the Río Las Leñas and Río Cachapoal drainages. In the Cachapoal Valley, steeply-dipping beds have yielded fossils of Tinguirirican age, including a polydolopine marsupial and an interatheriid notoungulate. The results of this study, the first 40Ar/39Ar analysis from the Cachapoal Valley, are a date of 11.1 ± 1.8 Ma, from stratigraphically higher levels loosely constraining the minimum age for fossiliferous deposits. Chapters five and six change tack a little, and investigate analytical methods in ancient-DNA phylogenomics, time-calibrated with radiocarbon dates, using as a study system Bootherium bombifrons, the endemic North American helmeted musk ox that went extinct, along with most of its megafaunal colleagues, at the end of the Pleistocene. This switch to a North American, rather than South American, endemic mammal was driven by availability of molecular comparative material and the presence of hundreds of helmeted musk ox specimens in the American Museum paleontology collections, which I was able to sample destructively. I present the first complete mitochondrial genome of the extinct musk ox Bootherium bombifrons, and new mitochondrial sequence data from seven individuals referred to Bootherium bombifrons Additionally, I obtained new radiocarbon dates from these same specimens, of 38580 ± 720, 30240 ± 260, 44570 ± 190, 41040 ± 910, 44240 ± 1500, 39080 ± 770, 28370 ± 210, and 47190 ± 2100 calibrated 14C years BP. Phylogenetic analysis supports placement of Bootherium as sister to the living musk ox, Ovibos moschatus, in agreement with morphological taxonomy. I also tested hypotheses regarding the impact that locus choice has on divergence date estimates using tip calibrations in this taxon. Estimates of the root age of three different gene trees for Bootherium specimens ranged from 19 ka, for cytochrome B, to over 50 ka for the mitochondrial control region. The final study in this dissertation (chapter seven) is a superordinal scale approach to fitting Notoungulata into the picture of all placental mammals through synthesis and expansion of existing total-evidence matrices. Results presented here corroborate previous findings that notoungulates are most closely related to Afrotheria, but do not support afrothere and notoungulate reciprocal monophyly, suggesting a complex biogeographic relationship between South America and Africa persisting from Late Cretaceous through mid-Paleocene.

Protein translation has emerged as an important mediator of cellular activity. Here, we discuss efforts to develop and apply new technologies designed to gain insights into translational control. We begin with the application of ribosome profiling to a RiboTag Glioma mouse model which enables translational profiling of transformed cellular populations. This approach demonstrates a number of abnormalities of translation in transformed cells. We go on to report the development of an inexpensive and rapid library preparation methodology which enables high-throughput sequencing of ribosome-protected footprints from small amounts of input material. We apply this technique to a CAMKII RiboTag mouse model to make new insights into cell-type specific translation. Finally, we describe efforts to investigate translation regulatory networks through the development of a technique which couples large-scale perturbation with a genome-wide readout of translation. Molecular dissection of tissues through the ectopic expression of modified ribosomal proteins commonly relies on tissue-specific genes which act as drivers. In the case of glioma, a gene specific to transformed tissue, but not expressed in normal brain tissue, has not been identified. Chapter 2 focuses on efforts to bypass this through the development of a RiboTag Glioma mouse model which allows for concurrent transformation and the expression of an epitope-tagged ribosomal protein in virally infected cells. This model made possible the isolation of translating mRNA from transformed cellular populations and was used to demonstrate the existence of a number of translational abnormalities in transformed cells. Conventional ribosome profiling is a powerful tool which allows for the identification of ribosome-protected mRNA footprints. However, it is time-consuming, expensive, and difficult to implement. Based on our experiences with conventional ribosome profiling, we sought to develop a method which could decrease the overall number of enzymatic reactions and purification steps, thereby reducing the time and cost associated with the procedure; these efforts are discussed in Chapter 3. Utilizing a ligation-free library preparation process, which incorporates poly(A)-polymerase, template switching and bead-based purification, we reduced the time, costs and input requirements required to generate a ribosome profiling library while maintaining high library complexity. We applied our ligation-free ribosome profiling technique to a CAMKII RiboTag mouse model which enabled us to identify patterns of cell-type specific translation and the effects of mTOR inhibition in CAMKII-expressing excitatory neurons. Regulation of protein expression is an essential and highly complex cellular activity. Aberrations of translational control are central to a host of pathologies and have direct clinical relevance. However, our knowledge of the networks which control translation is limited. Chapter 4 details our efforts to develop a highly-scalable technology which enables the identification of gene-specific translational alteration in response to perturbation. Coupled with a large-scale perturbation screen, this technique could lead to the generation of a network for translational control, similar to efforts previously undertaken to understand transcriptional control. By combining the recently developed PLATE-Seq method, which utilizes unique barcode identifiers and pooled library construction, with a technique for the identification and isolation of ribosome associated mRNA, we are able to rapidly and inexpensively determine genome-wide translational states in a highly scalable

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For the past twenty years, taxonomy has been in a state of turmoil. This confusion brings along with it four distinct schools of thought, each of which offers a different concept of biological species. The thesis will show that these concepts are purely operational and have only a weak theoretical force. In turn, it will be argued that a sound definition of species uses the notion of natural kinds, which is itself defined in term of non-causal nomological regularities.

Diss. (sammanfattning) Uppsala : Univ., 2007. / Härtill 4 uppsatser.

No description available.

No description available.

A fundamental goal in neuroscience research is to comprehensively characterize cell populations within the brain in order to uncover the mechanisms governing brain states and behavior. This involves profiling specific cells through multiple biological aspects and discerning their contributions to the brain's circuitry. Characterizing cell populations at a large scale from multiple perspectives is crucial for advancing our understanding of brain function. Recently, experimental technologies for generating large datasets have become more accessible to neuroscience researchers working at different biological scales, including molecular, cellular, and functional levels. These new technologies, including high-throughput sequencing, multiplexed spatial transcriptomics, cellular tracing, and multimodal experiments, provide us with a large number of rich datasets from which we can discern the underlying mechanisms governing the biological processes. Despite the power of these new technologies and how much information these datasets contain, such new data presents new computational challenges which prevent us from fully exploiting them to address critical biological questions. Specifically, this newly generated data differs substantially from traditional experimental data in terms of data size and captured dimensions. Traditional analytical approaches are either not applicable at all or need improvements that are specific to this type of new dataset. This in turn necessitates the development of robust and scalable analysis techniques that are specifically designed for the new data, as well as the exploration of the potential applications for these novel datasets. This thesis introduces computational tools we developed to analyze three distinct types of complex datasets. These datasets were meticulously collected using cutting-edge experimental techniques which investigate biological phenomena at multiple levels of resolution. Our methods utilize statistical modeling, image analysis, and computer vision techniques to better analyze such data and equip researchers with scalable and robust tools for the new data they generate. The first work in this thesis presents a demixing tool to accurately decipher high-throughput spatial transcriptomics signals in order to better understand molecular diversity among neurons. In the second work we propose a blind demixing method and use carefully simulated data to assess the feasibility of using cellular barcoding technology to reconstruct neural morphology. In the final work we develop a three-dimensional volumetric image registration pipeline and a semi-automatic registration framework, in order to map neuronal functional activity information to the molecular profiles of these neurons. We extensively validate our proposed methods using both simulations and real datasets that were generated in experimental laboratories, demonstrating the robustness of our methods and highlighting their potential utilization in future high-throughput experiments. In summary, this thesis provides three computational tools for facilitating analysis of advanced datasets at various biological levels. Addressing the computational challenges for these new datasets lays the foundation for a comprehensive understanding of cellular functions and brain functions, and the underlying mechanisms thereof. Though our methods were developed and validated using neuroscience data, we envision that these versatile tools can be seamlessly adapted and effectively applied to other fields, including but not limited to immunology and cancer research.

The evolution of snakes (Squamata: Serpentes) represents a major transition in squamate reptiles and involves extensive modifications in the body plan. Functional morphology and phylogeny are integrated to discuss adaptive morphological traits in the origin of snakes and in the evolution of the venom-injecting apparatus among lizards, which is convergent with that in snakes. The focus of these analyses is to determine how to best optimize morphological traits on the phylogeny and to use character distribution in terminal taxa to estimate ecological adaptation in ancestral nodes. To study the locomotion transition from lizards to snakes, 45 virtual models of the squamate bony labyrinth in the inner ear were reconstructed. The results show morphological diversifications of the vestibular region among burrowers, generalists and marine swimmers. The vestibule is enlarged in fossorial species, and reduced in marine snakes and the marine lizard Platecarpus coryphaeus. To quantify the morphological differences and provide tractable methods to reconstruct locomotion in ancestral snakes, I performed three-dimensional geometric morphometric analysis of the 45 samples, using six landmarks and 22 semilandmarks. ANOVA tests on the Procrustes coordinates supported differences among fossorial, generalist, and aquatic locomotion categories. Phylogenetic signal was insignificant in the Procrustes coordinates. Using a phylogeny with the 44 extant samples as terminal taxa, I reconstructed shape coordinates of all internal nodes. Reconstructed shape of the vestibular region of key ancestral nodes in snakes preferred a terrestrial origin for crown-group snakes. Morphological proxies used in this study can be applied to stem snakes to estimate locomotion. Methodologically, this study provides a novel approach to resolve the question whether snakes become limbless on land or in the oceans. Evolution of the venom-injection apparatus in lizards is discussed, based on new material of Estesia mongoliensis. A total-evidence phylogenetic analysis of anguimorph lizards is performed with 86 anguimorph taxa coded to 435 morphological characters and four genes. The matrix includes eight new morphological characters. The total-evidence phylogeny suggests that Estesia Mongoliensis is a basal monstersaur whose crown groups are Heloderma, the only living venomous lizards. Presence of tooth flanges with venom grooves is recognized as a new monstersaurian synapomorphy in this study. Estesia mongoliensis has venom grooves comparable to extant Heloderma, but has a deeper root-to-tip groove in the caudal tooth carina, revealing morphological variations of lizard venom delivery apparatus that are not recorded in extant species.