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Investigating the Function of Selfish Satellite Sequences through Expression Profiling in the Jewel Wasp testisBrody, Hanna F 01 January 2014 (has links)
Highly repetitive, non-protein-coding satellite DNAs are ubiquitous among eukaryotes. In some cases, these sequences make up entire chromosomes, and as much as half of most eukaryotic genomes. Currently, very little is known about the possible roles of satellite DNAs in genome function. In this study I have begun to address the critical issue of satellite DNA function through two different approaches. First, I have used quantitative-RTPCR to address transcriptional levels of three different satellites known to express transcripts in the male germ tissue of the jewel wasp, Nasonia vitripennis. Two of these satellites are located uniquely on a supernumerary (‘extra’), non-essential chromosome known as PSR (for paternal sex ratio), while the third satellite is located on a normal chromosome. These experiments are suggesting that all three of these satellites are not expressed at consistent levels across individuals, arguing against a functional role. Instead, this finding supports the longstanding view of satellites as truly parasitic agents, and their expression may be artifactual. Second, I initiated experiments to determine if conditional mutagenesis through RNA interference is possible; development of this method in the wasp male germ line would be an invaluable tool for further assessing the function of satellite expression. Specifically, I tested the ability of RNAi to deplete the wasp ortholog of cannonball, which plays a testis-specific role in sperm formation in other insects. These experiments resulted in a trend of lower cannonball levels, although non-significant, in RNAi-treated males. These findings suggest that RNAi may be a potentially effective method for conditional mutagenesis in this tissue, but will require further optimization.
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Developing a Gene Editing System to Study Haplodiploidy in the Jewel Wasp, Nasonia VitripennisMuller, Emily A. 01 January 2015 (has links)
Hymenopteran insects, which include all ants, bees and wasps, reproduce through a poorly understood form of reproduction known as haplodiploidy. A promising experimental system for understanding this developmental process is the jewel wasp, Nasonia vitripennis. A critical aspect of using Nasonia as a model is establishing an effective means for editing specific genes of interest so that their functions can be studied through genetic means. For my thesis research, I performed a pilot study of the gene editing method known as CRISPR in Nasonia. I targeted the single heterochromatin protein 1 (HP1) gene present in the Nasonia genome in order to assess the feasibility of this gene editing approach. Targeting HP1 would provide a clear phenotype when this gene is mutated due to its essential functions in early development known from studies in other eukaryotes. Additionally, creating a mutant of this gene will provide a means for studying the role of HP1 in wasp spermatogenesis, an aim that interlinks with the broader chromatin-based goals of our laboratory. Through this study I worked out a streamlined procedure for injecting CRISPR molecules into young wasp embryos, conducting genetic crosses with injected wasps, and screening through their progeny for potential mutants. I observed no mutant phenotypes in injected wasps, but instead, I isolated four potential mutants in F1 progeny. My work has helped to create a solid framework for improving this procedure in Nasonia, and they allow for a better overall understanding of the limitations of producing mutants through CRISPR gene editing in non-model organisms such as Nasonia.
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The Role of Neural Crest Cells in Vertebrate Cardiac Outflow DevelopmentAlonzo-Johnsen, Martha January 2014 (has links)
<p>Throughout vertebrate evolution, the cardiac outflow vasculature has changed from a branchial arch system to a systemic and pulmonary circulatory system. However, all vertebrate hearts and outflow tracts still develop from a single heart tube. In the chick and mouse, cardiac neural crest cells divide the single outflow tract into the aorta and pulmonary arteries. Additionally, cardiac neural crest cells provide the smooth muscle of the aortic arch arteries, help to remodel the aortic arch arteries into asymmetrical structures, and contribute cardiac ganglia. I review the major contributions of cardiac neural crest cells to the outflow vasculature of the chick and mouse and apply this information to study cardiac neural crest cell contributions to vertebrates that lack a divided circulatory system. I re-evaluate the role of cardiac neural crest cells in zebrafish vasculature and find that these cells do contribute to the gill arch arteries, the ventral aorta and cardiac ganglia, but they do not contribute to myocardium. I also study the outflow tract development of the turtle Trachemys scripta to understand the process of outflow septation in a vertebrate that has a divided outflow tract but an incomplete division of the ventricle. I compare the chick outflow tract to the turtle. The formation of the proximal versus distal cushions and the appearance of smooth muscle cells within the distal cushions of the turtle are very similar to the cushion position and cell types within the cushions of the chick. In the chick, the smooth muscle positive cells in the distal cushions are derived from cardiac neural crest cells. I hypothesize that cardiac neural crest cells are also responsible for the outflow tract septation of reptiles. These results demonstrate that the pattern of cardiac neural crest cell contribution to vertebrate vasculature remains predictable and consistent, enabling future studies to focus on changes in vascular patterning caused by cardiac neural crest cells among different vertebrate lineages.</p> / Dissertation
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Tissue interaction and spatial pattern formationCruywagen, Gerhard C. January 1992 (has links)
The development of spatial structure and form on vertebrate skin is a complex and poorly understood phenomenon. We consider here a new mechanochemical tissue interaction model for generating vertebrate skin patterns. Tissue interaction, which plays a crucial role in vertebrate skin morphogenesis, is modelled by reacting and diffusing signal morphogens. The model consists of seven coupled partial differential equations, one each for dermal and epidermal cell densities, four for the signal morphogen concentrations and one for describing epithelial mechanics. Because of its complexity, we reduce the full model to a small strain quasi-steady-state model, by making several simplifying assumptions. A steady state analysis demonstrates that our reduced system possesses stable time-independent steady state solutions on one-dimensional spatial domains. A linear analysis combined with a multiple time-scale perturbation procedure and numerical simulations are used to examine the range of patterns that the model can exhibit on both one- and two-dimensions domains. Spatial patterns, such as rolls, squares, rhombi and hexagons, which are remarkably similar to those observed on vertebrate skin, are obtained. Although much of the work on pattern formation is concerned with synchronous spatial patterning, many structures on vertebrate skin are laid down in a sequential fashion. Our tissue interaction model can account for such sequential pattern formation. A linear analysis and a regular perturbation analysis is used to examine propagating epithelial contraction waves coupled to dermal cell invasion waves. The results compare favourably with those obtained from numerical simulations of the model. Furthermore, sequential pattern formation on one-dimensional domains is analysed; first by an asymptotic technique, and then by a new method involving the envelopes of the spatio-temporal propagating solutions. Both methods provide analytical estimates for the speeds of the wave of propagating pattern which are in close agreement with those obtained numerically. Finally, by numerical simulations, we show that our tissue interaction model can account for two-dimensional sequential pattern formation. In particular, we show that complex two-dimensional patterns can be determined by simple quasi-one-dimensional patterns.
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Short-Range Inter-Blastomere Signaling Specifies Ectodermal Fate and is Required for Skeletal Patterning in the Sea UrchinMcIntyre, Daniel Clifton January 2012 (has links)
<p>Sea urchin larvae possess a beautiful, intricately patterned, calcium-carbonate skeleton. Formation of this complex structure results from two independent processes within the developing embryo: specification of the mesenchymal cells that produce the skeletal rods, and patterning inputs from the ectoderm, which secretes signals directing the growth and shape of the skeleton. To understand patterning of the skeleton therefore, the specification events behind these two processes must be understood separately, and then connected in order to understand how ectoderm signaling directs skeletal growth. While the former processes of mesenchyme specification and mineralization are under study elsewhere, the means by which ectodermal cues directing skeletal growth are activated and localized is not known. Using molecular genetics, including gene knock downs and mis-expression, as well as microsurgical manipulations of early cleavage embryos, I show how a previously undescribed territory within the ectoderm, the border ectoderm (BE) is specified with short range signaling inputs. Then, experiments show that the BE provides signals that initiate, and contribute to the propagation of skeletogenesis. From this dataset, and from biological experiments I outline a model for how the BE patterns and contributes to the directed growth of the skeleton. I also discuss challenges to this model that need to be addressed in future research. In principle, the mechanism proposed herein depends on the integration of information from both the primary and secondary embryonic axes. It requires both short-range signaling by Wnt5 from the endoderm to establish the BE fate, and TGFß signaling from the oral and aboral ectoderm which subdivides the BE into four territories. These findings demonstrate that the short-range signaling cascade that subdivides the embryo into first mesoderm and then endoderm also specifies ectodermal fates. Ultimately, this research paves the way for understanding how the larval skeleton is patterned during embryogenesis and may provide a paradigm for understanding other, more complex, developmental problems.</p> / Dissertation
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The Utilization of Mouse Models to Study Gene Functions: The Role of Foxn3 and Chd2 in Murine Development and CancerSamaan, George Azaz 01 December 2011 (has links)
Murine model organisms are an essential tool in the scientific community quest to decipher the molecular etiology of human diseases. Currently, several methods are used to induce or reproduce human diseases in mouse models using advanced genetic engineering techniques to mutate the wild-type genes. We utilized the Baygenomics gene-trap method to study the effects of two mammalian genes: FOXN3 and CHD2. The Forkhead Box (FOX) family of transcription factors shares a common DNA-binding domain and has been associated with organ development, differentiation, cell growth and proliferation, and cancer. Meanwhile, the CHD (Chromodomain helicase DNA binding protein) family of proteins is known to be involved in chromatin remodeling and regulation of gene expression. Phenotypic analysis of Foxn3 mutant animals revealed its indispensible role in craniofacial and embryonic development, embryonic lethality, expression of bone morphogenetic proteins, and spontaneous development of cancers in heterozygous and homozygous mutant mice. Preliminary evaluation of molecular mechanisms of FOXN3 signifies deregulation of cell-cycle checkpoint proteins Cyclin-B1 and CDK2 as the underlying etiology of tumors. Chd2 mutant mice exhibit spontaneous thymic and splenic lymphomas and reduced lifespan which can be restored through Chd2 re-expression in the thymus. At the molecular level, CHD2 deficiency reduces Puma (p53-upregulated modulator of apoptosis) induction after DNA damage in mouse thymocytes and HCT116 cells. Additionally, CHD2 is enriched at the Puma locus after DNA damage. CHD2-deficient cells also exhibit global reduction of active transcription markers H3K9-Acetylated and H4K8-Acetylated.
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Molecular interactions of latent transforming growth Factor-β binding Protein-2 (LTBP-2) with fibrillins and other extracellular matrix macromolecules [electronic resource]: LTBP-2 competes with LTBP-1 for binding to Fibrillin-1 suggesting that LTBP-2 may modulate latent TGF-β storageHirani, Rena M January 2006 (has links)
Elastic fibres, a major component of many connective tissues, are composed of an amorphous elastin core surrounded by fibrillin - containing microfibrils. The function of these microfibrils appears to require the co - ordinated interactions of fibrillins with a range of extracellular matrix ( ECM ) macromolecules including, latent transforming growth factor - β ( TGF - β ) binding proteins ( LTBPs ). LTBPs share a high degree of structural similarity to fibrillins, since they both contain unique 8 - cysteine motifs. Of the four members of the LTBP family, LTBPs - 1, - 3 and - 4 covalently bind to latent forms of TGF - β. LTBP - 1 has been shown to interact with the N - terminal domains of fibrillin - 1 and - 2 and LTBP - 4 interacts with the N - terminal domains of fibrillin - 1, suggesting that fibrillin - containing microfibrils may act as TGF - β stores and localise latent TGF - β complexes to the ECM. LTBP - 2 differs from other members of the LTBP family since it does not covalently bind latent TGF - β. However, LTBP - 2 strongly co - localises with fibrillin - containing microfibrils in a number of tissues suggesting that LTBP - 2 could have a structural role associated with these elements presumably independent of TGF - β storage, or could act to mediate specific microfibril - ECM interactions. To understand more about the function of LTBP - 2, this study involved screening for potentially important molecular interactions of LTBP - 2 with fibrillins and a variety of ECM proteins. Human recombinant LTBP - 2 ( r - LTBP - 2 ) was cloned, expressed and purified using a mammalian cell culture system. Solid phase binding assays were used to screen for interactions between r - LTBP - 2 and continguous fragments of fibrillin - 1 and - 2 as well as MAGPs, tropoelastin, collagens and proteoglycans. A cation dependant interaction was found between the C - terminal domains of LTBP - 2 and the N - terminal domains of fibrillin - 1, but not with the analogous region of fibrillin - 2. Thus, LTBP - 2 seems to have an exclusive role associated with fibrillin - 1 - containing microfibrils. Further studies found that the C - terminal region of LTBP - 2 competes with LTBP - 1 for binding to fibrillin - 1, suggesting that the binding site for LTBP - 2 on fibrillin - 1 is the same or in close proximity to that for LTBP - 1. Immunohistochemical analysis of LTBP - 1 and - 2 within developing human aorta indicated that both LTBPs co - localised with fibrillin - 1. However, the two LTBPs did have distinct distribution patterns in relation to each other, in that LTBP - 2 was found throughout the medial layer whereas LTBP - 1 was mainly located in patches of the outer medial layer. No regions of strong co - localisation of the two LTBPs were found. Thus, these findings suggest that LTBP - 2 could indirectly modulate the presence of TGF - β upon the fibrillin - containing microfibrils by competing for binding with the LTBP- 1 / TGF - β complex to these structures. Other binding studies showed a cation independent interaction between r - LTBP - 2 and an as yet unidentified component of a crude bovine collagen - IV extract. Since collagen - IV is a major component of basement membranes, an interaction between r - LTBP - 2 and a protein within this bovine collagen - IV preparation suggests LTBP - 2 may have a further function involving a basement membrane component. It will be interesting to determine if LTBP - 2 acts as a bridging molecule between basement membrane structures and fibrillin - containing microfibrils or if it has another function independent of these microfibrils. / Thesis (Ph.D.)--School of Medical Sciences, 2006.
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The role of FGF signaling in retinal developmentHartge, Abbie A., January 2008 (has links) (PDF)
Thesis (M.S.)--University of Tennessee Health Science Center, 2008. / Title from title page screen (viewed on January 29, 2009). Research advisor: Dr. Michael A. Dyer, Ph.D. Document formatted into pages (vi, 47 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 41-44).
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The effects of extracellular matrix compliance and protein expression on neuronsPrevitera, Michelle L., January 2010 (has links)
Thesis (Ph. D.)--Rutgers University, 2010. / "Graduate Program in Cell and Developmental Biology." Includes bibliographical references (p. 101-119).
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Understanding the molecular mechanism of the fat signaling pathway in Drosophila melanogasterFeng, YongQiang, January 2009 (has links)
Thesis (Ph. D.)--Rutgers University, 2009. / "Graduate Program in Cell and Developmental Biology." Includes bibliographical references (p. 185-197).
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