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Small Steps and Grand Leaps: A Study of Micro- and Macroevolutionary ProcessesTzika, Athanasia C. 14 March 2008 (has links)
Evolutionary biology is not a specialty, like genetics or development - it is an explanation of what is investigated by all biological specialties. Thus, the goal of this dissertation was to study both micro- and macroevolutionary processes in a multi-disciplinary framework.
Population genetics, conservation, and phylogeny inference. The Jamaican boa (Epicrates subflavus) is an endemic species, whose natural populations greatly and constantly declined since the late 19th century, mainly due to predation by introduced species, human persecution, and habitat destruction. Using species-specific nuclear microsatellite loci and mitochondrial sequences, we investigated the population structure of this endangered reptile. All analyses pinpointed to an Eastern versus (Western+Central) pattern of differentiation in agreement with geological data and patterns of differentiation uncovered in other vertebrate and invertebrate Jamaican species. The same molecular markers were employed on 80 Jamaican boas of the European captive breeding program. This approach allowed us to (i) clarify all ambiguities in the studbook, (ii) correct parental allocation errors and (iii) assess the genetic diversity and the level of inbreeding of the current captive population. These results provide important insights for guiding the development of proper ex-situ and in-situ species survival and habitat management plans for this vulnerable snake. In the same framework of classical evolutionary genetics, we performed preliminary analyses of cytochrome b-like sequences in representatives of all cetacean families (but one), and revealed the presence of at least four nuclear mitochondrial pseudogenes that were independently inserted into the nuclear genome.
Evo-Devo. The emergence of Evolutionary Developmental biology has caused a partial shift in the criteria for the selection of model species. Thus far, the main criterion was the relevance of a species for understanding human biology, whereas in the frame of the new discipline, it is the understanding of the generative mechanisms underlying biological diversity that is put forward. We discussed a few criteria and limitations of major relevance to the choice of model species for Evo-Devo studies, and applied a pragmatic approach to identify possible model species within Amniotes.
Moreover, we developed MANTiS, an application pipeline that aims at integrating genomic, functional and expression data with evolutionary concepts, thus constituting the missing link between multi-species genome comparisons and functional analyses. Using MANTiS, we proceeded in the analysis of 35 metazoan full genomes for identifying all lineage-specific gene gains and losses. These results were combined with functional and expression analyses, and we demonstrated the much higher performance of MANTiS against popular databases of ortholog clusters (InParanoid, OrthoMCL, RoundUp).
Finally, preliminary results of our attempt to adapt the new revolutionary technology of DNA sequencing in microfabricated high-density picoliter reactors (developed by 454/Roche) to the ultra-fast sequencing of brain full transcriptomes in multiple reptilian species are highly promising. As an example, the Crocodylus sample generated more than 72 Mbases (per run), which were successfully assembled in approximately 31,000 contigs. One third of the latter could be matched to known sequences in the transcriptome of related species. After fine-tuning of the in silico analyses, and incorporation of genomic sequence data, we expect our approach to provide important insights not only in the evolution of central nervous system novelties in vertebrates, but in transcriptomes in general as the brain transcriptome is one of the most complex among all organs.
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Chromatin regulators and transcriptional control of Drosophila developmentDai, Qi January 2007 (has links)
The development of a multicellular organism is programmed by complex patterns of gene expression. In eukaryotic cells, genes are packaged by histone proteins into chromatin. Chromatin regulators often function as transcription co-factors. In this study, we have investigated the function of four co-factors, dAda2b, Reptin, Ebi and Brakeless during development of the fruit fly Drosophila melanogaster. dAda2b and Reptin belong to histone acetyl transferase (HAT) complexes, a SAGA-like complex and the Tip60 complex, respectively. We generated dAda2b mutants and found that lack of dAda2b strongly affects global histone acetylation and viability. We further propose that Ada2 may be involved in DNA repair. Our studies revealed new roles of Reptin and other Tip60 complex components in Polycomb Group mediated repression and heterochromatin formation, thereby promoting generation of silent chromatin. During embryogenesis, transcriptional repressors establish localized and tissue-specific patterns of gene expression. In this thesis, we identified two novel co-repressors in the early embryo, Ebi and Brakeless. Ebi genetically and physically interacts with the Snail repressor. The Ebi-interaction motif in the Snail protein is essential for Snail function in vivo and is evolutionarily conserved in insects. We further demonstrated that Ebi associates with histone deacetylase 3 (HDAC3) and that histone deacetylation is part of the mechanism by which Snail mediates transcriptional repression. We isolated Brakeless in a genetic screen for novel regulators of gene expression during embryogenesis. We found that mutation of brakeless impairs function of the Tailless repressor. Brakeless associates with Atrophin, another Tailless corepressor, and they function together in Tailless-mediated repression. In summary, transcription co-factors, including chromatin regulators, are selectively required in distinct processes during development.
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Roles for UNC-6/Netrin Signaling During Cell Invasion in C. ElegansZiel, Joshua W. January 2011 (has links)
<p>Basement membranes are dense, sheet-like forms of extracellular matrix that</p><p>surround the epithelial tissues of metazoan organisms. While these structures are</p><p>critical for epithelial support and tissue organization, basement membranes also pose</p><p>formidable barriers to most cells. However, certain specialized cells are able to breach</p><p>these barriers and move between tissues. Acquisition of cell invasive behavior by some</p><p>tumor cells is thought be an important step in cancer progression. Due to the clear basic</p><p>and clinical importance of understanding the mechanisms underlying cell invasion</p><p>through basement membranes, cell invasive behaviors has been an area of intense study.</p><p>In this work I examine a developmentally regulated model of cell invasive behavior in</p><p>the nematode worm, C. elegans. In this system a single proto-epithelial cell remodels</p><p>basement membrane to connect two epithelial tissues, the uterus and vulva. Using this</p><p>model I identify a novel role for UNC-6/Netrin signaling during this process through basement membranes. I show that Netrin signaling is a third regulatory input for AC invasion that functions partially in parallel to fos-1a and the vulval signal. Further I link netrin signaling to the formation of invasive protrusions that penetrate basement membrane.</p> / Dissertation
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Hormones and Cuscuta Development Abscisic Acid And Its Conjugates-Endogenous Levels And Metabolism During Growth And Haustoria FormationVasanthi, G. 10 1900 (has links)
Cuscuta or dodder is one of the best known higher plant parasite with around 158 species over 5 continents parasitising a wide variety of host plants (Yuncker,1932) Dodders are characterised by their interesting parasitic behaviour and extraordinary appearance among the obligate parasitic flowering plants (Kuijt, 1969)
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The role of poly (ADP-ribose) polymerase-1 in the celluar response to several marine-derived compoundsPatel, Brijesh B. January 2009 (has links)
Thesis (M.S.)--Rutgers University, 2009. / "Graduate Program in Cell and Developmental Biology." Includes bibliographical references (p. 45-53).
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Functional analyses of type IIA procollagen in embryo developmentLeung, Wai-lun, Alan., 梁瑋倫. January 2006 (has links)
Type II collagen is the major extracellular matrix (ECM) protein present in
cartilage and is detected in non-chondrogenic tissues such as the heart and the
neural tube during developmental stages involving rapid tissue morphogenesis
indicating an active role played by the collagen in embryogenesis. Type II
collagen is synthesized as a procollagen precursor which has amino- and
carboxyl-terminal globular extensions (N- and C-propeptides) flanking a central
triple helical domain. Two isoforms of type II procollagen are generated by
alternative mRNA splicing of the exon 2: IIA and IIB. Sequence present in the
N-propeptide of IIA, translated from the spliced-in exon 2, encodes a von
Willebrand factor-like C cysteine rich (CR) domain. This domain is homologous
to those present in regulators of the bone morphogenetic protein (BMP) signaling
such as chordin (Chd), twisted gastrulation (Tsg) and crossveinless (Cv).
Previous in vitro binding assays and overexpression studies in frog embryo
suggest that the CR domain of IIA antagonized BMP signaling. In order to give
a better understanding of the function of IIA in embryonic development and
cellular signaling, several approaches including expression pattern analyses,
phenotypic analyses of null mutant and gain of function studies are employed in
this study. Expression studies of IIA mRNA in early postimplantation mouse
embryos find that it is present in the axial mesendoderm (including the anterior
definitive endoderm [ADE] and the prechordal plate) which is a critical head
organizer at neural plate (E7.5) and head process (E8.0) stages. Characterization
of the IIA deficient mice (IIA-/-), constructed by removing exon 2 from type II
collagen (Col2a1) gene by homologous recombination, indeed reveals that the
anterior-most neural tissue is deficient at early somitogenesis denoted by
reduction/loss of the forebrain/optic cup markers. Marker studies indicate that
the ADE may already be affected at the neural plate stage in IIA-/-. The neural
phenotype of IIA-/- displays significant similarities with mutants deficient in BMP
pathway components such as Chd-/-;Nog+/-, Tsg-/- and Tsg-/-;BMP4+/- suggesting
that IIA plays a role in maintaining the specification and/or regulating the
signaling properties of the anterior midline tissue which involves regulation of
BMP signaling. Results of ectopic expression of IIA in Xenopus laevis embryos
suggest that IIA regulate BMP and the related Nodal signaling pathways in a
context dependent manner which has significant implications in normal anterior
neural plate development. Based on the work described in this thesis and the
body of existing evidence, a model is presented which suggests that IIA
promote/maintain anterior neural plate development by regulating the range and
extent of BMP signaling in the anterior neural plate. This study sheds light on
the role of an ECM component in regulating tissue patterning and cellular
signaling during early mouse development and also provides putative function for
the CR domain of other fibrillar procollagens including type I, III and V which is
poorly understood currently. This work will provide the framework for the
design of subsequent studies in re-examining the role of these fibrillar
procollagens in embryogenesis. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy
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Functional Analysis of Notch Signaling during Vertebrate Retinal DevelopmentMizeracka, Karolina 21 June 2013 (has links)
The process of cell fate determination, which establishes the vastly diverse set of neural cell types found in the central nervous system, remains poorly understood. During retinal development, multipotent retinal progenitor cells generate seven major cell types, including photoreceptors, interneurons, and glia, in an ordered temporal sequence. The behavior of these progenitor cells is influenced by the Notch pathway, a widely utilized signal during embryogenesis which can regulate proliferation and cell fate decisions. To examine the underlying genetic changes that occur when Notch1 is removed from individual retinal cells, microarray analysis of single cells from wild type or Notch1 conditional knockout retinas was performed. Notch1 deficient cells downregulated progenitor and cell cycle marker genes, while robustly upregulating genes associated with rod genesis. Single wild type cells expressed markers of both rod photoreceptors and interneurons, suggesting that these cells were in a transitional state. In order to examine the role of Notch signaling in cell fate specification separate from its role in proliferation, Notch1 was genetically removed specifically from newly postmitotic cells. Notch1 deficient cells preferentially became cone photoreceptors at embryonic stages, and rod photoreceptors at postnatal stages. In both cases, this cell fate change occurred at the expense of the other cell types normally produced at that time. In addition, single cell profiling revealed that Inhibitor of differentiation 1 and 3 genes were robustly downregulated in Notch1 deficient cells. Ectopic expression of these genes during postnatal development in wild type retinas was sufficient to drive production of progenitor/Müller glial cells. Moreover, Id1 and 3 partially rescued the production of Müller glial cells and bipolar cells in the absence of Notch1, even in newly postmitotic cells. We propose that after cell cycle exit, retinal precursor cells transition through a period in which they express marker genes of several different cell types as they commit to a fate, likely endowed by their progenitor cell. Specifically, cells that will become bipolars or Müller glia depend on Id-mediated Notch signaling during this transitional state to take on their respective fates.
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Evidence for a Novel Multipotent Mammary Progenitor with Pregnancy-Specific ActivityKaanta, Alice 20 December 2012 (has links)
The mouse mammary gland has emerged as a model system for studying processes involved in the development of epithelial tissues. Current evidence suggests the existence of a differentiation hierarchy in the mammary gland, consisting of a stem cell capable of reconstituting the tissue, progenitors with the capacity to produce specific functional cell types, and differentiated cells with limited or no repopulation potential. Although markers for mammary stem cells and progenitors have been identified, these populations have not been isolated to purity and our understanding of how they function in different stages of mammary development remains incomplete. Many adult stem cells are mitotically quiescent and can therefore retain a DNA or histone label significantly longer than differentiated cells. In an attempt to identify mammary stem cells/progenitors by histone label retention, I crossed a mouse carrying the tetracycline-inducible histone 2b/eGFP (H2BGFP) gene with tetracycline transactivator strains expected to induce H2BGFP in the mammary gland. H2BGFP expression was induced in the mammary gland until puberty and then chased for 6-8 weeks; \(H2BGFP^+\) label retaining cells were isolated and assayed. Transplantation experiments comparing MMTVrtTA/H2BGFP MECs isolated after induction to MMTVrtTA/H2BGFP MECs retaining label post-chase failed to prove that label retention enriches for stem cells/progenitors in the MMTVrtTA/H2BGFP system. During the course of these experiments, I unexpectedly discovered that MMTVrtTA induced H2BGFP expression exclusively in the \(CD24^+/CD29^+\) and \(CD24^+/CD29^{lo}\) populations, which contain stem cells and progenitors, respectively. Interestingly, I also discovered that H2BGFP+/CD24+/CD29lo MECs developed limited mammary outgrowths in vivo and that pregnancy increased the repopulation ability of these cells by 5-10-fold. H2BGFP+/CD24+/CD29lo outgrowths contained all mammary lineages and produced milk, but were unable to self-renew in serial transplant assays. Furthermore, \(H2BGFP^+/CD24^+/CD29^{lo}\) and \(H2BGFP^-/CD24^+/CD29^{lo}\) MECs had distinct gene expression profiles, with H2BGFP+/CD24+/CD29lo MECs expressing lower levels of transcripts involved in mammary development and differentiation. These data provide evidence for the existence of a multipotent, pregnancy-activated mammary progenitor and suggests that different progenitor populations are responsible for mammary expansion during puberty and pregnancy. Future studies may identify FACS markers for purification of pregnancy-activated progenitors and further elucidate the role of different mammary cell types during pregnancy.
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Mechanistic Studies of Polycomb Group ProteinsGrau, Daniel James 20 December 2012 (has links)
Most cells within multicellular organisms contain the same genetic information, yet the appropriate tissue-specific expression of genes is required for the proper formation of adult tissues. Genes can either be “turned on” or “turned off” from the initial zygotic state and maintained during subsequent cell divisions. Maintaining the correct expression profiles during cell divisions is accomplished by a number of different nuclear factors. One of the key families of proteins that maintains the repression of target genes during development is the Polycomb group (PcG) of proteins. PcG proteins form a number of different multi-subunit protein complexes that interact with specific regions of chromatin and direct the repression of nearby genes by reducing transcription. One PcG complex, Polycomb repressive complex 1 (PRC1), inhibits transcription and nucleosome remodeling as well as compacts chromatin, both in vivo and in vitro. The in vitro repressive activities map mainly to one subunit of Drosophila PRC1—the Posterior sex combs (PSC) protein. The PRC1 complex is conserved in many other organisms including mammals. To better understand the mechanisms involved in PcG mediated repression we undertook a biochemical structure/function analysis of mouse PRC1. In chapter one, I review the current understanding of PcG biology and a rationale for the dissertation is provided. In chapter two, data are presented that argues that a mouse PRC1 protein, M33/Cbx2, which is non-homologous to PSC, is responsible for chromatin compaction and repression of nucleosome remodeling. Data are presented that suggests these activities are localized to a basic, natively unfolded region of M33/Cbx2. In chapter three, we extend the findings from chapter two in an attempt to predict whether homologous PcG proteins from other species besides fly and mouse have biochemical activity. In agreement with predictions, a panel of recombinant PcG proteins was generated and data are presented that shows the predicted active PcG proteins are capable of both inhibition of nucleosome remodeling and compaction of chromatin. Finally, in chapter four, the implications of the data presented are discussed, and directions for further inquiry are explored.
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Early Cell Fate Determination in ZebrafishXu, Cong January 2012 (has links)
ESC/iPSC-derive somatic cells may be ideal for treating disorders caused by cellular deficiency or dysfunction. To form a lineage-specific cell population, ESCs/iPSCs undergo a multi-step process that recapitulates embryonic development. ESC/iPSC differentiation protocols are hampered by the limitation of our understanding in development. Zebrafish embryos are fertilized and developed externally, a feature facilitates the observation and manipulation of embryonic development. To explore the zebrafish as a system to study cell lineage determination, in this thesis, I 1) identified an ortholog of the key pluripotency regulator Nanog in zebrafish and examined its role in early cell fate determination; 2) developed a high-throughput image-based chemical screening system in zebrafish blastomere cell culture that is very similar to, but much faster than, ESC/iPSC differentiation screens. Specifically, in an effort to examine the role of Nanog in vivo, I identified a zebrafish Nanog ortholog, and found that its knockdown impaired endoderm formation. Genome-wide transcription analysis revealed that nanog-like morphants fail to develop the extra-embryonic yolk syncytial layer (YSL), which produces Nodal required for endoderm induction. I examined the genes that were regulated by Nanog-like, and identified the homeobox gene mxtx2, which is both necessary and sufficient for YSL induction. Chromatin immunoprecipitation assays and genetic studies indicated that Nanog-like directly activates mxtx2, which in turn specifies the YSL lineage by directly activating YSL genes. The study identifies a Nanog-like-Mxtx2-Nodal pathway and establishes a role for Nanog-like in regulating the formation of the extra-embryonic tissue required for endoderm induction. In the second part of the thesis, I developed a system that allows high-throughput image-based chemical screening using cultured zebrafish blastomere cells. To demonstrate its potential, this system is utilized to study skeletal muscle development. I screened 2,400 chemicals, finding 11 chemicals that block mature muscle cell differentiation and 17 chemicals that block skeletal muscle progenitor formation. The subsequent studies of these hits illustrate an RTK-PI3K-mTOR- GSK3 signaling cascade that is critical for skeletal muscle development. Preliminary data in mouse Skeletal Muscle Precursors (SMPs) suggest the pathway is conserved in murine adult muscle stem cells. This system, which can be modified for any cell lineage, promises to enhance our understanding of fundamental biology and to identify chemicals for cell-based therapies for many diseases.
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