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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
11

From bacteria to humans: the CRISPR/Cas9 editing system, a novel therapeutic

Millman, Sarah 12 March 2016 (has links)
CRISPR (clustered regulatory interspaced short palindromic repeats) is a bacterial adaptive immune system, which can target specific sequences of foreign nucleic acids. The system is made up of unique spacer sequences flanked by short, repetitive, palindromic sequences, and sequences encoding Cas (CRISPR associated) proteins. The CRISPR-Cas9 system has been utilized from S. pyrogenes for use in eukaryotes for gene editing. Many years were spent uncovering the components of the system and its mechanism. Now the RNA-guided endonuclease abilities of the system are utilized to induce double-strand breaks in targeted DNA sequences. Through the advent of double-strand breaks researchers can now induce mutations, knock out genes through non-homologous end joining, and knock-in DNA sequences through homologous directed repair. Additionally, the system has been utilized in gene regulation to activate or repress protein expression. Studies have shown that CRISPR-Cas9 as a gene-editing tool is highly efficient and specific. The system only requires a Cas9 protein and guideRNA to function and can alter multiple genes with the addition of different guideRNA. Most scientists have reported minimal off-target mutations caused by Cas9, and many strategies have been initiated to reduce off-target effects even further. Already, the system has been shown to have therapeutic applications both in vitro in human cells and in vivo in animals (including non-human primates). Future applications in therapeutics and gene-editing have the potential to change research and medicine.
12

Forensic science applications of commercially available Mawi DNA technologies' iSWAB-ID lytic buffer and still in-development iSWAB-Cells-400 non-lytic buffer

Yamamoto, Lisa 25 July 2018 (has links)
In forensic science, it is important to have the capability to collect, stabilize and store deoxyribonucleic acid (DNA) in an efficient manner so that successful and probative DNA profiles can be generated. The success of generating downstream profiles is dependent on viable DNA extracts, thus the preservation and protection of the samples from degradation or loss is imperative. According to the National Institute for Standards and Technology (NIST) Biological Evidence Preservation Handbook, dried biologically stained samples are best stored in a temperature controlled environment, which is defined as less than 60% humidity with a temperature maintained between 15.5°C and 24°C. Presently, long-term forensic evidence samples, in the form of DNA extracts, have a preservative added and are either refrigerated (2°C – 8°C) or stored in freezers with temperatures typically set at or below -10°C[1]. However, using freezers as a means of sample storage can result in limited storage space, high cost, and risk of electrical failure[2]. Alternative storage methods that can be employed in ambient conditions have been implemented in some forensic laboratories and are suitable to protect and preserve DNA evidence. Different stabilizing agents such as SampleMatrix™ and DNAstable™ have been studied to assess their ability at storing and stabilizing extracted DNA at ambient temperatures. Research by Lee et al., showed DNA stored in SampleMatrix™ was protected against degradation for up to 12 months and maintained its integrity, resulting in a 2-fold increase of recoverability over samples stored in a freezer for the same amount of time. This study also showed that DNA stored in SampleMatrix™ did not affect the PCR amplification process and samples that were desiccated and stored in SampleMatrix™ resulted in similar completeness of STR profiles with samples stored at -20°C for 0.125 ng and 0.25 ng DNA concentrations over the course of 12 months[3]. DNAstable™ was also shown to have similar storage and stabilizing capabilities over a long period of time under various temperature conditions. Mawi DNA Technologies’ iSWAB™-ID collection system and its associated buffer is capable of being a single step process for collection, extraction, and stabilization of biological specimens at room temperature for long periods of time[4]. In addition to the known properties of the iSWAB™-ID buffer, this research examines the potential of using the iSWAB™-ID buffer to perform a single tube differential extraction. Various conditions of the iSWAB™-Cells-400 non-lytic buffer were also analyzed for their potential of stabilizing and protecting DNA stored within whole and intact cells under ambient conditions for up to 4 weeks. The following parameters of the iSWAB™-Cells-400 non-lytic buffer were tested: 1) ability to extract DNA from the intact cells with various methods after cells have been stored in the buffer; 2) compatibility with downstream polymerase chain reaction (PCR)-based analyses; and 3) ability to stabilize and store intact cells and their DNA for extended periods of time. Overall, both epithelial cells (e-cells) and sperm cells were lysed within minutes of being added to the iSWAB™-ID buffer, eliminating the use of this buffer from being used for differential extraction. However, it was determined that the addition of dithiothreitol (DTT) aided in the recovery of both e-cell and sperm DNA. The iSWAB™-Cells-400 non-lytic buffer did not protect DNA for periods longer than a week, as degradation of DNA extracts was seen in cells stored in the buffer at room temperature for a week or more. Additional modifications to the iSWAB™-Cells-400 buffer should be explored before cells stored in this buffer can be successfully analyzed using short tandem repeat (STR) analysis.
13

Temporal control of vulval precursor cell fate patterning in Caenorhabditis elegans

Li, Ji January 2011 (has links)
Development of a multicellular organism requires precise coordination of temporal and spatial cues to ensure that developmental events occur at the correct time and place. C. elegans vulval development offers a convenient experimental system for investigating the temporal and spatial regulation of multiple developmental decisions in response to different patterning signals. In this thesis, I present my studies on the temporal control of Vulval Precursor Cell (VPC) fate patterning through analyses of VPC development defects in heterochronic mutants. I show that loss of the miRNA lin-4 inhibits LIN-12/Notch activity through persistence of LIN-14, but not LIN-28 or HBL-1. Persistent lin-14 blocks LIN-12 activity without interfering with the key events of LIN-12/Notch signal transduction, and lin-14 activity in the second larval stage is sufficient to prevent premature LIN-12 activation. I also present evidence that persistent lin-14 activity impedes extension of the VPC apical domains, and that ectopic Wnt signaling prevents the daughters of uninduced VPCs from fusing with the major hypodermal syncytium in lin-4 null mutants. Finally, through characterization of heterochronic mutants that exhibit precocious or delayed vulval induction, I provide clues to possible mechanisms underlying the temporal control of vulval induction.
14

Examining the Role and Regulation of Cell-Cell Adhesion in Aggressive Prostate Cancer

Barber, Alison G. January 2011 (has links)
Prostate cancer is the second leading cause of cancer death in American men, yet despite the prevalence of this disease, there is a distinct lack of prognostic biomarkers for estimating the likelihood of prostate cancer aggressiveness. The loss of cell-cell adhesion is frequently associated with the progression of prostate cancer to a metastatic state. While both adherens junctions and desmosomes are involved in establishing and maintaining this adhesion, previous studies of cell-cell adhesion in prostate cancer have focused solely on the role of adherens junctions, leaving the role of desmosomal adhesion unexplored. The goals of this thesis were to perform a functional analysis of the role and regulation of adherens junctions and desmosomes in aggressive prostate cancer, and to examine the efficacy of classical and desmosomal cadherins as prognostic biomarkers of aggressive prostate cancer. I began this study by characterizing the expression profile of desmosomal cadherins in normal human prostate and metastatic prostate cancer cell lines. This study revealed that DSG2, DSC2, and DSG4 were consistently expressed at a high level in the luminal cells of the prostate. Further, analysis of metastatic prostate cancer cell lines showed that the expression of DSG2 is present in most cell lines examined, while the expression of DSG4 is absent. Following this characterization, I examined the role of E-cadherin and DSG2 in metastatic prostate cancer cell lines. Interestingly, the loss of E-cadherin resulted in the inhibition of extensive primary and metastatic tumor formation, suggesting that E-cadherin may have a role in promoting the progression of prostate cancer in addition to its well-established role as a tumor suppressor. Additionally, the loss of E-cadherin based adherens junctions was not associated with the reciprocal loss of DSG2 based desmosomes, challenging the common belief that the formation of adherens junctions is a prerequisite for the formation of desmosomes. I then examined the regulatory effects of PI3K/AKT signaling on E-cadherin and DSG2 expression in metastatic prostate cancer cell lines. The expression of activated AKT was found to be associated with the inhibition of E-cadherin expression, while the expression of DSG2 was relatively unperturbed in the presence of activated AKT expression. These results suggest that aberrantly activated PI3K/AKT signaling in prostate cancer may result in the loss of E-cadherin expression, and that the loss of E-cadherin and DSG2 expression in prostate cancer may be regulated by separate pathways. Finally, I examined the expression of E-cadherin and DSG2 in a large cohort of patients with prostate cancer to determine whether these cadherins were associated with prostate cancer aggressiveness. Interestingly, the loss of these cadherins was found to be significantly associated with biochemical recurrence demonstrating their potential utility as prognostic markers of aggressive prostate cancer.
15

Structure-function analysis of the essential islet regulatory factor Nkx2.2

Papizan, James January 2013 (has links)
The specification and differentiation of the pancreatic beta cell lineage requires guidance by spatiotemporally regulated signaling cues and a highly orchestrated set of transcription factors. Defining the factors and their regulatory functions that are required for proper beta cell development will enhance our ability to recapitulate these developmental events in vitro to generate beta cells from alternate cell sources. The homeodomain transcription factor Nkx2.2 is essential for pancreatic endocrine cell development; Nkx2.2-/- mice lack all beta cells and have reductions in alpha and pancreatic polypeptide (PP) cells. In place of these cell populations, the Nkx2.2-/- null islet is replete with ghrelin-producing epsilon cells. An Nkx2.2-repressor fusion protein derivative (Pdx1:Nkx2.2-EnR) expressed in the Nkx2.2-/- background can fully rescue the alpha cell population, but can only specify a few immature beta cells, suggesting that Nkx2.2 must contain both repressor and activator functions to properly guide beta cell development. Accordingly, Nkx2.2 has been shown to be an activator of several beta-cell targets. It has also been demonstrated that the corepressor Grg3 is expressed in the endocrine population and can physically interact with Nkx2.2, which points toward a mechanism by which Nkx2.2 confers transcriptional repression; however, the genes targeted by Nkx2.2/Grg3 are unknown. Additionally, how Nkx2.2 can both repress and activate genes in the same cellular context, and differentially regulate the same gene in different cellular contexts, is not understood. In this dissertation, I sought to determine the regulatory role of Nkx2.2 in the developing pancreas and its dependence on Grg interactions, and to elucidate whether post-translational modifications play a role in modulating Nkx2.2 regulatory activities. By analyzing mice carrying knock-in mutations in the Nkx2.2 Grg-interaction domain (Nkx2.2TNmut/TNmut), I show that the interaction between Nkx2.2 and Grg protein is required at two developmental stages of beta cell development: 1) Grg-mediated Nkx2.2 repression is necessary for correct beta-cell specification, and 2) the recruitment of Grg by Nkx2.2 is required to repress Arx in the beta cells to prevent beta-to-alpha cell reprogramming. Additionally, by analyzing the Nkx2.2TNmut/TNmut and Nkx2.2TNmut/TNmut;Ins:Cre;Arxfl/fl mice, I have identified several additional genes that may be regulated by Grg-mediated Nkx2.2 repression. Finally, I also present data to suggest that Nkx2.2 protein is phosphorylated, and that the phosphorylation state determines whether Nkx2.2 functions as an activator or a repressor in a promoter-specific context. These studies have begun to elucidate the complex regulatory roles that Nkx2.2 plays in specifying and maintaining the beta-cell lineage. Future analyses will help us to better understand the spatiotemporal regulatory activities that are required to make and maintain functional beta cells.
16

Regulating Distinct Cell Lineages in the Pancreatic Islet

Levine, Joshua January 2013 (has links)
Type I and type II diabetes mellitus are associated with a loss of functioning insulin-producing β cells in the pancreas. Understanding the mechanism of normal islet and β cell development will be an important step in developing possible treatments for the disease. Nkx2.2 is essential for proper β cell differentiation. Nkx2.2 mice show a complete absence of insulin-producing β cells, a 90% reduction of glucagon-producing α cells, and an increase in ghrelin-producing cells. Nkx2.2 contains three conserved domains: the tinman domain (TN), homeodomain (HD), and NK2-specific domain (SD). The SD domain is highly conserved among Nk2 family members and across species. However, its function remains largely unknown. In order to further understand the molecular interactions involving Nkx2.2 in the developing mouse pancreas, we have generated a mouse line containing mutations in the NK2-SD domain. We show that SD mutant mice have a decrease in β cell numbers as well as a decrease in the β cell markers, NeuroD, Nkx6.1, Ins1 and Ins2. However, there is no change in α cell numbers or the α cell markers, Glucagon and Irx2. Unlike the persistent upregulation of Ghrelin in the Nkx2.2 mice, Nkx2.2SD/SD mice display a transient increase in Ghrelin expression, which normalizes by birth. Additionally, polyhormonal cells are seen as early as E12.5 and persist postnatally. Postnatally, the mice show morphological changes in islet size and the proximity of their islets to the ducts. Moreover, they show a continuing loss of β cells and the persistence of polyhormonal cells resulting in severe hyperglycemia. Mechanistically, Nkx2.2 has been shown to interact in a protein complex involving several methylation factors. We show that the SD domain is necessary for the interaction of Nkx2.2 and Dnmt1, the maintenance methyltransferase. We further show that there is a loss of methylation in the α cell gene Arx in sorted β cells of the Nkx2.2 SD/SD mice as well as global hypomethylation in the Nkx2.2 SD/SD mice. These data suggest that Nkx2.2 is responsible for proper methylation patters of islet specific genes in the developing pancreas, which is important for β cell development and the formation of normal islet cell identities.
17

The role of Ultrabithorax negative autoregulation in Drosophila melanogaster

Ranade, Vikram January 2013 (has links)
One of the more striking features of animal development is that a limited set of developmental control genes is used repeatedly, in different contexts (within an organism and between species), to form different structures. To achieve this, gene regulatory networks must be versatile. Transcription factors regulate target genes by acting combinatorially, and must be deployed with spatial, temporal, and quantitative precision. In addition to being versatile, gene regulatory networks are robust, enabling animal development to yield reproducible outcomes despite environmental and genetic variation. Focusing on the D. melanogaster Hox gene Ultrabithorax (Ubx), I explore how cis-regulatory elements of developmental control genes contribute to these two hallmarks of developmental biology: versatility and robustness. Ubx specifies the identity of the third thoracic (T3) segment along the anterior-posterior axis of the developing fly. It is required for the development of T3 appendages including the haltere - a dorsal appendage that helps the fly balance during flight. Not only is Ubx presence required, but its levels are also important: Ubx levels are inversely correlated with haltere size. In Chapter 2, we describe how Ubx negative autoregulation establishes different Ubx levels in two different spatial domains of the developing haltere: the proximal haltere (which forms the joint and body wall in the adult) and the distal haltere (which forms the capitellum - the appendage proper). Ubx directly represses its own transcription with the aid of Homothorax (Hth) and Extradenticle (Exd) in the developing proximal haltere. Distally, Hth is absent, Exd is cytoplasmic, and Ubx levels are high. We identify an enhancer that captures this regulation and identify a binding site for Ubx/Exd/Hth. In Chapter 3, we describe another function for Ubx negative autoregulation: promoting developmental robustness by buffering haltere size against changes in Ubx levels. Haltere size is inversely correlated with Ubx levels, but neither haltere size nor Ubx levels change in step with changes in Ubx copy number, suggesting the possibility of phenotypic buffering. Consistently, certain Ubx enhancer traps are silenced in response to increases in Ubx gene dose. Here, we show that functional Ubx protein must exceed a certain threshold to silence Ubx enhancer traps, confirming the idea that it reflects Ubx negative autoregulation at work. Together with the results from Chapter 2, this shows that a single gene can employ the same mechanism to achieve two seemingly opposing purposes: conferring variation and robustness to its expression. Finally, we investigate Ubx enhancer trap silencing in response to naturally occurring genetic variation. We previously described that the same Ubx enhancer traps that are silenced by increases in Ubx copy number are also silenced in F1 offspring of outcrosses to certain wild populations of D. melanogaster. Although it is unclear if this is due to Ubx negative autoregulation or an independent mechanism, our data argue that the Ubx locus, and not the P-element insertions themselves, are being silenced. Interestingly, we find that i) silencing is suppressed by a gain-of-function mutation in a gene that opposes the spread of heterochromatin and ii) the expression of Position Effect Variegation reporters also changes when outcrossed to certain wild populations of D. melanogaster. Together, these results suggest that there are considerable fluctuations in the transcriptional landscape between different populations of a given species.
18

The Fate and Behavior of Ret-expressing Tip Cells in Kidney Development

Riccio, Paul January 2013 (has links)
The mammalian kidney is a complex structure composed of many highly differentiated cell types. The spatial distribution of these cells, however, emerges from elaboration of an earlier program of epithelial and mesenchymal interactions in which a tubular epithelium, the ureteric bud, undergoes successive rounds of branching within the metanephric mesenchyme. Signaling through the receptor tyrosine kinase RET is required for the normal progression of this developmental program. A complete portrait of the transcriptional changes effected by RET activation has emerged through microarray profiling. Genetic studies in the mouse have further elucidated the roles of many of these downstream genes in mediating particular inductive events, or in directing a cell toward a particular differentiation program. Comparatively little is known, however, about the fates of Ret-expressing tip cells, themselves, and of the cell-level manipulations required to sustain branching of the ureteric bud. The research presented in this thesis is an attempt to broach these questions by harnessing the growing precision of cell-level, mosaic genetic manipulations. An inducible Cre allele under control of the Ret promoter served as a tool to unambiguously confirm that Ret-expressing tip cells are multipotent progenitors that give rise to the entirety of the renal collecting system. The Ret-expressing tip cells form a self-renewing niche that furthermore gives rise to the "trunk" regions of collecting system. As these tip-derived progeny differentiate, they are competent to assume both principal and intercalated phenotypes. The RetCreERT2 allele, itself a null allele, was used to mosaically ablate Ret within the tip domain by crossing to a conditional Ret line. Loss of Ret from a portion of the tip cells severely disrupted normal branching, yielding hypomorphic kidneys with dysplastic tips. This occurred even upon later stage Ret deletion, suggesting a continued role for Ret in maintaining the branching program. Surprisingly, the mosaic loss of Ret from the tip domain is more disruptive to the branching program than is mosaic deletion of tip cells themselves. In the latter set of experiments, kidney growth was reduced, however, the morphology of the tips and of the collecting system remained normal. Mosaic analysis with double markers (MADM) was utilized to follow the fates of individual cells that lost Ret activity. This genetic tool proved incredibly powerful, revealing that tip cells that lose Ret are near completely excluded from the tip domain. Initial results suggest that this sorting behavior might be fairly rapid, which would reject the hypothesis that Ret confers a proliferative advantage to the cells. Ret activity might instead confer an ability to undergo cell rearrangements or migration-like movements that keep a cell positioned at the tip. Collectively, these findings augment our appreciation of the Ret-expressing tip domain as a special compartment within the branching ureteric bud. Ret plays a continued role in maintaining branching past its previously known role in primary ureteric bud formation. Finally, the observations made using the MADM technique compel the hypothesis that Ret-dependent cell rearrangements sculpt and continually refine the branching ureteric bud.
19

Development of Vessels, Airways and Cartilage Rings: The role of T-box genes

Arora, Ripla January 2012 (has links)
Tbx4 and Tbx5 are two closely related genes that belong to the T-box family of transcription factor genes. Loss of Tbx4 results in absence of chorio-allantoic fusion and a failure of formation of the primary vascular plexus of the allantois leading to embryonic death at E10.5. Using a candidate gene approach we identified a number of genes downstream of Tbx4 in the allantois including, extracellular matrix molecules Vcan, Has2, Itgα5; transcription factors Snai1 and Twist, and signaling molecules Bmp2, Bmp7, Notch2, Jag1 and Wnt2In addition, we show that the canonical Wnt signaling pathway contributes to the vessel-forming potential of the allantois. Ex vivo, the Tbx4 mutant phenotype can be rescued using agonists of the Wnt signaling pathway and an inhibitor of the canonical Wnt signaling pathway phenocopies the Tbx4mutant phenotype in wildtype allantoises. In vivo, Tbx4 and Wnt2 double heterozygous placentas show decreased vasculature suggesting interactions between Tbx4 and the canonical Wnt signaling pathway in the process of allantois-derived blood vessel formation. Both Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing respiratory system. Normal development of the respiratory system is essential for survival and is regulated by multiple genes and signaling pathways. Although many genes are known to be required in the epithelium, only Fgfs have been well studied in the mesenchyme. We investigated the roles of Tbx4 and Tbx5 in lung and trachea development using conditional mutant alleles and two different Cre recombinase transgenic lines. Loss of Tbx5 leads to a unilateral loss of lung bud specification and absence of tracheal specification in organ culture. Mutants deficient in Tbx4 and Tbx5 show severely reduced lung branching at mid-gestation. Concordant with this defect, the expression of mesenchymal markers Wnt2 and Fgf10, as well as Fgf10 target genes in the epithelium, Bmp4 and Spry2, is downregulated. Lung branching undergoes arrest ex vivo when Tbx4 and Tbx5 are both completely lacking. Lung-specific Tbx4 heterozygous; Tbx5 conditional null mice die soon after birth due to respiratory distress. These pups have small lungs and show severe disruptions in tracheal-bronchial cartilage rings. Sox9 a master regulator of cartilage formation, is expressed in the trachea but mesenchymal cells fail to condense and consequently do not develop cartilage normally at birth. Tbx4;Tbx5 double heterozygous mutants show decreased lung branching and fewer tracheal cartilage rings, suggesting a genetic interaction. Finally, we show that Tbx4 and Tbx5 interact with Fgf10 during the process of lung growth and branching but not during tracheal bronchial cartilage development.
20

Genetic analysis of novel regulators of neuronal migration in Caenorhabditis elegans: the insulin/IGF-1 signaling pathway, a chromatin-binding factor ZFP-1 (AF10) and endogenous RNAi

Kennedy, Lisa Michelle January 2013 (has links)
The generation of functional neural circuitries requires neuronal migration, a central component of proper nervous system development. When defective, it can lead to devastating conditions including epilepsy and mental retardation. In the nematode C. elegans, neurons undergo both short- and long-range migrations that are regulated by conserved pathways. In my thesis study, I explore novel roles for both the insulin/IGF-1 signaling pathway and RNAi factors in neuronal migration by using the embryonic anterior migrations of the hermaphrodite-specific neurons (HSNs) of C. elegans as a model. I demonstrate that the insulin/IGF-1 signaling pathway modulates the activity of the conserved DAF-16/FOXO transcription factor non-autonomously in the hypodermis to regulate HSN migration. Furthermore, I identify PAK-1, a p21-activated kinase, as a downstream target of DAF-16 in the hypodermis. This study is the first to demonstrate a non-autonomous role for both FOXO and Pak1 in neuronal migration. I also implicate a conserved PHD zinc finger protein ZFP-1/AF10 and endogenous RNAi in the regulation of HSN migration. I determine that ZFP-1 affects HSN migration in part through its negative effect on the transcription of the conserved insulin/IGF-1 signaling kinase gene pdk-1 and the modulation of downstream DAF-16 activity. This study expands the limited understanding of the normal developmental roles of both ZFP-1/AF10 and RNAi. Combined, this thesis highlights a requirement for the coordinated activities of DAF-16/FOXO, ZFP-1/AF10 and endogenous RNAi in the establishment of proper neuronal positioning during development.

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