256 research outputs found
Samira Bellil: Dans l'enfer des tournantes - commented translation extended by analysis of translation complexities and reception of literature of the "beurs"
No full textThis master's thesis consists of a Czech language translation and commentary for selected chapters of Dans l'enfer des tournantes, an autobiography by Samira Bellil, a French author of Algerian descent. The commentary is divided into two parts: a presentation of literature written by French authors of Maghrebi descent, the so-called beurs, the language they use in literature and reception of their works; and an analysis of the source text and its reception, the problems encountered during the process of translation and their solutions as well as the chosen translation method. Key words: Samira Bellil, autobiography, beurs, translation, translation analysis, translation method, translation problem, translation shift, suburbs of Pari
CRISPR transcriptional repression devices and layered circuits in mammalian cells
No full textA key obstacle to creating sophisticated genetic circuits has been the lack of scalable device libraries. Here we present a modular transcriptional repression architecture based on clustered regularly interspaced palindromic repeats (CRISPR) system and examine approaches for regulated expression of guide RNAs in human cells. Subsequently we demonstrate that CRISPR regulatory devices can be layered to create functional cascaded circuits, which provide a valuable toolbox for engineering purposes.National Institutes of Health (U.S.) (Grant 5R01CA155320-04)National Institutes of Health (U.S.) (Grant P50 GM098792)Korea (South). Ministry of Science, Information and Communication Technolgy. Intelligent Synthetic Biology Center of Global Frontier Project (2013M3A6A8073557
Social media and the Arab Spring
No full textIn late of 2010 and during 2011, the Arab countries saw a series of large scale political uprisings. Facebook, Twitter, YouTube, and other forms of social media have played a major role in the planning, acceleration, and even the preparation of some of the uprisings and revolutions that too place in the Middle East at this time. Social media was employed effectively to awaken the Arab people and to mobilize them to fight against repressive regimes in their drive for greater freedom and independence. This paper will discuss the cause of the uprisings. In addition, the role of social media in the Middle East before the revolutions and the impact it had on the uprisings and revolutions known as the Arab Springs will be examined.M.A.L.S.Includes bibliographical referencesby Samira F. Hassa
Genetically engineering self-organization of human pluripotent stem cells into a liver bud-like tissue using Gata6
No full textHuman induced pluripotent stem cells (hiPSCs) have potential for personalized and regenerative medicine. While most of the methods using these cells have focused on deriving homogenous populations of specialized cells, there has been modest success in producing hiPSC-derived organotypic tissues or organoids. Here we present a novel approach for generating and then co-differentiating hiPSC-derived progenitors. With a genetically engineered pulse of GATA-binding protein 6 (GATA6) expression, we initiate rapid emergence of all three germ layers as a complex function of GATA6 expression levels and tissue context. Within 2 weeks we obtain a complex tissue that recapitulates early developmental processes and exhibits a liver bud-like phenotype, including haematopoietic and stromal cells as well as a neuronal niche. Collectively, our approach demonstrates derivation of complex tissues from hiPSCs using a single autologous hiPSCs as source and generates a range of stromal cells that co-develop with parenchymal cells to form tissues.National Science Foundation (U.S.). Emergent Behaviors of Integrated Cellular Systems (NSF CBET-0939511)Synthetic Biology Engineering Research CenterNational Institutes of Health (U.S.) (P50GM098792)Ernst Schering FoundationSwiss National Science Foundatio
Using Bioengineering Approaches to Generate a Three-Dimensional Human Induced Pluripotent Stem-Cell Based Model of Alzheimer's Disease
No full textabstract: The pathophysiology of Alzheimer’s disease (AD) remains difficult to precisely ascertain in part because animal models fail to fully recapitulate many aspects of the disease and postmortem studies do not allow for the study of the pathophysiology. In vitro models of AD generated with patient derived human induced pluripotent stem cells (hiPSCs) could provide new insight into disease mechanisms. Although many protocols exist to differentiate hiPSCs to neurons, standard practice relies on two-dimensional (2-D) systems, which do not accurately mimic the complexity and architecture of the in vivo brain microenvironment. This research aims to create three-dimensional (3-D) models of AD using hiPSCs, which would enhance the understanding of AD pathophysiology thereby, enabling the generation of effective therapeutics.Dissertation/ThesisMasters Thesis Bioengineering 201
Germ Cell Specific Safety Switches: Cell Isolation
No full textabstract: The process of spermatogenesis, the differentiation of sperm stem cells into spermatozoa, produces a diverse array of descendent cells which express varied morphological and genetic traits throughout their maturation. Beginning with primordial germ cells, these sperm progenitors experience twelve stages of differentiation before maturation into their final stage. During their differentiation, these cells reside in the seminiferous tubules within the testes. These tubules are surrounded by somatic cells, primarily Sertoli, Leydig, myoid, and epithelial cells. These cells provide the germ cells with necessary signaling proteins for their progression as well as protection from exterior toxins through the formation of the blood-testis barrier (BTB). However, their close association with germ cells makes extracting these sperm progenitors difficult. Here, I convey the results for an initial trial of harvesting germ cells from two mice. Due to inconclusive qRT-PCR amplification data from the first experiment, future iterations of this harvest will explore other previously published methods. These will include Magnetic-Activated Cell Sorting which will target individual sperm progenitor populations using cell-surface receptors such as GFRα-1 and THY1 to obtain sperm stem cells. Additionally, Fluorescence-Activated Cell Sorting may be useful for obtaining multiple groups of meiotic cell types from a heterogenous cell suspension harvested from the seminiferous tubules through the use of Hoechst 33342 staining. Finally, extraction of spermatozoa from the Cauda Epididymis, a storage site for these mature sperm, can be performed either in conjunction with testes collection during necropsy or as an in vivo technique intended for serial sampling of sperm cells over time. Regardless, it is necessary for these methods to produce populations from spermatogonia to spermatozoa with high purity in order to produce representative qRT-PCR results downstream, indicating either presence or lack of genetic mutation enacted by future CRISPR-Cas9 experiments
Developing safe and controllable Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based therapies with design principles of synthetic biology
No full textabstract: The CRISPR/Cas9 gene-editing tool is currently in clinical trials as the excitement about its therapeutic potential is exponentially growing. However, many of the developed CRISPR based genome engineering methods cannot be broadly translated in clinical settings due to their unintended consequences. These consequences, such as immune reactions to CRISPR, immunogenic adverse events following receiving of adeno-associated virus (AAV) as one of the clinically relevant delivery agents, and CRISPR off-target activity in the genome, reinforces the necessity for improving the safety of CRISPR and the gene therapy vehicles. Research into designing more advanced CRISPR systems will allow for the increased ability of editing efficiency and safety for human applications. This work 1- develops strategies for decreasing the immunogenicity of CRISPR/Cas9 system components and improving the safety of CRISPR-based gene therapies for human subjects, 2- demonstrates the utility of this system in vivo for transient repression of components of innate and adaptive immunity, and 3- examines an inducible all-in-one CRISPR-based control switch to pave the way for controllable CRISPR-based therapies.Dissertation/ThesisDoctoral Dissertation Biological Design 202
The Effect of GATA6 Expression and Its Neighborhood Impact Factor on Regulating Cell Fate
No full textabstract: A genetically engineered line of human induced pluripotent stem cells was used to study the effects of gene expression on cell fate. These cells were designed to activate expression of the gene GATA6 when exposed to the small molecule doxycycline. This gene was chosen because it plays an important role in the developmental biology stages of liver formation. Because of the way the cells were engineered, a given population would have a heterogeneous expression of GATA6 because each cell could have a different copy number of the exogenous gene. This variation allows for the differentiation of multiple cell types, and is used to grow liver organoids. The early liver organoid samples were studied via immunofluorescent staining, imaging, and quantitative image analysis. It was originally hypothesized that absolute gene expression was not the most important factor in determining cell fate, but relative gene expression was. This meant that the spatial location of the cells and their local environment were critical in determining cell fate. In other words, the level of GATA6 of a cell is important, but so is the level of GATA6 in the surrounding cells, or neighborhood, of that cell. This hypothesis was analyzed with the creation of various Neighborhood Impact Factor (NIF) methods. Multiple time points of growth were analyzed to study the temporal effect, in addition to the gene expression and NIF influence on a cell’s fate. Direct gene expression level showed correlation with certain cell fate markers. In addition to GATA6 expression levels, NIF results from early and late time point experiments show statistical significance with relatively small neighborhood radii. The NIF analysis was useful for examining the effect of neighboring cells and determining the size of the neighborhood – how far cells influence one another. While these systems are complex, the NIF analysis provides a way to look at gene expression and its influence in spatial context.Dissertation/ThesisPowerpoint presentation used in the defense.Masters Thesis Bioengineering 201
CRISPR/Cas9 Mediated Mutation in the ATP-ase Domain of XPB to Study its Role in Pancreatic Ductal Adenocarcinoma
No full textabstract: Pancreatic ductal adenocarcinoma (PDAC) is a form of pancreatic cancer that affects the exocrine function of the pancreas. PDAC is often hard to diagnose and has shown to also be as difficult to treat. Xeroderma pigmentosum type B (XPB), is a protein can be found in Transcription Factor II Human (TFIIH). It is known to have ATP-ase and helicase activities. The ATP-ase activities could be used to regulate the transcription within super enhancer (SE) networks. Knocking out the ATP-ase activity in XPB in the same way that triptolide does would offer a more individualized therapeutic regiment. A loss of function mutation was tested to identify whether or not the mutation was present within the strand of DNA. In order to explore the role of XPB in pancreatic cancer, a knockout clone was made through the use of the CRISPR/Cas9 genome editing technology to induce a clone in exon 2 of XPB using a plasmid with Green Fluorescent Protein (GFP) selection marker. Once the clones were successfully made, they underwent testing through the use of a Surveyor Mutation Detection Kit for standard electrophoresis. The confirmation of a functional clone lead to GFP, which contained the mutation, being chosen for further testing be compared to the wild type GFP. After the GFP D54H mutation was chosen for further testing, it was then cultured from bacteria and wild type GFP and GFP D54H underwent a restriction enzyme digest. The digest resulted in showing that GFP and GFP D54H were the same on a larger level, and that one of the only ways to prove that the mutation was present was through amplification and analysis using the mutation detection kit
Validating a Novel CRISPR/Cas9 System for Simultaneous Gene Modification and Transcriptional Regulation
No full textabstract: A novel clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) tool for simultaneous gene editing and regulation was designed and tested. This study used the CRISPR-associated protein 9 (Cas9) endonuclease in complex with a 14-nucleotide (nt) guide RNA (gRNA) to repress a gene of interest using the Krüppel associated box (KRAB) domain, while also performing a separate gene modification using a 20-nt gRNA targeted to a reporter vector. DNA Ligase IV (LIGIV) was chosen as the target for gene repression, given its role in nonhomologous end joining, a common DNA repair process that competes with the more precise homology-directed repair (HDR).
To test for gene editing, a 20-nt gRNA was designed to target a disrupted enhanced green fluorescent protein (EGFP) gene present in a reporter vector. After the gRNA introduced a double-stranded break, cells attempted to repair the cut site via HDR using a DNA template within the reporter vector. In the event of successful gene editing, the EGFP sequence was restored to a functional state and green fluorescence was detectable by flow cytometry. To achieve gene repression, a 14-nt gRNA was designed to target LIGIV. The gRNA included a com protein recruitment domain, which recruited a Com-KRAB fusion protein to facilitate gene repression via chromatin modification of LIGIV. Quantitative polymerase chain reaction was used to quantify repression.
This study expanded upon earlier advancements, offering a novel and versatile approach to genetic modification and transcriptional regulation using CRISPR/Cas9. The overall results show that both gene editing and repression were occurring, thereby providing support for a novel CRISPR/Cas system capable of simultaneous gene modification and regulation. Such a system may enhance the genome engineering capabilities of researchers, benefit disease research, and improve the precision with which gene editing is performed.Dissertation/ThesisMasters Thesis Molecular and Cellular Biology 201
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