Browsing by Author "Wagner, Daniel"

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    Caenorhabditis elegans as a tool for high-throughput screening and gene network analysis
    (2021-12-01) Anderson, Quinton Levi; Kirienko, Natasha V.; Wagner, Daniel
    Often, we think of mitochondria as simply as the powerhouse of the cell, but we have come to understand that they are crucial to multiple facets of cellular health. Mitochondria are involved in the production of ROS (reactive oxygen species), fatty acid metabolism, and calcium storage. Mitochondria are also extremely dynamic organelles that undergo fusion, fission, and biogenesis. While mitochondria are often involved in the health of the cell, mitochondrial dysfunction has been linked to serval pathologies from cancer to neurodegenerative diseases such as Parkinson’s and Alzheimer’s. In order to understand the genetic bias of these diseases, high throughput screening assays needs to be used. In my thesis I will discuss the development of a high throughput assay that can be utilized to understand interactions in a host-pathogen system, or an activation of the fluorescent reporter. This assay may allow us to understand the genetics that leads to observable phenotypes. These observations can be catalogued and placed into a pipeline that becomes a predictive model to treat human diseases. This advent will allow for a more personalized form of treatment options for patients.
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    Enhancing Stability and Expression of Recombinant Human Hemoglobin in E. Coli: Progress in the Development of a Recombinant HBOC Source
    (2008) Graves, Philip Edwar; Olson, John S.; MacKenzie, Kevin; Tao, Yitzi Jane; Wagner, Daniel
    The commercial feasibility of recombinant human Hb (rHb) as an O2 delivery pharmaceutical is limited by the production yield of holoprotein in E. coli. Currently the production ofrHb is not cost effective for use as a source in the development of third and fourth generation Hb-based oxygen carriers (HBOCs). The major problems appear to be aggregation and degradation of apoglobin at the nominal expression temperatures, 28-37° C, and the limited amount of free heme that is available for holohemoglobin assembly. One approach to solve the first problem is to inhibit apoglobin precipitation by a comparative mutagenesis strategy to improve apoglobin stability. aGlyl5 to Ala and P Glyl6 to Ala mutations have been constructed to increase the stability of the A helices of both subunits of adult human hemoglobin (HbA), based on comparison with the sequences of the more stable sperm whale hemoglobin subunits. Human fetal hemoglobin is also known to be more stable than HbA, and comparisons between human P and y (fetal Hb) chains indicate several substitutions that stabilize the aiPi interface, one of which, pHisllb to lie, increases resistance to denaturation and enhances expression in E. coli. These favorable effects of enhanced globin stability can be augmented by co-expression of bacterial membrane heme transport systems to increase the rate and extent of heme uptake through the bacterial cell membranes. The combination of increased apoglobin stability and active heme transport may enhance holohemoglobin production to levels that may make rHb a plausible starting material for all extracellular Hb-based oxygen carriers.
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    Expanding the Enzymatic Activity of the Programmable Endonuclease Cas9
    (2018-08-09) Rivera Longsworth, Gia Francesca; Wagner, Daniel
    The CRISPR-Cas9 gene editing system has revolutionized our ability to make targeted mutations in a variety of organisms. In zebrafish, we are able to use microinjections of Cas9 protein or mRNA alongside guide RNAs (gRNA) to create targeted insertions and deletions (indels) at a frequency sufficient for recovery of loss of function alleles. Combined with the many practical advantages of working with zebrafish, CRISPR-Cas9 has enabled many labs to study gene function at a lowered cost. However, there are improvements that can be made to facilitate the knock in of sequences into the genome, a process which remains inefficient in zebrafish due to a low frequency of homology directed repair (HDR), a mechanism necessary for knock in. Our aim is to increase the rate of HDR in zebrafish by modifying Cas9 using two approaches. First, we hypothesize that the long latency period of the Cas9-DNA complex taken with the short cell cycle of the early zebrafish embryo is biasing the repair mechanism against HDR, so I have engineered a CL1 degron-tagged Cas9 (DegCas9) to destabilize the Cas9 protein. A second approach to increasing the rate of knock in is to reduce the number of molecules involved in the HDR process by using an RNA template for recombination. We hypothesize that the rate of HDR can be improved if the recombination template remains in proximity to the DSB. To this aim, I have created a reverse-transcriptase Cas9 fusion and modified gRNAs that include the targeting sequence for recombination. Our goal is to create a Cas9 system that efficiently uses an RNA template for recombination. Results indicate that DegCas9 is capable of inducing mutations at a reduced rate, and that the modified gRNAs for the RTCas9 system do not reduce the effectiveness of Cas9 function. Further studies will include analyzing the spectrum of lesions induced by DegCas9 and creating alternative versions of DegCas9 and RTCas9 using different domains. If successful, these Cas9 constructs will expand the toolkit available for genetic engineering in zebrafish.
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    The role of MK2/TTP pathway in regulating early development and innate immunity in zebrafish
    (2018-11-30) Tandon, Bhavna; Silberg, Jonathan; Wagner, Daniel
    The origin of novel structures and the genes that regulate their formation is a challenging question that has been difficult to address. One such novel structure is the yolk syncytial layer (YSL) in teleost fishes like zebrafish. I studied the genetic pathways operating in the YSL using the betty boop (bbp) mutant. betty boop encodes mitogen activated protein kinase activated protein kinase 2a (mapkapk2a), also known as mk2a. One of the downstream targets of MK2a is an RNA binding protein Tristetraprolin (TTP). bbp mutants lose the expression of YSL specific genes mxtx1 and mxtx2. Here, we show that these genes are regulated through their 3’untranslated region (UTR) by MK2a/TTP pathway required in the YSL during early zebrafish development to limit their expression to the YSL. I developed an in vivo reporter assay to detect spatial regulation of mRNA stability by the MK2/TTP pathway in early zebrafish embryos which can also be used to test other potential TTP targets. MK2a is the zebrafish homolog of mammalian MK2. In mammals, MK2 is not active during early development but rather is required to regulate genes that are responsible for inflammatory response. I show that MK2/TTP reporters are not regulated in early Xenopus embryo either, implying that the early requirement for MK2 arose after the division of tetrapod and teleost lineages. The role of mk2a and its gene duplicate mk2b in regulating immune response has not been previously studied in zebrafish. I developed zebrafish as a model infection system for infection by multiple species of Candida, an opportunistic pathogen and cause of an increasing number of hospital acquired infections. I developed novel transgenic zebrafish lines that allow inducible ablation of different phagocyte classes and identified different inflammatory responses to different Candida species. Preliminary results using these tools show that mk2b-/- mutants show a higher susceptibility to Candida hyphal infection, demonstrating that mk2b plays a role in innate immunity. Whether that role is the same that MK2 plays in mammals in regulating inflammatory cytokines still need to be determined. Taken together, all these findings suggest a model in which the ancestral MK2/TTP pathway was co-opted in teleosts to restrict expression of the mxtx genes to the yolk cell, but also retains its function in innate immune response.