Matching Items (16)

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Improving the Valley Fever Gene Annotation Through Proteogenomic Analysis

Description

Valley Fever, also known as coccidioidomycosis, is a respiratory disease that affects 10,000 people annually, primarily in Arizona and California. Due to a lack of gene annotation, diagnosis and treatment

Valley Fever, also known as coccidioidomycosis, is a respiratory disease that affects 10,000 people annually, primarily in Arizona and California. Due to a lack of gene annotation, diagnosis and treatment of Valley Fever is severely limited. In turn, gene annotation efforts are also hampered by incomplete genome sequencing. We intend to use proteogenomic analysis to reannotate the Coccidioides posadasii str. Silveira genome from protein-level data. Protein samples extracted from both phases of Silveira were fragmented into peptides, sequenced, and compared against databases of known and predicted proteins sequences, as well as a de novo six-frame translation of the genome. 288 unique peptides were located that did not match a known Silveira annotation, and of those 169 were associated with another Coccidioides strain. Additionally, 17 peptides were found at the boundary of, or outside of, the current gene annotation comprising four distinct clusters. For one of these clusters, we were able to calculate a lower bound and an estimate for the size of the gap between two Silveira contigs using the Coccidioides immitis RS transcript associated with that cluster's peptides \u2014 these predictions were consistent with the current annotation's scaffold structure. Three peptides were associated with an actively translated transposon, and a putative active site was located within an intact LTR retrotransposon. We note that gene annotation is necessarily hindered by the quality and level of detail in prior genome sequencing efforts, and recommend that future studies involving reannotation include additional sequencing as well as gene annotation via proteogenomics or other methods.

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Created

Date Created
  • 2016-12

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Can Akt3 Decrease Tumorigenicity in Glioblastoma Multiforme Through a Cell Cycle Mechanism?

Description

Glioblastoma multiforme is associated with a very low survival rate and is recognized as the most vicious form of intracranial cancer. The Akt gene pathway has three different isoforms, each

Glioblastoma multiforme is associated with a very low survival rate and is recognized as the most vicious form of intracranial cancer. The Akt gene pathway has three different isoforms, each of which has a different role in the tumors of GBM. Preliminary data suggests that Akt3 may work to decrease tumorigenicity. A produced image that visualizes the subcellular localization of Akt3 led the author to believe that Akt3 may reduce tumorigenicity by decreasing genomic instability caused by the cancer. To explore this, flow cytometry was performed on GBM cell lines with Akt3v1 over-expression, Akt3v2 over-expression, and a control glioma cell line.

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Created

Date Created
  • 2012-12

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Going Beyond the Diagnosis in Rare Childhood Disorders: Development of Personalized Treatment Methods for Mitochondrial Disease in Patients with a Nonsynonymous MTFMT Mutation

Description

A single splice site mutation in the mitochondrial methionyl-tRNA formyltransferase (MTFMT) gene is described in three patients with mitochondrial disease from two unrelated families. Nuclear-encoded MTFMT localized to the mitochondria

A single splice site mutation in the mitochondrial methionyl-tRNA formyltransferase (MTFMT) gene is described in three patients with mitochondrial disease from two unrelated families. Nuclear-encoded MTFMT localized to the mitochondria is responsible for the formylation of Met-tRNAMet necessary for the initiation of translation in the mitochondria. This mutation has been associated with mitochondrial disease (oxidative phosphorylation deficiencies due to a decreased expression of MTFMT), Leigh syndrome, and developmental delay. However, there is significant phenotypic variation between patients, which is not uncommon in mitochondrial disease. Though the variation was not clearly elucidated through analysis of gene expression, this data supported two potential gene modifiers as well as proposed an alternative energy producing pathway in the cell—glutamine metabolism. This nonsynonymous mutation at site c.626C>T generates a splicing suppressor in the coding region on exon 4 resulting exon skipping in almost all transcripts in homozygotes during splicing. It is hypothesized that antisense oligotherapy will be effective in rescuing this mutation by inhibiting the splice silencer and promoting exon inclusion as well as an increased expression of MTFMT protein in affected patients. Patient fibroblast cells were treated with MTFMT Oligo 3, which was shown to be promising in previous experiments. Real-Time qPCR was used to measure mRNA expression showing a significant up-regulation of wild-type MTFMT with treatment. In order to test whether this therapy increases mitochondrial function as well, three mitochondrial functional assays measuring superoxide species in the mitochondria, the mitochondrial membrane potential, and calcium uptake in the mitochondria were tested for optimization of results. Success has been shown in the measurement of superoxide species and mitochondrial membrane potential in patient cells without treatment. Oligotherapy will hopefully be considered as a viable therapeutic option in the future as further testing is conducted and perfected.

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Agent

Created

Date Created
  • 2016-05

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It's All In the Genes: Designing and Administering a Brief Survey to Local Dairymen In Order to Gauge their Interest In Genomics

Description

One of the newest technologies available for agricultural use is the sequencing of the bovine genome and the identification of specific genes that would ensure favorable physical traits in the

One of the newest technologies available for agricultural use is the sequencing of the bovine genome and the identification of specific genes that would ensure favorable physical traits in the herd. An easy way for this technology to be utilized is in the milking herds of dairies, the herd has already been bred for specific traits and any change due to a genomic influence would be easily seen. Dairy cattle are commonly bred through artificial insemination, and this would be a perfect place for the genomic programs to prove themselves. In order to determine the attitudes of local dairymen toward genomics, I designed and administered a survey to gauge their opinions. The survey was given to a meeting of the United Dairymen of Arizona at their Tempe offices. The survey covered the current breeding methods used by the dairies, the desired attributes in a milking herd and a breeding program, and a place for the dairymen to give their own opinions on genomics. The results indicated that the dairymen are interested of using genomics, but they are unsure of the cost. Dairymen are often looking for new methods to increase their milk production and herd value, but are reluctant to pay a high amount. One recommendation is for these dairymen to utilize bulls that have had their genome analyzed when they are breeding their cows. This would allow the dairymen to see the effects and benefits of genomics on their herd without the dairymen having to front the large start up cost for their own genomic program.

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Agent

Created

Date Created
  • 2016-05

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Big Data Network Analysis of Genetic Variation and Gene Expression in Individuals with Breast Cancer

Description

The advent of big data analytics tools and frameworks has allowed for a plethora of new approaches to research and analysis, making data sets that were previously too large or

The advent of big data analytics tools and frameworks has allowed for a plethora of new approaches to research and analysis, making data sets that were previously too large or complex more accessible and providing methods to collect, store, and investigate non-traditional data. These tools are starting to be applied in more creative ways, and are being used to improve upon traditional computation methods through distributed computing. Statistical analysis of expression quantitative trait loci (eQTL) data has classically been performed using the open source tool PLINK - which runs on high performance computing (HPC) systems. However, progress has been made in running the statistical analysis in the ecosystem of the big data framework Hadoop, resulting in decreased run time, reduced storage footprint, reduced job micromanagement and increased data accessibility. Now that the data can be more readily manipulated, analyzed and accessed, there are opportunities to use the modularity and power of Hadoop to further process the data. This project focuses on adding a component to the data pipeline that will perform graph analysis on the data. This will provide more insight into the relation between various genetic differences in individuals with breast cancer, and the resulting variation - if any - in gene expression. Further, the investigation will look to see if there is anything to be garnered from a perspective shift; applying tools used in classical networking contexts (such as the Internet) to genetically derived networks.

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Created

Date Created
  • 2016-12

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Informatics approaches for integrative analysis of disparate high-throughput genomic datasets in cancer

Description

The processes of a human somatic cell are very complex with various genetic mechanisms governing its fate. Such cells undergo various genetic mutations, which translate to the genetic aberrations that

The processes of a human somatic cell are very complex with various genetic mechanisms governing its fate. Such cells undergo various genetic mutations, which translate to the genetic aberrations that we see in cancer. There are more than 100 types of cancer, each having many more subtypes with aberrations being unique to each. In the past two decades, the widespread application of high-throughput genomic technologies, such as micro-arrays and next-generation sequencing, has led to the revelation of many such aberrations. Known types and subtypes can be readily identified using gene-expression profiling and more importantly, high-throughput genomic datasets have helped identify novel sub-types with distinct signatures. Recent studies showing usage of gene-expression profiling in clinical decision making in breast cancer patients underscore the utility of high-throughput datasets. Beyond prognosis, understanding the underlying cellular processes is essential for effective cancer treatment. Various high-throughput techniques are now available to look at a particular aspect of a genetic mechanism in cancer tissue. To look at these mechanisms individually is akin to looking at a broken watch; taking apart each of its parts, looking at them individually and finally making a list of all the faulty ones. Integrative approaches are needed to transform one-dimensional cancer signatures into multi-dimensional interaction and regulatory networks, consequently bettering our understanding of cellular processes in cancer. Here, I attempt to (i) address ways to effectively identify high quality variants when multiple assays on the same sample samples are available through two novel tools, snpSniffer and NGSPE; (ii) glean new biological insight into multiple myeloma through two novel integrative analysis approaches making use of disparate high-throughput datasets. While these methods focus on multiple myeloma datasets, the informatics approaches are applicable to all cancer datasets and will thus help advance cancer genomics.

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Agent

Created

Date Created
  • 2014

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Insights towards developing regenerative therapies: the lizard, Anolis carolinensis, as a genetic model for regeneration in amniotes

Description

Damage to the central nervous system due to spinal cord or traumatic brain injury, as well as degenerative musculoskeletal disorders such as arthritis, drastically impact the quality of life. Regeneration

Damage to the central nervous system due to spinal cord or traumatic brain injury, as well as degenerative musculoskeletal disorders such as arthritis, drastically impact the quality of life. Regeneration of complex structures is quite limited in mammals, though other vertebrates possess this ability. Lizards are the most closely related organism to humans that can regenerate de novo skeletal muscle, hyaline cartilage, spinal cord, vasculature, and skin. Progress in studying the cellular and molecular mechanisms of lizard regeneration has previously been limited by a lack of genomic resources. Building on the release of the genome of the green anole, Anolis carolinensis, we developed a second generation, robust RNA-Seq-based genome annotation, and performed the first transcriptomic analysis of tail regeneration in this species. In order to investigate gene expression in regenerating tissue, we performed whole transcriptome and microRNA transcriptome analysis of regenerating tail tip and base and associated tissues, identifying key genetic targets in the regenerative process. These studies have identified components of a genetic program for regeneration in the lizard that includes both developmental and adult repair mechanisms shared with mammals, indicating value in the translation of these findings to future regenerative therapies.

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Agent

Created

Date Created
  • 2015

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Genome-driven targeted cancer therapy

Description

Cancer is a heterogeneous disease with discrete oncogenic mechanisms. P53 mutation is the most common oncogenic mutation in many cancers including breast cancer. This dissertation focuses on fundamental genetic alterations

Cancer is a heterogeneous disease with discrete oncogenic mechanisms. P53 mutation is the most common oncogenic mutation in many cancers including breast cancer. This dissertation focuses on fundamental genetic alterations enforced by p53 mutation as an indirect target. p53 mutation upregulates the mevalonate pathway genes altering cholesterol biosynthesis and prenylation. Prenylation, a lipid modification, is required for small GTPases signaling cascades. Project 1 demonstrates that prenylation inhibition can specifically target cells harboring p53 mutation resulting in reduced tumor proliferation and migration. Mutating p53 is associated with Ras and RhoA activation and statin prevents this activity by inhibiting prenylation. Ras-related pathway genes were selected from the transcriptomic analysis for evaluating correlation to statin sensitivity. A gene signature of seventeen genes and TP53 genotype (referred to as MPR signature) is generated to predict response to statins. MPR signature is validated through two datasets of drug screening in cell lines. As advancements in targeted gene modification are rising, the CRISPR-Cas9 technology has emerged as a new cancer therapeutic strategy. One of the important risk factors in gene therapy is the immune recognition of the exogenous therapeutic tool, resulting in obstruction of treatment and possibly serious health consequences. Project 2 describes a method development that can potentially improve the safety and efficacy of gene-targeting proteins. A cohort of 155 healthy individuals was screened for pre-existing B cell and T cell immune response to the S. pyogenes Cas9 protein. We detected antibodies against Cas9 in more than 10% of the healthy population and identified two immunodominant T cell epitopes of this protein. A de-immunized Cas9 that maintains the wild-type functionality was engineered by mutating the identified T cell epitopes. The gene signature and method described here have the potential to improve strategies for genome-driven tumor targeting.

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Agent

Created

Date Created
  • 2017

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Development of CRISPR-RNA guided recombinases for genome engineering

Description

Recombinases are powerful tools for genome engineering and synthetic biology, however recombinases are limited by a lack of user-programmability and often require complex directed-evolution experiments to retarget specificity. Conversely, CRISPR

Recombinases are powerful tools for genome engineering and synthetic biology, however recombinases are limited by a lack of user-programmability and often require complex directed-evolution experiments to retarget specificity. Conversely, CRISPR systems have extreme versatility yet can induce off-target mutations and karyotypic destabilization. To address these constraints we developed an RNA-guided recombinase protein by fusing a hyperactive mutant resolvase from transposon TN3 to catalytically inactive Cas9. We validated recombinase-Cas9 (rCas9) function in model eukaryote Saccharomyces cerevisiae using a chromosomally integrated fluorescent reporter. Moreover, we demonstrated cooperative targeting by CRISPR RNAs at spacings of 22 or 40bps is necessary for directing recombination. Using PCR and Sanger sequencing, we confirmed rCas9 targets DNA recombination. With further development we envision rCas9 becoming useful in the development of RNA-programmed genetic circuitry as well as high-specificity genome engineering.

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Agent

Created

Date Created
  • 2018

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Promoter Identification in Daphnia Populations Revealed by Transcription Start Site Profiling

Description

Regulation of transcription initiation is a critical factor in the emergence of diverse biological phenotypes, including the development of multiple cell types from a single genotype, the ability of organisms

Regulation of transcription initiation is a critical factor in the emergence of diverse biological phenotypes, including the development of multiple cell types from a single genotype, the ability of organisms to respond to environmental cues, and the rise of heritable diseases. Transcription initiation is regulated in large part by promoter regions of DNA. The identification and characterization of cis-regulatory regions, and understanding how these sequences differ across species, is a question of interest in evolution. To address this topic, I used the model organism Daphnia pulex, a well-characterized microcrustacean with an annotated genome sequence and selected a distribution of well-defined populations geographically located throughout the Midwestern US, Oregon, and Canada. Using isolated total RNA from adult, female Daphnia originating from the selected populations as well as a related taxon, Daphnia pulicaria (200,000 years diverged from D. pulex), I identified an average of over 14,000 (n=14,471) promoter regions using a novel transcription start site (TSS) profiling method, STRIPE-seq. Through the identification of sequence architecture, promoter class, conservation, and transcription start region (TSR) width, of cis-regulatory regions across the aforementioned Daphnia populations, I constructed a system for the study of promoter evolution, enabling a robust interpretation of promoter evolution in the context of the population-genetic environment. The methodology presented, coupled with the generated dataset, provides a foundation for the study of the evolution of promoters across both species and populations.

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Agent

Created

Date Created
  • 2020