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- All Subjects: Genetics
- Creators: School of Life Sciences
Description
Nucleic acids encode the information required to create life, and polymerases are the gatekeepers charged with maintaining the storage and flow of this genetic information. Synthetic biologists utilize this universal property to modify organisms and other systems to create unique traits or improve the function of others. One of the many realms in synthetic biology involves the study of biopolymers that do not exist naturally, which is known as xenobiology. Although life depends on two biopolymers for genetic storage, it may be possible that alternative molecules (xenonucleic acids – XNAs), could be used in their place in either a living or non-living system. However, implementation of an XNA based system requires the development of polymerases that can encode and decode information stored in these artificial polymers. A strategy called directed evolution is used to modify or alter the function of a protein of interest, but identifying mutations that can modify polymerase function is made problematic by their size and overall complexity. To reduce the amount of sequence space that needs to be samples when attempting to identify polymerase variants, we can try to make informed decisions about which amino acid residues may have functional roles in catalysis. An analysis of Family B polymerases has shown that residues which are involved in substrate specificity are often highly conserved both at the sequence and structure level. In order to validate the hypothesis that a strong correlation exists between structural conservation and catalytic activity, we have selected and mutated residues in the 9°N polymerase using a loss of function mutagenesis strategy based on a computational analysis of several homologues from a diverse range of taxa. Improvement of these models will hopefully lead to quicker identification of loci which are ideal engineering targets.
ContributorsHaeberle, Tyler Matthew (Author) / Chaput, John (Thesis director) / Chen, Julian (Committee member) / Larsen, Andrew (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Introduction: Human papillomavirus (HPV) infection is seen in up to 90% of cases of cervical cancer, the third leading cancer cause of death in women. Current HPV screening focuses on only two HPV types and covers roughly 75% of HPV-associated cervical cancers. A protein based assay to test for antibody biomarkers against 98 HPV antigens from both high and low risk types could provide an inexpensive and reliable method to screen for patients at risk of developing invasive cervical cancer. Methods: 98 codon optimized, commercially produced HPV genes were cloned into the pANT7_cGST vector, amplified in a bacterial host, and purified for mammalian expression using in vitro transcription/translation (IVTT) in a luminescence-based RAPID ELISA (RELISA) assay. Monoclonal antibodies were used to determine immune cross-reactivity between phylogenetically similar antigens. Lastly, several protein characteristics were examined to determine if they correlated with protein expression. Results: All genes were successfully moved into the destination vector and 86 of the 98 genes (88%) expressed protein at an adequate level. A difference was noted in expression by gene across HPV types but no correlation was found between protein size, pI, or aliphatic index and expression. Discussion: Further testing is needed to express the remaining 12 HPV genes. Once all genes have been successfully expressed and purified at high concentrations, DNA will be printed on microscope slides to create a protein microarray. This microarray will be used to screen HPV-positive patient sera for antibody biomarkers that may be indicative of cervical cancer and precancerous cervical neoplasias.
ContributorsMeshay, Ian Matthew (Author) / Anderson, Karen (Thesis director) / Magee, Mitch (Committee member) / Katchman, Benjamin (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
The Intercellular Adhesion Molecule-1 (ICAM-1, known as CD54) is a cell surface type I transmembrane glycoprotein with a molecular weight of 85 to 110 kDa. The primary function of ICAM-1 is to provide adhesion between endothelial cells and leukocytes after injury or stress. ICAM-1 is used as a receptor for various pathogens such as rhinoviruses, coxsackievirus A21 and the malaria parasite Plasmodium falciparum. ICAM-1 contains five immunoglobulin (Ig) domains in its long N-terminal extracellular region, a hydrophobic transmembrane domain, and a small C-terminal cytoplasmic domain. The Ig domains 1-2 and Ig domains 3-4-5 have been crystallized separately and their structure solved, however the full ICAM-1 structure has not been solved. Because ICAM-1 appears to be important for the mediation of cell-to-cell communication in physiological and pathological conditions, gaining a structural understanding of the full-length membrane anchored ICAM-1 is desirable. In this context, we have transiently expressed a plant-optimized gene encoding human ICAM-1 in Nicotiana benthamiana plants using the MagnICON expression system. The plant produced ICAM-1 is forming aggregates according to previous data. Thus, the current extraction and purification protocols have been altered to include TCEP, a reducing agent. The protein was purified using TALON metal affinity resin and partially characterized using various biochemical techniques. Our results show that there is a reduction in aggregation formation with the use of TCEP.
ContributorsPatel, Heeral (Author) / Mor, Tsafrir (Thesis director) / Mason, Hugh (Committee member) / Kannan, Latha (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Variants of human butyrylcholinesterase (BChE) have been designed to have high cocaine hydrolytic activity. These variants have potential pharmacological applications toward treating cocaine overdose and addiction. These enzymes must be stable in the human body over fairly long periods of time in order to be effective at treating cocaine addiction. Recombinantly expressed BChE, however, tends to be in monomer or dimer oligomeric forms, which are far less stable than the tetramer form of the enzyme. When BChE is transiently expressed in Nicotiana benthamiana, it is produced mainly as monomers and dimers. However, when the protein is expressed through stable transformation, it produces much greater proportions of tetramers. Tetramerization of WT human plasma derived BChE is facilitated by the binding of a proline rich peptide. In this thesis, I investigated if a putative plant-derived analog of the mammalian proline-rich attachment domain caused stably expressed cocaine hydrolase variants of human BChE to undergo tetramerization. I also examined if co-expression of peptides with known proline-rich attachment domains further shifted the monomer-tetramer ratio toward the tetramer.
ContributorsKendle, Robert Player (Author) / Mor, Tsafrir (Thesis director) / Mason, Hugh (Committee member) / Larrimore, Kathy (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
The Dorrance Center for Rare Childhood Disorders is a unique research division at TGen (The Translational Genomics Research Institute) that provides personalized care to children and young adults facing rare, undiagnosed diseases. TGen scientists believe that the answers to these enigmatic disorders can often be found in a person's genetic code. They aim to solve these genetic mysteries using whole exome sequencing, a method that prioritizes the protein-coding portion of the genome in the search for disease-causing variants. Unfortunately, a communication gap sometimes exists between the TGen scientists and the patients they serve. I have seen, first hand, the kind of confusion that this study elicits in the families of its participants. Therefore, for my thesis, I decided to create a booklet that is meant to provide some clarity as to what exactly The Dorrance Center for Rare Childhood Disorders does to help diagnose children with rare disorders. The purpose of the booklet is to dispel any confusion regarding the study by providing a general review of genetics and an application of these lessons to the relevant sequencing technology as well as a discussion of the causes and effects of genetic mutations that often times are linked to rare childhood disorders.
ContributorsCambron, Julia Claire (Author) / LaBelle, Jeffrey (Thesis director) / Huentelman, Matt (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Efforts to quantify the diversity of the T cell repertoire have generally been unsuccessful because not all factors accounting for diversity have been considered. In order to get an accurate representation of the T cell repertoire, one must incorporate analysis of germline gene diversity, diversity from somatic recombination, joining diversity from N- and P- nucleotides, and TCR chain pairing diversity. Because of advances in high-throughput sequencing techniques, estimates have been able to account for diversity from TCR genes. However the ability to account for chain pairing diversity has been more difficult. In order to do so, single cell sorting techniques must be employed. These techniques, though effective, are time consuming and expensive. For this reason, no large-scale analyses have been done on the immune repertoires using these techniques. In this study, we propose a novel method for linking the two TCR chain sequences from an individual cell. DNA origami nanostructure technology is employed to capture and bind the TCRγ and TCRδ chain mRNA inside individual cells using probe strands complementary to the C-region of those sequences. We then use a dual-primer RT and ligation molecular strategy to link the two sequences together. The result is a single amplicon containing the CDR3 region of the TCRγ and TCRδ. This amplicon can then be easily PCR amplified using sequence specific primers, and sequenced. DNA origami nanostructures offer a rapid, cost-effective method alternative to conventional single cell sorting techniques, as both TCR mRNA can be captured on one origami molecule inside a single cell. At present, this study outlines a proof-of-principle analysis of the method to determine its functionality. Using known TCRγ and TCRδ sequences, the DNA origami and RT/PCR method was tested and resulting sequence data proved the effectiveness of the method. The original TCRγ and TCRδ sequences were linked together as a single amplicon containing both CDR3 regions of the genes. Thus, this method can be employed in further research to elucidate the γδ T cell repertoire. This technology is also easily adapted to any gene target or cell type and therefore presents a large opportunity to be used in other immune repertoire analysis and other immunological studies (such as the rapid identification and subsequent production of antibodies).
ContributorsPoindexter, Morgan Elizabeth (Author) / Blattman, Joseph (Thesis director) / Yan, Hao (Committee member) / Schoettle, Louis (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Duchenne Muscular Dystrophy (DMD) is an X-linked recessive disease characterized by progressive muscle loss and weakness. This disease arises from a mutation that occurs on a gene that encodes for dystrophin, which results in observable muscle death and inflammation; however, the genetic changes that result from dystrophin's dysfunctionality remain unknown. Current DMD research uses mdx mice as a model, and while very useful, does not allow the study of cell-autonomous transcriptome changes during the progression of DMD due to the strong inflammatory response, perhaps hiding important therapeutic targets. C. elegans, which has a very weak inflammatory response compared to mdx mice and humans, has been used in the past to study DMD with some success. The worm ortholog of the dystrophin gene has been identified as dys-1 since its mutation phenocopies the progression of the disease and a portion of the human dystrophin gene alleviates symptoms. Importantly, the extracted RNA transcriptome from dys-1 worms showed significant change in gene expression, which needs to be further investigated with the development of a more robust model. Our lab previously published a method to isolate high-quality muscle-specific RNA from worms, which could be used to study such changes at higher resolution. We crossed the dys-1 worms with our muscle-specific strain and demonstrated that the chimeric strain exhibits similar behavioral symptoms as DMD patients as characterized by a shortened lifespan, difficulty in movement, and a decrease in speed. The presence of dys-1 and other members of the dystrophin complex in the body muscle were supported by the development of a resulting phenotype due to RNAi knockdown of each component in the body muscle; however, further experimentation is needed to reinforce this conclusion. Thus, the constructed chimeric C. elegans strain possesses unique characteristics that will allow the study of genetic changes, such as transcriptome rearrangements and dysregulation of miRNA, and how they affect the progression of DMD.
ContributorsNguyen, Thuy-Duyen Cao (Author) / Mangone, Marco (Thesis director) / Newbern, Jason (Committee member) / Duchaine, Thomas (Committee member) / School of Social Transformation (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
ContributorsSavarese, Erica (Author) / Robert, Jason (Thesis director) / Hurlbut, Ben (Committee member) / Verrelli, Brian (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2013-05
Description
In medical field today, current diagnostic tools for neurodegenerative diseases fail to diagnose patients prior to the occurrence of damaging neuronal loss. Oftentimes, this means that by the time a patient has been diagnosed with a disease such as Alzheimer's disease (AD) or Parkinson's disease (PD), they have already suffered severe, irreversible neurodegeneration. One of the significant weaknesses in the diagnosis and treatment of patients with AD and PD is the lack of viable biomarkers. Biomarkers are vital tools that can be utilized to identify patients who are in presymptomatic stages of a disease, track and quantify disease progression, and also determine whether or not a patient is responding to a particular treatment. RNAs are involved in all cellular processes, and due to their very specific spatial, temporal, and even cellular-level expression, abnormal expression signatures serve as key indicators of many diseases. Recently, cells have been shown to secrete nanometer-sized microvesicles, called exosomes, which moderate the horizontal transfer of mRNAs and miRNAs between cells. We hypothesize that exosomes obtained from human biofluids, such as cerebral spinal fluid (CSF) and blood plasma, can be used to determine extracellular RNA (exRNA) expression signatures associated with neurodegenerative disease. This experiment used pooled samples of CSF and plasma in order to investigate which of 3 sample enrichment methods would be most conducive to studying exRNA contained within exosomes. The results from this preliminary investigation will be used in later investigations that will seek to determine exRNA biomarkers of neurodegenerative disease.
ContributorsBeecroft, Taylor Alexandria (Author) / Capco, David (Thesis director) / Van Keuren-Jensen, Kendall (Committee member) / Huentelman, Matt (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2013-05
Description
It is important to consider factors that contribute to successful fertilization and the development of viable offspring. Better understanding the factors that contribute to infertility can be used to assist in the development of viable offspring, especially for human beings looking to successfully reproduce. Identifying paternal effect genes, genes that come from the father, introduces more targets that can be manipulated to produce specific reproductive effects. Use of Drosophila melanogaster as a model to study reproduction has increased, in part, due to the use of the GAL4 system. In this system, the GAL4 gene encodes an 881 amino acid protein that binds to the 4-site Upstream Activating Sequence (UAS) to induce transcription of the gene of interest. These sequences constitute the two components of the system: the driver (GAL4) and the responder (gene of interest) \u2014 each of which is maintained as a separate parental line. Effects of the GAL4 driver line "driving" transcription of the responder can be assessed by examining the offspring. One of the more common uses of the GAL4 system involves analyzing phenotypic effects of reducing or eliminating expression of a target gene through the induction of RNAi transcription, which often results in toxicity, lethality, or reduced viability. Utilizing these principles, we strove to demonstrate the effect of knocking down the expression of testis-specific sperm-leucyl-aminopeptidases gene CG13340 on progeny by inducing expression of RNAi with two distinct GAL4 driver lines - one with a nonspecific actin-binding activation sequence and the other with a testis-specific activation sequence. Comparison of both GAL4 driver lines to crosses using N01 wild type ("wt") flies verify that inducing RNAi transcription using the GAL4 system results in reduction of proper offspring development. Further studies using D. melanogaster and the GAL4 system can improve knowledge of factors contributing to male fertility and also be applied to better understand mammalian, specifically human, fertility.
ContributorsEvans, Donna Marie (Author) / Karr, Timothy L. (Thesis director) / Roland, Kenneth (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / Department of English (Contributor)
Created2014-05