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- Creators: School of Molecular Sciences
- Resource Type: Text
- Status: Published
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 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.
ContributorsMoskowitz, Abby Mae (Author) / Huentelman, Matt (Thesis director) / Schrauwen, Isabelle (Committee member) / Rangasamy, Sampath (Committee member) / School of Human Evolution and Social Change (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Identifying immunoreactive cytotoxic T lymphocytes (CTLs) by current technologies (cytokine secretion, intracellular cytokine, ELISPOT, and MHC tetramer assays) is often difficult when probing for multiple target antigens. CTLs activate and induce apoptosis of pathogenic cells when T-cell receptors (TCRs) specifically bind to antigenic peptides and major histocompatibility complexes (pMHCs) presented on the target cell’s surface. Flow cytometric MHC class I tetramer assays allow for the direct quantification and sorting of most CD8+ T lymphocytes whose TCRs recognize bound peptides, regardless of effector function. Class I tetramers are traditionally produced using BL21-DE3 E. coli expression, denaturation and folding in vitro, which is technically challenging, time-consuming, and low-throughput. We are developing an assay amenable to rapid, high-throughput screening of peptide libraries to characterize and quantitate antigen-specific CTLs in peripheral blood mononuclear cells (PBMCs). Baculovirus expression systems, utilizing host eukaryotic chaperones and isomerases, are capable of producing soluble, properly-folded protein complexes with high yields. The HLA-A*0201 heavy chain and beta-2-microglobulin genes were cloned into pIEx baculovirus expression vectors. Recombinant HLA-A*0201 and β2m viruses were synthesized using the BacMagic-3 DNA/pIEx method and transfected into Spodoptera frugiperda (Sf9) cells, and protein expression was confirmed by Western blot. To prepare T cells for testing, PBMCs from a healthy HLA-A2+ donor were collected and pulsed with DMSO control or CEF peptide pool (a mixture of CMV-, EBV-, and Flu-specific HLA class I epitopes). After 5 days, the CD8+ and CD8- fractions were sorted by MACS-based magnetic separation, and the frequency of FluM1-specific lymphocytes in the CD8+ populations was determined (0.1% of DMSO control vs. 0.772% of CEF-pulsed cells) using a commercial tetramer. We are optimizing HLA-A*0201 and β2m baculovirus co-infection ratios and evaluating the efficiency of intracellular MHC folding.
ContributorsRoesler, Alexander Scott (Author) / Anderson, Karen (Thesis director) / Blattman, Joseph (Committee member) / School of Molecular Sciences (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Current studies in Multiple Myeloma suggest that patient tumors and cell lines cluster separately based on gene expression profiles. Hyperdiploid patients are also extremely underrepresented in established human myeloma cell lines (HMCLs). This suggests that the average HMCL model system does not accurately represent the average myeloma patient. To investigate this question we performed a combined CNA and SNV evolutionary comparison between four myeloma tumors and their established HMCLs (JMW-1, VP-6, KAS-6/1-KAS-6/2 and KP-6). We identified copy number changes shared between the tumors and their cell lines (mean of 74 events - 59%), those unique to patients (mean of 21.25 events - 17%), and those only in the cell lines (mean of 30.75 events \u2014 24%). A relapse sample from the JMW-1 patient showed 58% similarity to the primary diagnostic tumor. These data suggest that, on the level of copy number abnormalities, HMCLs show equal levels of evolutionary divergence as that observed within patients. By exome sequencing, patient tumors were 71% similar to their representative HMCLs, with ~12.5% and ~16.5% of SNVs unique to the tumors and HMCLs respectively. The HMCLs studied appear highly representative of the patient from which they were derived, with most differences associated with an enrichment of sub-populations present in the primary tumor. Additionally, our analysis of the KP-6 aCGH data showed that the patient's hyperdiploid karyotype was maintained in its respective HMCL. This discovery confirms the establishment and validation of a novel and potentially clinically relevant hyperdiploid HMCL that could provide a major advance in our ability to understand the pathogenesis and progression of this prominent patient population.
ContributorsBenard, Brooks Avery (Author) / Keats, Jonathan (Thesis director) / Anderson, Karen (Committee member) / Jelinek, Diane (Committee member) / School of Molecular Sciences (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Glycosaminoglycan (GAG) binding by the cytokine pleiotrophin (PTN) was examined by expressing both thrombospondin 1 type-1 repeat domains of PTN separately, as PTN-N and PTN-C. PTN-N contains residues 31-89, and PTN-C contains residues 90-146. Nuclear magnetic resonance (NMR) experiments were conducted on both PTN-N and PTN-C to elucidate GAG binding regions. Titration with heparin dp6 showed a twofold increase in affinity when expressing PTN-N and PTN-C separately rather than as intact PTN. Paramagnetic relaxation rate enhancement experiments and surface paramagnetic relaxation enhancement (PRE) perturbation experiments were used to determine which residues were involved in GAG binding. One binding site was observed in PTN-N, around residue T82, and two binding sites were observed in PTN-C, one around residue K93 and the other around residue G142. These observed binding sites agree with the binding sites already proposed by the Wang lab group and other studies. Future work on the subject could be done on confirming that other varieties and length GAGs bind at the same sites, as well as examining the effect longer GAG fragments have on the affinity of intact PTN versus separate domains.
ContributorsKuch, Nathaniel Jacob (Author) / Wang, Xu (Thesis director) / Van Horn, Wade (Committee member) / School of Molecular Sciences (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2015-12
Description
Collective decision making in social organism societies involves a large network of communication systems. Studying the processes behind the transmission of information allows for greater understanding of the decision making capabilities of a group. For Temnothorax rugatulus colonies, information is commonly spread in the form of tandem running, a linear recruitment pattern where a leading ant uses a short-ranged pheromone to direct a following ant to a target location (in tandem).The observed phenomenon of reverse tandem running (RTR), where a follower is lead from a target back to the home nest, has not been as extensively studied as forward tandem running and transportation recruitment activities. This study seeks to explain a potential reason for the presence of the RTR behavior; more specifically, the study explores the idea that reverse tandem run followers are being shown a specific route to the home nest by a highly experienced and efficient leading ant. Ten colonies had migrations induced experimentally in order to generate some reverse tandem running activity. Once an RTR has been observed, the follower and leader were studied for behavior and their pathways were analyzed. It was seen that while RTR paths were quite efficient (1.4x a straight line distance), followers did not experience a statistically significant improvement in their pathways between the home and target nests (based on total distance traveled) when compared to similar non-RTR ants. Further, RTR leading ants were no more efficient than other non-RTR ants. It was observed that some followers began recruiting after completion of an RTR, but the number than changed their behavior was not significant. Thus, the results of this experiment cannot conclusively show that RTR followers are utilizing reverse tandem runs to improve their routes between the home and target nests.
ContributorsColling, Blake David (Author) / Pratt, Stephen (Thesis director) / Liebig, Juergen (Committee member) / Sasaki, Takao (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2014-12
Description
Communication amongst eusocial insect is key to their success. Ants rely on signaling to mediate many different functions within a colony such as policing and nest mate recognition. Camponotus floridanus uses chemosensory signaling in the form of cuticular hydrocarbons to regulate these functions. Each cuticular hydrocarbon profile contains numerous hydrocarbons, however it is yet to be seen if Camponotus floridanus can discriminate between linear hydrocarbons of similar length. Individual specimens were conditioned in three different ways: 5 conditioning with high concentration of sugar water (1;1 ratio), 1 conditioning with high concentration of sugar water, and 5 conditioning with low concentration of sugar water (1;4). Two linear hydrocarbons were use, C23 and C24, with C23 always being the conditioned stimulus. Specimens who were conditioned 5 times with high concentration of sugar water were the only group to show a significant response to the conditioned stimulus with a p-value of .008 and exhibited discrimination behavior 46% of the time. When compared 5 conditioning with high concentration to the other two testing conditioning groups, 1 conditioning with high concentration produced an insignificant p-value of .13 was obtained whereas when comparing it with 5 conditioning low concentration of sugar a significant p-value of .0132 was obtained. This indiciates that Camponotus floridanus are capable of discrimination however must be conditioned with high concentration of sugar water, while number of conditioning is insignificant.
ContributorsDamari, Ben Aviv (Author) / Liebig, Juergen (Thesis director) / Ghaninia, Majid (Committee member) / Pratt, Stephen (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2014-05
Description
Cuticular hydrocarbons (CHCs) play a crucial role in social insect recognition systems. In this study we investigated mate choice in the red harvester ant, Pogonomyrmex barbatus. In Phoenix, this species has two lineages, J1 and J2, which look identical, but are genetically isolated. In the genetic caste determination (GCD) system workers and queens are determined by their genotype (i.e., workers develop from interlineage crosses, queens from intralineage crosses). As such, J1 and J2 lineages are dependent on each other in order for colonies to produce both workers and reproductive queens. Given their genetic isolation and interdependence, we hypothesized that the CHCs of alate males and queens are affected by lineage, and that differences in the CHC profile are used for mate recognition. We tested these hypotheses by analyzing the lineage distributions of actively mating pairs (n=65), and compared them with the overall distribution of male and female sexuals (n=180). We additionally analyzed the five most abundant CHC compounds for 20 of the actively mating P. barbatus alate male and queen pairs to determine how variable the two lineages are between each sex. We found that mating pair distributions did not significantly differ from those expected under a random mating system (�2= 1.4349, P= 0.6973), however, CHC profiles did differ between J1 and J2 lineages and sexes for the five most abundant CHC compounds. Our results show that random mating is taking place in this population, however given the differences observed in CHC profiles, mate recognition could be taking place.
ContributorsTula Del Moral Testai, Pedro Rafael (Co-author) / Cash, Elizabeth (Co-author) / Gadau, Juergen (Thesis director) / Liebig, Juergen (Committee member) / Barrett, The Honors College (Contributor) / School of International Letters and Cultures (Contributor) / School of Life Sciences (Contributor)
Created2014-05
Description
Evolutionary theory predicts that animal behavior is generally governed by decision rules (heuristics) which adhere to ecological rationality: the tendency to make decisions that maximize fitness in most situations the animal encounters. However, the particular heuristics used by ant colonies of the genus Temnothorax and their propensity towards ecological rationality are up for debate. These ants are adept at choosing a nest site, making a collective decision based on complex interactions between the many individual choices made by workers. Colonies will migrate between nests either upon the destruction of their current home or the discovery of a sufficiently superior nest. This study offers a descriptive analysis of the heuristics potentially used in nest-site decision-making. Colonies were offered a choice of nests characterized by the Ebbinghaus Illusion: a perceptual illusion which effectively causes the viewer to perceive a circle as larger when it is surrounded by small circles than when that same circle is surrounded by large circles. Colonies were separated into two conditions: in one, they were given the option to move to a high-quality nest surrounded by poor-quality nests, and in the other they were given the option to move to a high-quality nest surrounded by medium-quality nests. The colonies in the poor condition were found to be more likely to move to the good nest than were colonies in the medium condition at a statistically significant level. That is, they responded to the Ebbinghaus Effect in the way that is normally expected. This result was discussed in terms of its implications for the ecological rationality of the nest-site choice behavior of these ants.
ContributorsTalken, Lucas Warren (Author) / Pratt, Stephen (Thesis director) / Sasaki, Takao (Committee member) / Liebig, Juergen (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Department of Psychology (Contributor) / Economics Program in CLAS (Contributor)
Created2014-05
Description
About 1% of the United States adult population currently suffers from schizophrenia. The symptoms of schizophrenia can be broken down into three main categories including: positive symptoms such as psychoses, negative symptoms such as anhedonia, and cognitive symptoms such as memory difficulties. The early growth response 3 (Egr3) is part of a family of genes known as the immediate early genes (IEGs), which are zinc-finger transcription factors. IEGs are not protein synthesis dependent, which means that they can be activated quickly, within 30-45 minutes, in response to certain environmental stimuli such as sleep deprivation. Egr3, an activity dependent gene, may be up-regulated by both genetic and environmental cues. Egr3 is thought to play an integral role in a biochemical pathway that may explain the onset of schizophrenia. However, the exact causes of schizophrenia remain unknown. Egr3 is not only activated in response to environmental factors, but has also been linked to many genes that are associated with schizophrenia in humans (Huentelman et al., 2015). Post-mortem brain tissue studies of patients with schizophrenia have decreased levels of EGR3 in their prefrontal cortex (PFC) and mice lacking Egr3 (Egr3 -/-) exhibit schizophrenia-like phenotypes such as locomotor hyperactivity. Egr 3 -/- mice also exhibit a diminished head twitch response to 2,5-Dimethoxy-4-iodoamphetamine (DOI), a 5-HT2A agonist (Yamada, et al., 2007; Gallitano-Mendel, et al., 2008). A link was established between schizophrenia patients and the serotonin 2A receptor (5-HT2AR) upon recognizing that 5-HT2AR agonists like lysergic acid diethylamide (LSD) create hallucinations similar to those in schizophrenic patients and 5-HT2AR antagonists such as the second-generation antipsychotic clozapine can reverse those hallucinations (Sommer, 2012). Paradoxically, however, post-mortem studies of schizophrenia patients have actually shown a decrease in PFC 5-HT2ARs as well as a 70% decrease found in the PFC of Egr3 -/- mice (Rasmussen, et al., 2010; Williams, et al., 2012). Therefore, we hypothesize that EGR3 directly regulates expression of 5-HT2ARs. To test this we will use virus-mediated overexpression of 5-HT2ARs in the PFCs of mice to see if we can rescue the schizophrenia-like phenotypes of the Egr3 -/- mice. After bilateral PFC stereotaxic injection of herpes simplex virus (HSV) with enhanced green fluorescent protein (EGFP) or HSV-Htr2a-EGFP in both wild type (WT) and Egr3 -/- mice, the mice were behaviorally tested using locomotor activity and DOI-induced head twitch response. We found that Egr3-/- mice, compared to WT mice, demonstrated locomotor hyperactivity and a decreased DOI-induced head twitch response, confirming prior findings, but no significant main effect of virus. A significant effect of the HSV-Htr2a-EGFP was seen when comparing DOI-induced head twitch response in WT mice to Egr3 -/- mice. WT mice showed a higher number of head twitches in comparison to the knockout mice. These findings suggest further research must be conducted in order to investigate whether a functional 5-HT2AR is being translated and correctly transported to the membrane. These findings may also point to an unknown factor mediating the regulation between Egr3 and 5-HT2ARs.
ContributorsHoebee, Shelby Marie (Author) / Van Horn, Wade (Thesis director) / Gallitano, Amelia (Committee member) / Department of Psychology (Contributor) / School of Molecular Sciences (Contributor) / School of Criminology and Criminal Justice (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Living a healthy and balanced life can be difficult for college students at Arizona State<br/>University due to the barrier of high cost of healthy food. To understand more about this problem<br/>we conducted research on the Tempe campus through surveys and virtual focus groups. We<br/>discovered that the cost of healthy food is one of the main barriers preventing students from<br/>eating healthy. We also learned that the students would be more willing to eat healthier if they<br/>could access healthy foods at a more reasonable price. Our solution to this problem was Eunoia,<br/>a service that allows students to receive discounts on healthy food and incentivize them to eat<br/>healthier in the future. Our company creates an innovative relationship between our customers,<br/>their private health insurance companies and local grocery stores throughout the Phoenix Metro<br/>area. Students at Arizona State University will be able to purchase healthy food items discounted<br/>by up to 30%. These discounts will be funded by their health insurance companies as well as the<br/>local grocery stores they purchase from. Our business model allows our customers to live<br/>healthier lives while also providing value to partnered health insurance companies and grocery<br/>stores. Once we established our business model, we spoke with students at Arizona State<br/>University and representatives from health insurance companies. Through these demographics,<br/>we received positive feedback and early traction with our idea. Our goal is to be able to<br/>implement our product in the Arizona State University community and then expand this product<br/>into a more general market to help all people live a pure and balanced life.
ContributorsEvans, Hayden Curtis (Co-author) / Wijesinghe, Megan (Co-author) / Zimprich, Preston (Co-author) / Byrne, Jared (Thesis director) / Hall, Rick (Committee member) / School of Molecular Sciences (Contributor) / Dean, W.P. Carey School of Business (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05