Matching Items (10)
Filtering by
- Creators: Barrett, The Honors College
- Creators: Kusumi, Kenro
Description
Vitellogenin (vg) is a precursor protein of egg yolk in honeybees, but it is also known to have immunological functions. The purpose of this experiment was to determine the effect of vg on the viral load of deformed wing virus (DWV) in worker honey bees (Apis mellifera). I hypothesized that a reduction in vg expression would lead to an increase in the viral load. I collected 180 worker bees and split them into four groups: half the bees were subjected to a vg gene knockdown by injections of double stranded vg RNA, and the rest were injected with green fluorescent protein (gfp) double stranded RNA. Half of each group was thereafter injected with DWV, and half given a sham injection. The rate of mortality in all four groups was higher than expected, leaving only 17 bees total. I dissected these bees' fat bodies and extracted their RNA to test for vg and DWV. PCR results showed that, out of the small group of remaining bees, the levels of vg were not statistically different. Furthermore, both groups of virus-injected bees showed similar viral loads. Because of the high mortality rate bees and the lack of differing levels of vg transcript between experimental and control groups, I could not draw conclusions from these results. The high mortality could be caused by several factors: temperature-induced stress, repeated stress from the two injections, and stress from viral infection. In addition, it is possible that the vg dsRNA batch I used was faulty. This thesis exemplifies that information cannot safely be extracted when loss of sampling units result in a small datasets that do not represent the original sampling population.
ContributorsCrable, Emma Lewis (Author) / Amdam, Gro (Thesis director) / Wang, Ying (Committee member) / Dahan, Romain (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-12
Description
The development of skeletal muscle during embryogenesis and repair in adults is dependent on the intricate balance between the proliferation of myogenic progenitor cells and the differentiation of those cells into functional muscle fibers. Recent studies demonstrate that the Drosophila melanogaster transcription factor CG9650 is expressed in muscle progenitor cells, where it maintains myoblast numbers. We are interested in the Mus musculus orthologs Bcl11a and Bcl11b (C2H2 zinc finger transcription factors), and understanding their role as molecular switches that control proliferation/differentiation decisions in muscle progenitor cells. Expression analysis revealed that Bcl11b, but not Bcl11a, is expressed in the region of the mouse embryo populated with myogenic progenitor cells; gene expression studies in muscle cell culture confirmed Bcl11b is also selectively transcribed in muscle. Furthermore, Bcl11b is down-regulated with differentiation, which is consistent with the belief that the gene plays a role in cell proliferation.
ContributorsDuong, Brittany Bach (Author) / Rawls, Alan (Thesis director) / Wilson-Rawls, Jeanne (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor) / School of Life Sciences (Contributor)
Created2014-05
Description
A major goal of the Center for Biosignatures Discovery Automation (CBDA) is to design a diagnostic tool that detects novel cancer biosignatures at the single-cell level. We designed the Single-cell QUantitative In situ Reverse Transcription-Polymerase Chain Reaction (SQUIRT-PCR) system by combining a two-photon laser lysis (2PLL) system with a microfluidic reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) platform. It is important to identify early molecular changes from intact tissues as prognosis for premalignant conditions and develop new molecular targets for prevention of cancer progression and improved therapies. This project analyzes RNA expression at the single-cell level and presents itself with two major challenges: (1) detecting low levels of RNA and (2) minimizing RNA absorption in the polydimethylsiloxane (PDMS) microfluidic channel. The first challenge was overcome by successfully detecting picogram (pg) levels of RNA using the Fluidigm (FD) BioMark™ HD System (Fluidigm Corporation, South San Francisco, CA) for RT-qPCR analysis. This technology incorporates a highly precise integrated fluidic circuit (IFC) that allows for high-throughput genetic screening using microarrays. The second challenge entailed collecting data from RNA flow-through samples that were passed through microfluidic channels. One channel was treated with a coating of polyethylene glycol (PEG) and the other remained untreated. Various flow-through samples were subjected to RT-qPCR and analyzed using the FD FLEXsix™ Gene Expression IFC. As predicted, the results showed that the treated PDMS channel absorbed less RNA than the untreated PDMS channel. Once the optimization of the PDMS microfluidic platform is complete, it will be implemented into the 2PLL system. This novel technology will be able to identify cell populations in situ and could have a large impact on cancer diagnostics.
ContributorsBlatt, Amy Elissa (Author) / Meldrum, Deirdre R. (Thesis director) / Tran, Thai (Committee member) / Chao, Joseph (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor)
Created2014-05
Description
Objective: Isoforms of insulin-like growth factor-1 (IGF-1) gene encodes different IGF-1 isoforms by alternative splicing, and which are known to play distinct roles in muscle growth and repair. These isoforms in humans exist as IGF-1Ea, IGF-1Eb and IGF-1Ec (the latter is also known as mechano-growth factor). We sought to determine whether mRNA expression of any of these isoforms is impaired in skeletal muscle of humans with obesity, and given that humans with obesity display reduced protein synthesis in muscle. Methods: We studied 10 subjects (3 females/7 males) with obesity (body mass index: 34 ± 1 kg/m2) and 14 subjects (6 females/8 males) that were lean (body mass index: 24 ± 1 kg/m2) and served as controls. The groups represented typical populations of individuals that differed (P < 0.05) in body fat (obese: 32 ± 2; lean: 22 ± 2) and insulin sensitivity (Matsuda insulin sensitivity index, obese: 5 ± 1; lean 11 ± 2). Total RNA was extracted from 20-30 mg of tissue from muscle biopsies performed after an overnight fast. Isolated RNA was used to perform cDNA synthesis. Real-time PCR was performed using predesigned TaqMan® gene expression assays (Thermo Fisher Scientific Inc) for IGF-1Ea (assay ID: Hs01547657_m1), IGF-1Eb (assay ID: Hs00153126_m1) and IGF-1Ec (assay ID: Hs03986524_m1), as well as ACTB (assay ID: Hs01060665_g1), which was used to adjust the IGF-1 isoform mRNA expression. Responses for mRNA expression were calculated using the comparative CT method (2-ΔΔCT). Results: IGF-1Eb mRNA expression was lower in the subjects with obesity compared to the lean controls (0.67 ± 0.09 vs 1.00 ± 0.13; P < 0.05) but that of IGF-1Ea (0.99 ± 0.16 vs 1.00 ± 0.33) or IGF-1Ec (0.83 ± 0.14 vs 1.00 ± 0.21) were not different between groups (P > 0.05). Conclusions: Among the IGF-1 mRNA isoforms, IGF-1Eb mRNA is uniquely decreased in humans with obesity. Lower IGF-1Eb mRNA expression in muscle of humans with obesity may explain the lower protein synthesis observed in these individuals. Furthermore, these findings may have direct implications for muscle growth and repair in humans with obesity.
ContributorsSon, John Lee (Author) / Katsanos, Christos (Thesis director) / Gu, Haiwei (Committee member) / College of Health Solutions (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Agassiz’s desert tortoise (Gopherus agassizii) is a long-lived species native to the Mojave Desert and is listed as threatened under the US Endangered Species Act. To aid conservation efforts for preserving the genetic diversity of this species, we generated a whole genome reference sequence with an annotation based on deep transcriptome sequences of adult skeletal muscle, lung, brain, and blood. The draft genome assembly for G. agassizii has a scaffold N50 length of 252 kbp and a total length of 2.4 Gbp. Genome annotation reveals 20,172 protein-coding genes in the G. agassizii assembly, and that gene structure is more similar to chicken than other turtles. We provide a series of comparative analyses demonstrating (1) that turtles are among the slowest-evolving genome-enabled reptiles, (2) amino acid changes in genes controlling desert tortoise traits such as shell development, longevity and osmoregulation, and (3) fixed variants across the Gopherus species complex in genes related to desert adaptations, including circadian rhythm and innate immune response. This G. agassizii genome reference and annotation is the first such resource for any tortoise, and will serve as a foundation for future analysis of the genetic basis of adaptations to the desert environment, allow for investigation into genomic factors affecting tortoise health, disease and longevity, and serve as a valuable resource for additional studies in this species complex.
Data Availability: All genomic and transcriptomic sequence files are available from the NIH-NCBI BioProject database (accession numbers PRJNA352725, PRJNA352726, and PRJNA281763). All genome assembly, transcriptome assembly, predicted protein, transcript, genome annotation, repeatmasker, phylogenetic trees, .vcf and GO enrichment files are available on Harvard Dataverse (doi:10.7910/DVN/EH2S9K).
ContributorsTollis, Marc (Author) / DeNardo, Dale F (Author) / Cornelius, John A (Author) / Dolby, Greer A (Author) / Edwards, Taylor (Author) / Henen, Brian T. (Author) / Karl, Alice E. (Author) / Murphy, Robert W. (Author) / Kusumi, Kenro (Author)
Created2017-05-31
Description
It is well documented that menopause and the related decline in circulatory steroid hormones estrogen and progesterone are associated with memory alterations. Rodent models of surgical menopause can be used to study these effects, including ovariectomy (Ovx), or the surgical removal of the ovaries. This thesis aimed to characterize the effects of surgical menopause on spatial working and reference memory in rats and examine profiles of uterine gene expression alterations that may serve as indications of mechanisms underlying this association. Eighteen female rats were randomly assigned to one of two surgical treatment groups, either Ovx (the surgical menopause group) or sham (the control group). All subjects underwent testing on the water version of the radial arm maze (WRAM) which allows for the assessment of reference memory errors and two types of working memory errors. After behavioral testing, rat uterine tissues were dissected and RNA sequenced. The results showed that Ovx impaired spatial reference memory performance during a maze learning phase, with Ovx rats making reference memory failures earlier in the day, even before working memory load increased, as compared to control rats. There were no surgical menopause effects on spatial working memory, which may be due to the low working memory load and the young age of the rats. Post-hoc analyses showed that reference memory performance was correlated with nerve growth factor (NGF) and acetylcholinesterase (AChE) gene expression in uterine tissues. These findings add to the literature on the impact of estrogen and female cyclicity on memory and cognition. The results suggest that Ovx impairment of the ability to learn long-term spatial memory information relates to uterine gene expression underlying cellular functioning and that NGF and AChE genes are involved in pathways that give way to underlying cellular functioning that impacts cognition. Future studies should continue to evaluate the effects of menopause on memory function and the effectiveness of hormone therapy.
ContributorsOyen, Emma (Author) / Bimonte-Nelson, Heather (Thesis director) / Corbin, William (Committee member) / Wilson, Melissa (Committee member) / Lizik, Camryn (Committee member) / Barrett, The Honors College (Contributor) / Department of Psychology (Contributor)
Created2024-05
Description
Rapid advancements in genomic technologies have increased our understanding of rare human disease. Generation of multiple types of biological data including genetic variation from genome or exome, expression from transcriptome, methylation patterns from epigenome, protein complexity from proteome and metabolite information from metabolome is feasible. "Omics" tools provide comprehensive view into biological mechanisms that impact disease trait and risk. In spite of available data types and ability to collect them simultaneously from patients, researchers still rely on their independent analysis. Combining information from multiple biological data can reduce missing information, increase confidence in single data findings, and provide a more complete view of genotype-phenotype correlations. Although rare disease genetics has been greatly improved by exome sequencing, a substantial portion of clinical patients remain undiagnosed. Multiple frameworks for integrative analysis of genomic and transcriptomic data are presented with focus on identifying functional genetic variations in patients with undiagnosed, rare childhood conditions. Direct quantitation of X inactivation ratio was developed from genomic and transcriptomic data using allele specific expression and segregation analysis to determine magnitude and inheritance mode of X inactivation. This approach was applied in two families revealing non-random X inactivation in female patients. Expression based analysis of X inactivation showed high correlation with standard clinical assay. These findings improved understanding of molecular mechanisms underlying X-linked disorders. In addition multivariate outlier analysis of gene and exon level data from RNA-seq using Mahalanobis distance, and its integration of distance scores with genomic data found genotype-phenotype correlations in variant prioritization process in 25 families. Mahalanobis distance scores revealed variants with large transcriptional impact in patients. In this dataset, frameshift variants were more likely result in outlier expression signatures than other types of functional variants. Integration of outlier estimates with genetic variants corroborated previously identified, presumed causal variants and highlighted new candidate in previously un-diagnosed case. Integrative genomic approaches in easily attainable tissue will facilitate the search for biomarkers that impact disease trait, uncover pharmacogenomics targets, provide novel insight into molecular underpinnings of un-characterized conditions, and help improve analytical approaches that use large datasets.
ContributorsSzelinger, Szabolcs (Author) / Craig, David W. (Thesis advisor) / Kusumi, Kenro (Thesis advisor) / Narayan, Vinodh (Committee member) / Rosenberg, Michael S. (Committee member) / Huentelman, Matthew J (Committee member) / Arizona State University (Publisher)
Created2015
Description
In the U.S., breast cancer (BC) incidences among African American (AA) and CA (CA) women are similar, yet AA women have a significantly higher mortality rate. In addition, AA women often present with tumors at a younger age, with a higher tumor grade/stage and are more likely to be diagnosed with the highly aggressive triple-negative breast cancer (TNBC) subtype. Even within the TNBC subtype, AA women have a worse clinical outcome compared to CA. Although multiple socio-economic and lifestyle factors may contribute to these observed health disparities, it is essential that the underlying biological differences between CA and AA TNBC are identified. In this study, gene expression profiling was performed on archived FFPE samples, obtained from CA and AA women diagnosed with early stage TNBC. Initial analysis revealed a pattern of differential expression in the AA cohort compared to CA. Further molecular characterization results showed that the AA cohort segregated into 3-TNBC molecular subtypes; Basal-like (BL2), Immunomodulatory (IM) and Mesenchymal (M). Gene expression analyses resulted in 190 differentially expressed genes between the AA and CA cohorts. Pathway enrichment analysis demonstrated that differentially expressed genes were over-represented in cytoskeletal remodeling, cell adhesion, tight junctions, and immune response in the AA TNBC -cohort. Furthermore, genes in the Wnt/β-catenin pathway were over-expressed. These results were validated using RT-qPCR on an independent cohort of FFPE samples from AA and CA women with early stage TNBC, and identified Caveolin-1 (CAV1) as being significantly expressed in the AA-TNBC cohort. Furthermore, CAV1 was shown to be highly expressed in a cell line panel of TNBC, in particular, those of the mesenchymal and basal-like molecular subtype. Finally, silencing of CAV1 expression by siRNA resulted in a significant decrease in proliferation in each of the TNBC cell lines. These observations suggest that CAV1 expression may contribute to the more aggressive phenotype observed in AA women diagnosed with TNBC.
ContributorsGetz, Julie (Author) / Baumbach-Reardon, Lisa L (Thesis advisor) / Lake, Douglas F (Thesis advisor) / Bussey, Kimberly (Committee member) / Kusumi, Kenro (Committee member) / Arizona State University (Publisher)
Created2015
Description
This research project investigated known and novel differential genetic variants and their associated molecular pathways involved in Type II diabetes mellitus for the purpose of improving diagnosis and treatment methods. The goal of this investigation was to 1) identify the genetic variants and SNPs in Type II diabetes to develop a gene regulatory pathway, and 2) utilize this pathway to determine suitable drug therapeutics for prevention and treatment. Using a Gene Set Enrichment Analysis (GSEA), a set of 1000 gene identifiers from a Mayo Clinic database was analyzed to determine the most significant genetic variants related to insulin signaling pathways involved in Type II Diabetes. The following genes were identified: NRAS, KRAS, PIK3CA, PDE3B, TSC1, AKT3, SOS1, NEU1, PRKAA2, AMPK, and ACC. In an extensive literature review and cross-analysis with Kegg and Reactome pathway databases, novel SNPs located on these gene variants were identified and used to determine suitable drug therapeutics for treatment. Overall, understanding how genetic mutations affect target gene function related to Type II Diabetes disease pathology is crucial to the development of effective diagnosis and treatment. This project provides new insight into the molecular basis of the Type II Diabetes, serving to help untangle the regulatory complexity of the disease and aid in the advancement of diagnosis and treatment. Keywords: Type II Diabetes mellitus, Gene Set Enrichment Analysis, genetic variants, KEGG Insulin Pathway, gene-regulatory pathway
ContributorsBucklin, Lindsay (Co-author) / Davis, Vanessa (Co-author) / Holechek, Susan (Thesis director) / Wang, Junwen (Committee member) / Nyarige, Verah (Committee member) / School of Human Evolution & Social Change (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Inhibitor of growth factor 4 (ING4) is a tumor suppressor of which low expression has been associated with poor patient survival and aggressive tumor progression in breast cancer. ING4 is characterized as a transcription regulator of inflammatory genes. Among the ING4-regulated genes is CXCL10, a chemokine secreted by endothelial cells during normal inflammation response, which induces chemotactic migration of immune cells to the site. High expression of CXCL10 has been implicated in aggressive breast cancer, but the mechanism is not well understood. A potential signaling molecule downstream of Cxcl10 is Janus Kinase 2 (Jak2), a kinase activated in normal immune response. Deregulation of Jak2 is associated with metastasis, immune evasion, and tumor progression in breast cancer. Thus, we hypothesized that the Ing4/Cxcl10/Jak2 axis plays a key role in breast cancer progression. We first investigated whether Cxcl10 affected breast cancer cell migration. We also investigated whether Cxcl10-mediated migration is dependent on ING4 expression levels. We utilized genetically engineered MDAmb231 breast cancer cells with a CRISPR/Cas9 ING4-knockout construct or a viral ING4 overexpression construct. We performed Western blot analysis to confirm Ing4 expression. Cell migration was assessed using Boyden Chamber assay with or without exogenous Cxcl10 treatment. The results showed that in the presence of Cxcl10, ING4-deficient cells had a two-fold increase in migration as compared to the vector controls, suggesting Ing4 inhibits Cxcl10-induced migration. These findings support our hypothesis that ING4-deficient tumor cells have increased migration when Cxcl10 signaling is present in breast cancer. These results implicate Ing4 is a key regulator of a chemokine-induced tumor migration. Our future plan includes evaluation of Jak2 as an intermediate signaling molecule in Cxcl10/Ing4 pathway. Therapeutic implications of these findings are targeting Cxcl10 and/or Jak2 may be effective in treating ING4-deficient aggressive breast cancer.
ContributorsArnold, Emily (Author) / Kim, Suwon (Thesis director) / Blattman, Joseph (Thesis director) / Mason, Hugh (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05