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- All Subjects: Molecular Biology
- Creators: Kusumi, Kenro
- Status: Published
Description
The majority of chronic myeloid leukemia (CML) and some of acute lymphocytic leukemia (ALL) cases are associated with possessing the BCR-Abl fusion protein from an oncogenic translocation, resulting in a constantly active form of Abl and rapid proliferation. CML and ALL cells that possess the BCR-Abl fusion protein are known as Philadelphia chromosome positive (Ph+). Currently, Imatinib (selective Abl inhibitor) is used as therapy against CML and ALL. However, some patients may have malignancies which show resistance to Imatinib. Previous work displays that the transformation of progenitor B cells with the v-Abl oncogene of Abelson murine leukemia virus results in cell cycle progression, rapid proliferation, and potentially malignant transformation while preventing any further differentiation. Progenitor B cells transformed with the temperature-sensitive form of the v-Abl oncogene have served as a model to study cellular response to Imatinib treatment. After some manipulation, very few cells were forced to progress to malignancy, forming tumor in vivo. These cells were no long sensitive to v-Abl inactivation, resembling the Imatinib resistant ALL. Autophagy is the process by which proteins and organelles are broken-down and recycled within the eukaryotic cell and has been hypothesized to play a part in cancer cell survival and drug-resistance. LC3 processing is a widely accepted marker of autophagy induction and progression. It has also been shown that Imatinib treatment of Ph+ leukemia can induce autophagy. In this study, we examined the autophagy induction in response to v-Abl inactivation in a Ph+-B-ALL cell model that shows resistance to Imatinib. In particular, we wonder whether the tumor cell line resistant to v-Abl inactivation may acquire a high level of autophagy to become resistant to apoptosis induced by v-Abl inactivation, and thus become addicted to autophagy. Indeed, this tumor cell line displays a high basal levels of LC3 I and II expression, regardless of v-Abl activity. We further demonstrated that inhibition of the autophagy pathway enhances the tumor line's sensitivity to Imatinib, resulting in cell cycle arrest and massive apoptosis. The combination of autophagy and Abl inhibitions may serve as an effective therapy for BCR-Abl positive CML.
ContributorsArkus, Nohea (Author) / Chang, Yung (Thesis advisor) / Kusumi, Kenro (Committee member) / Lake, Douglas (Committee member) / Jacobs, Bertram (Committee member) / Arizona State University (Publisher)
Created2011
Description
Well-established model systems exist in four out of the seven major classes of vertebrates. These include the mouse, chicken, frog and zebrafish. Noticeably missing from this list is a reptilian model organism for comparative studies between the vertebrates and for studies of biological processes unique to reptiles. To help fill in this gap the green anole lizard, Anolis carolinensis, is being adapted as a model organism. Despite the recent release of the complete genomic sequence of the A. carolinensis, the lizard lacks some resources to aid researchers in their studies. Particularly, the lack of transcriptomic resources for lizard has made it difficult to identify genes complete with alternative splice forms and untranslated regions (UTRs). As part of this work the genome annotation for A. carolinensis was improved through next generation sequencing and assembly of the transcriptomes from 14 different adult and embryonic tissues. This revised annotation of the lizard will improve comparative studies between vertebrates, as well as studies within A. carolinensis itself, by providing more accurate gene models, which provide the bases for molecular studies. To demonstrate the utility of the improved annotations and reptilian model organism, the developmental process of somitogenesis in the lizard was analyzed and compared with other vertebrates. This study identified several key features both divergent and convergent between the vertebrates, which was not previously known before analysis of a reptilian model organism. The improved genome annotations have also allowed for molecular studies of tail regeneration in the lizard. With the annotation of 3' UTR sequences and next generation sequencing, it is now possible to do expressional studies of miRNA and predict their mRNA target transcripts at genomic scale. Through next generation small RNA sequencing and subsequent analysis, several differentially expressed miRNAs were identified in the regenerating tail, suggesting miRNA may play a key role in regulating this process in lizards. Through miRNA target prediction several key biological pathways were identified as potentially under the regulation of miRNAs during tail regeneration. In total, this work has both helped advance A. carolinensis as model system and displayed the utility of a reptilian model system.
ContributorsEckalbar, Walter L (Author) / Kusumi, Kenro (Thesis advisor) / Huentelman, Matthew (Committee member) / Rawls, Jeffery (Committee member) / Wilson-Rawls, Norma (Committee member) / Arizona State University (Publisher)
Created2012
Description
Induced pluripotent stem cells (iPSCs) are an intriguing approach for neurological disease modeling, because neural lineage-specific cell types that retain the donors' complex genetics can be established in vitro. The statistical power of these iPSC-based models, however, is dependent on accurate diagnoses of the somatic cell donors; unfortunately, many neurodegenerative diseases are commonly misdiagnosed in live human subjects. Postmortem histopathological examination of a donor's brain, combined with premortem clinical criteria, is often the most robust approach to correctly classify an individual as a disease-specific case or unaffected control. We describe the establishment of primary dermal fibroblasts cells lines from 28 autopsy donors. These fibroblasts were used to examine the proliferative effects of establishment protocol, tissue amount, biopsy site, and donor age. As proof-of-principle, iPSCs were generated from fibroblasts from a 75-year-old male, whole body donor, defined as an unaffected neurological control by both clinical and histopathological criteria. To our knowledge, this is the first study describing autopsy donor-derived somatic cells being used for iPSC generation and subsequent neural differentiation. This unique approach also enables us to compare iPSC-derived cell cultures to endogenous tissues from the same donor. We utilized RNA sequencing (RNA-Seq) to evaluate the transcriptional progression of in vitro-differentiated neural cells (over a timecourse of 0, 35, 70, 105 and 140 days), and compared this with donor-identical temporal lobe tissue. We observed in vitro progression towards the reference brain tissue, supported by (i) a significant increasing monotonic correlation between the days of our timecourse and the number of actively transcribed protein-coding genes and long intergenic non-coding RNAs (lincRNAs) (P < 0.05), consistent with the transcriptional complexity of the brain, (ii) an increase in CpG methylation after neural differentiation that resembled the epigenomic signature of the endogenous tissue, and (iii) a significant decreasing monotonic correlation between the days of our timecourse and the percent of in vitro to brain-tissue differences (P < 0.05) for tissue-specific protein-coding genes and all putative lincRNAs. These studies support the utility of autopsy donors' somatic cells for iPSC-based neurological disease models, and provide evidence that in vitro neural differentiation can result in physiologically progression.
ContributorsHjelm, Brooke E (Author) / Craig, David W. (Thesis advisor) / Wilson-Rawls, Norma J. (Thesis advisor) / Huentelman, Matthew J. (Committee member) / Mason, Hugh S. (Committee member) / Kusumi, Kenro (Committee member) / Arizona State University (Publisher)
Created2013
Description
Postnatal skeletal muscle repair is dependent on the tight regulation of an adult stem cell population known as satellite cells. In response to injury, these quiescent cells are activated, proliferate and express skeletal muscle-specific genes. The majority of satellite cells will fuse to damaged fibers or form new muscle fibers, while a subset will return to a quiescent state, where they are available for future rounds of repair. Robust muscle repair is dependent on the signals that regulate the mutually exclusive decisions of differentiation and self-renewal. A likely candidate for regulating this process is NUMB, an inhibitor of Notch signaling pathway that has been shown to asymmetrically localize in daughter cells undergoing cell fate decisions. In order to study the role of this protein in muscle repair, an inducible knockout of Numb was made in mice. Numb deficient muscle had a defective repair response to acute induced damage as characterized by smaller myofibers, increased collagen deposition and infiltration of fibrotic cells. Satellite cells isolated from Numb-deficient mice show decreased proliferation rates. Subsequent analyses of gene expression demonstrated that these cells had an aberrantly up-regulated Myostatin (Mstn), an inhibitor of myoblast proliferation. Further, this defect could be rescued with Mstn specific siRNAs. These data indicate that NUMB is necessary for postnatal muscle repair and early proliferative expansion of satellite cells. We used an evolutionary compatible to examine processes controlling satellite cell fate decisions, primary satellite cell lines were generated from Anolis carolinensis. This green anole lizard is evolutionarily the closet animal to mammals that forms de novo muscle tissue while undergoing tail regeneration. The mechanism of regeneration in anoles and the sources of stem cells for skeletal muscle, cartilage and nerves are poorly understood. Thus, satellite cells were isolated from A. carolinensis and analyzed for their plasticity. Anole satellite cells show increased plasticity as compared to mouse as determined by expression of key markers specific for bone and cartilage without administration of exogenous morphogens. These novel data suggest that satellite cells might contribute to more than muscle in tail regeneration of A. carolinensis.
ContributorsGeorge, Rajani M (Author) / Wilson-Rawls, Jeanne (Thesis advisor) / Rawls, Alan (Committee member) / Whitfield, Kerr (Committee member) / Kusumi, Kenro (Committee member) / Arizona State University (Publisher)
Created2012
Description
New genomic resources allow for the investigation of gene family diversity in genome-enabled reptiles. The Toll-like Receptor (TLR) gene family recognizes pathogen-associated molecular patterns (PAMPs) and coevolves with environmental pathogens which makes it a strong candidate for looking at the interplay between gene family diversification and host-pathogen coevolution. Using a new orthology curation pipeline and phylogenetic reconstruction, a novel gene expansion event of TLR8 was identified to be exclusive to crocodilians and chelonians with species-specific pseudogenization events. A new gene, TLR21-like, was identified as a part of the TLR11 subfamily. These findings uncover reptile-specific gene family evolution and provide indications of the role of habitat in this process.
ContributorsMorales, Matheo (Author) / Kusumi, Kenro (Thesis director) / Dolby, Greer (Committee member) / Scott, Peter (Committee member) / School of Mathematical and Statistical Sciences (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Metabolomics focuses on the study of metabolic changes occurring in varioussystems and utilizes quantitative and semi-quantitative measurements of multiple metabolites in parallel. Mass spectrometry (MS) is the most ubiquitous platform in this field, as it provides superior sensitivity regarding measurements of complex metabolic profiles in biological systems. When combined with MS, multivariate statistics and advanced machine learning algorithms provide myriad opportunities for bioinformatics insights beyond simple univariate data comparisons. In this dissertation, the application of MS-based metabolomics is introduced with an emphasis on biomarker discovery for human disease detection. To advance disease diagnosis using MS-based metabolomics, numerous statistical techniques have been implemented in this research including principal component analysis, factor analysis, partial least squares-discriminant analysis (PLS-DA), orthogonal PLS-DA, random forest, receiver operating characteristic analysis, as well as functional pathway/enzyme enrichment analyses. These approaches are highly useful for improving classification sensitivity and specificity related to disease-induced biological variation and can help identify useful biomarkers and potential therapeutic targets. It is also shown that MS-based metabolomics can distinguish between clinical and prodromal disease as well as similar diseases with related symptoms, which may assist in clinical staging and differential diagnosis, respectively. Additionally, MS-based metabolomics is shown to be promising for the early and accurate detection of diseases, thereby improving patient outcomes, and advancing clinical care. Herein, the application of MS methods and chemometric statistics to the diagnosis of breast cancer, coccidioidomycosis (Valley fever), and senile dementia (Alzheimer's disease) are presented and discussed. In addition to presenting original research, previous efforts in biomarker discovery will be synthesized and appraised. A Comment will be offered regarding the state of the science, specifically addressing the inefficient model of repetitive biomarker discovery and the need for increased translational efforts necessary to consolidate metabolomics findings and formalize purported metabolic markers as laboratory developed tests. Various factors impeding the translational throughput of metabolomics findings will be carefully considered with respect to study design, statistical analysis, and regulation of biomedical diagnostics. Importantly, this dissertation will offer critical insights to advance metabolomics from a scientific field to a practical one including targeted detection, enhanced quantitation, and direct-to-consumer considerations.
ContributorsJasbi, Paniz (Author) / Johnston, Carol S (Thesis advisor) / Gu, Haiwei (Thesis advisor) / Lake, Douglas F (Committee member) / Sweazea, Karen (Committee member) / Tasevska, Natasha (Committee member) / Arizona State University (Publisher)
Created2022
Description
Small Cell Carcinoma of the Ovary Hypercalcemic Type (SCCOHT) is a rare and highly aggressive ovarian cancer that affects children and young women at a mean age of 24 years. Most SCCOHT patients are diagnosed at an advanced stage and do not respond to chemotherapy. As a result, more than 75% of patients succumb to their disease within 1-2 years. To provide insights into the biological, diagnostic, and therapeutic vulnerabilities of this deadly cancer, a comprehensive characterization of 22 SCCOHT cases and 2 SCCOHT cell lines using microarray and next-generation sequencing technologies was performed. Following histological examination, tumor DNA and RNA were extracted and used for array comparative genomic hybridization and gene expression microarray analyses. In agreement with previous reports, SCCOHT presented consistently diploid profiles with few copy number aberrations. Gene expression analysis showed SCCOHT tumors have a unique gene expression profile unlike that of most common epithelial ovarian carcinomas. Dysregulated cell cycle control, DNA repair, DNA damage-response, nucleosome assembly, neurogenesis and nervous system development were all characteristic of SCCOHT tumors. Sequencing of DNA from SCCOHT patients and cell lines revealed germline and somatic inactivating mutations in the SWI/SNF chromatin-remodeling gene SMARCA4 in 79% (19/24) of SCCOHT patients in addition to SMARCA4 protein loss in 84% (16/19) of SCCOHT tumors, but in only 0.4% (2/485) of other primary ovarian tumors. Ongoing studies are now focusing on identifying treatments for SCCOHT based on therapeutic vulnerabilities conferred by ubiquitous inactivating mutations in SMARCA4 in addition to gene and protein expression data. Our characterization of the molecular landscape of SCCOHT and the breakthrough identification of inactivating SMARCA4 mutations in almost all cases of SCCOHT offers the first significant insight into the molecular pathogenesis of this disease. The loss of SMARCA4 protein is a highly sensitive and specific marker of the disease, highlighting its potential role as a diagnostic marker, and offers the opportunity for genetic testing of family members at risk. Outstanding questions remain about the role of SMARCA4 loss in the biology, histogenesis, diagnosis, and treatment of SCCOHT.
ContributorsRamos, Pilar (Author) / Anderson, Karen (Thesis advisor) / Trent, Jeffrey (Committee member) / Kusumi, Kenro (Committee member) / Lake, Douglas (Committee member) / Arizona State University (Publisher)
Created2014
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
Thrombus (blood clot) formation is at the roots of hemostasis and pathological thrombosis. Although many studies have successfully elucidated the cellular and molecular mechanisms underlying thrombus formation, there is still a void in understanding the processes limiting thrombus growth beyond that needed for stabilization. As a hemostatic thrombus grows, its surface consisting primarily of platelets changes to that composed of fibrin, which mechanically stabilizes the thrombus. Formation of fibrin ceases after some time; however, it is unclear why this fibrin is non-thrombogenic. This is puzzling since fibrin is known to support strong integrin-mediated adhesion of both platelets and leukocytes in vitro. Therefore, it would be expected that the fibrin surface of hemostatic thrombi in the circulation also support accumulation of these cells and thus continuous thrombus growth or degradation. Nevertheless, many in vivo studies did not detect any accumulation of blood cells including platelets at the fibrin surfaces of thrombi. This finding suggests the existence of natural processes that modulate the adhesive properties of fibrin to ensure proper regulation of thrombus growth, stability and degradation. In this dissertation, I document and discuss the findings supporting the existence of anti-adhesive mechanisms and their physiological relevance in surface-mediated control of thrombus growth and stability. The studies discussed in my dissertation have the potential to establish a novel aspect of hemostasis. Furthermore, it may provide new insights into the intricate and dynamic interplay between the mechanisms underlying hemostatic balance, which is essential to understanding the dysfunction of this process during pathological conditions.
ContributorsOwaynat, Hadil (Author) / Chandler, Douglas E. (Thesis advisor) / Wilson-Rawls, Norma J (Committee member) / Lake, Douglas F (Committee member) / Baluch, Debra P (Committee member) / Arizona State University (Publisher)
Created2016
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