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- All Subjects: Cellular Biology
- Genre: Academic theses
- Creators: Jacobs, Bertram
- Creators: Kusumi, Kenro
- Member of: ASU Electronic Theses and Dissertations
- Resource Type: Text
Description
Cancer is one of the most serious global diseases. We have focused on cancer immunoprevention. My thesis projects include developing a prophylactic primary and metastatic cancer vaccines, early cancer detection and investigation of genes involved in tumor development. These studies were focused on frame-shift (FS) antigens. The FS antigens are generated by genomic mutations or abnormal RNA processing, which cause a portion of a normal protein to be translated out of frame. The concept of the prophylactic cancer vaccine is to develop a general cancer vaccine that could prevent healthy people from developing different types of cancer. We have discovered a set of cancer specific FS antigens. One of the FS candidates, structural maintenance of chromosomes protein 1A (SMC1A) FS, could start to accumulate at early stages of tumor and be specifically exposed to the immune system by tumor cells. Prophylactic immunization with SMC1A-FS could significantly inhibit primary tumor development in different murine tumor models and also has the potential to inhibit tumor metastasis. The SMC1A-FS transcript was detected in the plasma of the 4T1/BALB/c mouse tumor model. The tumor size was correlated with the transcript ratio of the SMC1A-FS verses the WT in plasma, which could be measured by regular RT-PCR. This unique cancer biomarker has a practical potential for a large population cancer screen, as well as clinical tumor monitoring. With a set of mimotope peptides, antibodies against SMC1A-FS peptide were detected in different cancer patients, including breast cancer, pancreas cancer and lung cancer with a 53.8%, 56.5% and 12.5% positive rate respectively. This suggested that the FS antibody could be a biomarker for early cancer detection. The characterization of SMC1A suggested that: First, the deficiency of the SMC1A is common in different tumors and able to promote tumor initiation and development; second, the FS truncated protein may have nucleolus function in normal cells. Mis-control of this protein may promote tumor development. In summary, we developed a systematic general cancer prevention strategy through the variety immunological and molecular methods. The results gathered suggest the SMC1A-FS may be useful for the detection and prevention of cancer.
ContributorsShen, Luhui (Author) / Johnston, Stephen Albert (Thesis advisor) / Chang, Yung (Committee member) / Miller, Laurence (Committee member) / Sykes, Kathryn (Committee member) / Jacobs, Bertram (Committee member) / Arizona State University (Publisher)
Created2012
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 species with highly heteromorphic sex chromosomes, the degradation of one of the sex chromosomes can result in unequal gene expression between the sexes (e.g., between XX females and XY males) and between the sex chromosomes and the autosomes. Dosage compensation is a process whereby genes on the sex chromosomes achieve equal gene expression which prevents deleterious side effects from having too much or too little expression of genes on sex chromsomes. The green anole is part of a group of species that recently underwent an adaptive radiation. The green anole has XX/XY sex determination, but the content of the X chromosome and its evolution have not been described. Given its status as a model species, better understanding the green anole genome could reveal insights into other species. Genomic analyses are crucial for a comprehensive picture of sex chromosome differentiation and dosage compensation, in addition to understanding speciation.
In order to address this, multiple comparative genomics and bioinformatics analyses were conducted to elucidate patterns of evolution in the green anole and across multiple anole species. Comparative genomics analyses were used to infer additional X-linked loci in the green anole, RNAseq data from male and female samples were anayzed to quantify patterns of sex-biased gene expression across the genome, and the extent of dosage compensation on the anole X chromosome was characterized, providing evidence that the sex chromosomes in the green anole are dosage compensated.
In addition, X-linked genes have a lower ratio of nonsynonymous to synonymous substitution rates than the autosomes when compared to other Anolis species, and pairwise rates of evolution in genes across the anole genome were analyzed. To conduct this analysis a new pipeline was created for filtering alignments and performing batch calculations for whole genome coding sequences. This pipeline has been made publicly available.
In order to address this, multiple comparative genomics and bioinformatics analyses were conducted to elucidate patterns of evolution in the green anole and across multiple anole species. Comparative genomics analyses were used to infer additional X-linked loci in the green anole, RNAseq data from male and female samples were anayzed to quantify patterns of sex-biased gene expression across the genome, and the extent of dosage compensation on the anole X chromosome was characterized, providing evidence that the sex chromosomes in the green anole are dosage compensated.
In addition, X-linked genes have a lower ratio of nonsynonymous to synonymous substitution rates than the autosomes when compared to other Anolis species, and pairwise rates of evolution in genes across the anole genome were analyzed. To conduct this analysis a new pipeline was created for filtering alignments and performing batch calculations for whole genome coding sequences. This pipeline has been made publicly available.
ContributorsRupp, Shawn Michael (Author) / Wilson Sayres, Melissa A (Thesis advisor) / Kusumi, Kenro (Committee member) / DeNardo, Dale (Committee member) / Arizona State University (Publisher)
Created2016
Description
Novel biological strategies for cancer therapy have recently been able to generate improved anti-tumor effects in the clinic. Of these new advancements, oncolytic virotherapy is a promising strategy through a dual mechanism of oncolysis and stimulation of tumor immunogenicity against the target cancer cells. Myxoma virus (MYXV) is an oncolytic poxvirus that has a natural tropism for Leporids, being nonpathogenic in humans and all other known vertebrates. MYXV is able to infect cancer cells due to mutations and defects in many innate signaling pathways, such as those involved in anti-viral responses. While MYXV alone infects and kills many classes of human cancer cells, recombinant techniques allow for the implementation of therapeutic transgenes, which have the potential of ‘arming’ the virus to enhance its potential as an oncolytic virus. The implementation of certain transgenes allows improved cancer cell killing and/or promotion of more robust anti-tumor immune responses. To investigate the potential of immune-inducing transgenes in MYXV, in vitro screening experiments were performed with several single transgene-armed recombinant MYXVs. As recent studies have shown the ability of MYXV to uniquely target malignant human hematopoietic stem cells, the potential of oncolytic MYXV armed with individual immune-enhancing transgenes was investigated through in vitro killing analysis using human acute myeloid leukemia (AML) and multiple myeloma (MM) cell lines. Additionally, in vitro experiments were performed using primary bone marrow (BM) cells obtained from human patients diagnosed with MM. Furthermore, the action of an engineered bispecific killer engager (huBIKE) was investigated through co-culture studies between the CD138 surface marker of target MM cells and the CD16 surface marker of primary effector peripheral blood mononuclear cells (PBMCs), particularly NK cells and neutrophils. In this study, several of the test armed MYXV-infected human AML and MM cell lines resulted in increased cell death compared to unarmed MYXV-infected cells. Additionally, increased killing of CD138+ MM cells from primary human BM samples was observed following infection with huBIKE-armed MYXV relative to infection with unarmed MYXV. Furthermore, analysis of co-culture studies performed suggests enhanced killing of target MM cells via engagement of NK cells with U266 MM cells by huBIKE.
ContributorsMamola, Joseph (Author) / McFadden, Grant (Thesis advisor) / Jacobs, Bertram (Committee member) / Blattman, Joseph (Committee member) / Arizona State University (Publisher)
Created2020
Description
Satellite cells are adult muscle stem cells that activate, proliferate, and differentiate into myofibers upon muscle damage. Satellite cells can be cultured and manipulated in vitro, and thus represent an accessible model for studying skeletal muscle biology, and a potential source of autologous stem cells for regenerative medicine. This work summarizes efforts to further understanding of satellite cell biology, using novel model organisms, bioengineering, and molecular and cellular approaches. Lizards are evolutionarily the closest vertebrates to humans that regenerate entire appendages. An analysis of lizard myoprogenitor cell transcriptome determined they were most transcriptionally similar to mammalian satellite cells. Further examination showed that among genes with the highest level of expression in lizard satellite cells were an increased number of regulators of chondrogenesis. In micromass culture, lizard satellite cells formed nodules that expressed chondrogenic regulatory genes, thus demonstrating increased musculoskeletal plasticity. However, to exploit satellite cells for therapeutics, development of an ex vivo culture is necessary. This work investigates whether substrates composed of extracellular matrix (ECM) proteins, as either coatings or hydrogels, can support expansion of this population whilst maintaining their myogenic potency. Stiffer substrates are necessary for in vitro proliferation and differentiation of satellite cells, while the ECM composition was not significantly important. Additionally, satellite cells on hydrogels entered a quiescent state that could be reversed when the cells were subsequently cultured on Matrigel. Proliferation and gene expression data further indicated that C2C12 cells are not a good proxy for satellite cells. To further understand how different signaling pathways control satellite cell behavior, an investigation of the Notch inhibitor protein Numb was carried out. Numb deficient satellite cells fail to activate, proliferate and participate in muscle repair. Examination of Numb isoform expression in satellite cells and embryonic tissues revealed that while developing limb bud, neural tube, and heart express the long and short isoforms of NUMB, satellite cells predominantly express the short isoforms. A preliminary immunoprecipitation- proteomics experiment suggested that the roles of NUMB in satellite cells are related to cell cycle modulation, cytoskeleton dynamics, and regulation of transcription factors necessary for satellite cell function.
ContributorsPalade, Joanna (Author) / Wilson-Rawls, Norma (Thesis advisor) / Rawls, Jeffrey (Committee member) / Kusumi, Kenro (Committee member) / Newbern, Jason (Committee member) / Stabenfeldt, Sarah (Committee member) / Arizona State University (Publisher)
Created2020
Description
Programmed cell death plays an important role in a variety of processes that promote the survival of the host organism. Necroptosis, a form of programmed cell death, occurs through a signaling pathway involving receptor-interacting serine-threonine protein kinase 3 (RIPK3). In response to vaccinia virus infection, necroptosis is induced through DNA-induced activator of interferon (DAI), which activates RIPK3, leading to death of the cell and thereby inhibiting further viral replication in host cells. DAI also localizes into stress granules, accumulations of mRNAs that have stalled in translation due to cellular stress. The toxin arsenite, a canonical inducer of stress granule formation, was used in this project to study necroptosis. By initiating necroptosis with arsenite and vaccinia virus, this research project investigated the roles of necroptosis proteins and their potential localization into stress granules. The two aims of this research project were to determine whether stress granules are important for arsenite- and virus-induced necroptosis, and whether the proteins DAI and RIPK3 localize into stress granules. The first aim was investigated by establishing a DAI and RIPK3 expression system in U2OS cells; arsenite treatment or vaccinia virus infection was then performed on the U2OS cells as well as on U2OSΔΔG3BP1/2 cells, which are not able to form stress granules. The second aim was carried out by designing fluorescent tagging for the necroptosis proteins in order to visualize protein localization with fluorescent microscopy. The results show that arsenite induces DAI-dependent necroptosis in U2OS cells and that this arsenite-induced necroptosis likely requires stress granules. In addition, the results show that vaccinia virus induces DAI-dependent necroptosis that also likely requires stress granules in U2OS cells. Furthermore, a fluorescent RIPK3 construct was created that will allowfor future studies on protein localization during necroptosis and can be used to answer questions regarding localization of necroptosis proteins into stress granules. This project therefore contributes to a greater understanding of the roles of DAI and RIPK3 in necroptosis, as well as the roles of stress granules in necroptosis, both of which are important in research regarding viral infection and cellular stress.
ContributorsGogerty, Carolina (Author) / Jacobs, Bertram (Thesis advisor) / Langland, Jeffrey (Committee member) / Jentarra, Garilyn (Committee member) / Arizona State University (Publisher)
Created2021