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- All Subjects: Molecular Biology
- Creators: Kusumi, Kenro
- Creators: Chang, Yung
- Status: Published
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
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
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
Telomerase enzyme is a truly remarkable enzyme specialized for the addition of short, highly repetitive DNA sequences onto linear eukaryotic chromosome ends. The telomerase enzyme functions as a ribonucleoprotein, minimally composed of the highly conserved catalytic telomerase reverse transcriptase and essential telomerase RNA component containing an internalized short template region within the vastly larger non-coding RNA. Even among closely related groups of species, telomerase RNA is astonishingly divergent in sequence, length, and secondary structure. This massive disparity is highly prohibitive for telomerase RNA identification from previously unexplored groups of species, which is fundamental for secondary structure determination. Combined biochemical enrichment and computational screening methods were employed for the discovery of numerous telomerase RNAs from the poorly characterized echinoderm lineage. This resulted in the revelation that--while closely related to the vertebrate lineage and grossly resembling vertebrate telomerase RNA--the echinoderm telomerase RNA central domain varies extensively in structure and sequence, diverging even within echinoderms amongst sea urchins and brittle stars. Furthermore, the origins of telomerase RNA within the eukaryotic lineage have remained a persistent mystery. The ancient Trypanosoma telomerase RNA was previously identified, however, a functionally verified secondary structure remained elusive. Synthetic Trypanosoma telomerase was generated for molecular dissection of Trypanosoma telomerase RNA revealing two RNA domains functionally equivalent to those found in known telomerase RNAs, yet structurally distinct. This work demonstrates that telomerase RNA is uncommonly divergent in gross architecture, while retaining critical universal elements.
ContributorsPodlevsky, Joshua (Author) / Chen, Julian (Thesis advisor) / Mangone, Marco (Committee member) / Kusumi, Kenro (Committee member) / Wilson-Rawls, Norma (Committee member) / Arizona State University (Publisher)
Created2015
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
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
V(D)J recombination is responsible for generating an enormous repertoire of immunoglobulins and T cell receptors, therefore it is a centerpiece to the formation of the adaptive immune system. The V(D)J recombination process proceeds through two steps, site-specific cleavage at RSS (Recombination Signal Sequence) site mediated by the RAG recombinase (RAG1/2) and the subsequent imprecise resolution of the DNA ends, which is carried out by the ubiquitous non-homologous end joining pathway (NHEJ). The V(D)J recombination reaction is obliged to be tightly controlled under all circumstances, as it involves generations of DNA double strand breaks, which are considered the most dangerous lesion to a cell. Multifaceted regulatory mechanisms have been evolved to create great diversity of the antigen receptor repertoire while ensuring genome stability. The RAG-mediated cleavage reaction is stringently regulated at both the pre-cleavage stage and the post-cleavage stage. Specifically, RAG1/2 first forms a pre-cleavage complex assembled at the boarder of RSS and coding flank, which ensures the appropriate DNA targeting. Subsequently, this complex initiates site-specific cleavage, generating two types of double stranded DNA breaks, hairpin-ended coding ends (HP-CEs) and blunt signal ends (SEs). After the cleavage, RAG1/2 proteins bind and retain the recombination ends to form post-cleavage complexes (PCC), which collaborates with the NHEJ machinery for appropriate transfer of recombination ends to NHEJ for proper end resolution. However, little is known about the molecular basis of this collaboration, partly attributed to the lack of sensitive assays to reveal the interaction of PCC with HP-CEs. Here, for the first time, by using two complementary fluorescence-based techniques, fluorescence anisotropy and fluorescence resonance energy transfer (FRET), I managed to monitor the RAG1/2-catalyzed cleavage reaction in real time, from the pre-cleavage to the post-cleavage stages. By examining the dynamic fluorescence changes during the RAG-mediated cleavage reactions, and by manipulating the reaction conditions, I was able to characterize some fundamental properties of RAG-DNA interactions before and after cleavage. Firstly, Mg2+, known as a physiological cofactor at the excision step, also promotes the HP-CEs retention in the RAG complex after cleavage. Secondly, the structure of pre-cleavage complex may affect the subsequent collaborations with NHEJ for end resolution. Thirdly, the non-core region of RAG2 may have differential influences on the PCC retention of HP-CEs and SEs. Furthermore, I also provide the first evidence of RAG1-mediated regulation of RAG2. Our study provides important insights into the multilayered regulatory mechanisms, in modulating recombination events in developing lymphocytes and paves the way for possible development of detection and diagnotic markers for defective recombination events that are often associated immunodeficiency and/or lymphoid malignancy.
ContributorsWang, Guannan (Author) / Chang, Yung (Thesis advisor) / Levitus, Marcia (Committee member) / Misra, Rajeev (Committee member) / Anderson, Karen (Committee member) / Arizona State University (Publisher)
Created2012
Description
Teleosts have the most primitive adaptive immune system. However, in terms of functionality the teleost immune system is similar to birds and mammals. On the other hand, enteric bacterial pathogens of mammals and birds present conserved regulatory mechanisms that control virulence factors. In this context, deletion of conserved genes that control virulence factors have been successfully used as measure to construct live attenuated bacterial vaccines for mammals and birds. Here, I hypothesize that evolutionary conserved genes, which control virulence factors or are essential for bacterial physiology in Enterobacteriaceae, could be used as universal tools to design live attenuated recombinant bacterial vaccines from fish to mammals. The evolutionary conserved genes that control virulence factors, crp and fur, and the essential gene for the synthesis of the cell wall, asd, were studied in Edwardsiella ictaluri to develop a live recombinant vaccine for fish host. The genus Edwardsiella is one of the most ancient represent of the Enterobacteriaceae family. E. ictaluri, a host restricted pathogen of catfish (Ictalurus punctatus), is the causative agent of the enteric septicemia and one of the most important pathogens of this fish aquaculture. Although, crp and fur control different virulence factors in Edwardsiella, in comparison to other enterics, individual deletion of these genes triggered protective immune response at the systemic and mucosal level of the fish. Deletion of asdA gene allowed the creation of a balanced-lethal system to syntheses heterologous antigens. I concluded that crp, fur and asd could be universally used to develop live attenuate recombinant Enterobacteriaceae base vaccines for different hosts.
ContributorsSantander Morales, Javier Alonso (Author) / Curtiss, Roy Iii (Thesis advisor) / Chandler, Douglas (Committee member) / Chang, Yung (Committee member) / Shi, Yixin (Committee member) / Arizona State University (Publisher)
Created2012
Description
Over the past decade, several high-value proteins have been produced using plant-based transient expression systems. However, these studies exposed some limitations that must be overcome to allow plant expression systems to reach their full potential. These limitations are the low level of recombinant protein accumulation achieved in some cases, and lack of efficient co-expression vectors for the production of multi-protein complexes. This study report that tobacco Extensin (Ext) gene 3' untranslated region (UTR) can be broadly used to enhance recombinant protein expression in plants. Extensin is the hydroxyproline-rich glycoprotein that constitutes the major protein component of cell walls. Using transient expression, it was found that the Ext 3' UTR increases recombinant protein expression up to 13.5- and 6-fold in non-replicating and replicating vector systems, respectively, compared to previously established terminators. Enhanced protein accumulation was correlated with increased mRNA levels associated with reduction in read-through transcription. Regions of Ext 3' UTR essential for maximum gene expression included a poly-purine sequence used as a major poly-adenylation site. Furthermore, modified bean yellow dwarf virus (BeYDV)-based vectors designed to allow co-expression of multiple recombinant genes were constructed and tested for their performance in driving transient expression in plants. Robust co-expression and assembly of heavy and light chains of the anti-Ebola virus monoclonal antibody 6D8, as well as E. coli heat-labile toxin (LT) were achieved with the modified vectors. The simultaneous co-expression of three fluoroproteins using the single replicon, triple cassette is demonstrated by confocal microscopy. In conclusion, this study provides an excellent tool for rapid, cost-effective, large-scale manufacturing of recombinant proteins for use in medicine and industry.
ContributorsRosenthal, Sun Hee (Author) / Mason, Hugh (Thesis advisor) / Mor, Tsafrir (Committee member) / Chang, Yung (Committee member) / Arntzen, Charles (Committee member) / Arizona State University (Publisher)
Created2012