Matching Items (23)
Filtering by
- All Subjects: Molecular Biology
- All Subjects: RNA
- Creators: Kusumi, Kenro
- Creators: Chen, Qiang
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
Ebola hemorrhagic fever (EHF) is a severe and often fatal disease in human and nonhuman primates, caused by the Ebola virus. Approximately 30 years after the first epidemic, there is no vaccine or therapeutic medication approved to counter the Ebola virus. In this dissertation, a geminiviral replicon system was used to produce Ebola immune complex (EIC) in plant leaves and tested it as an Ebola vaccine. The EIC was produced in Nicotiana benthamiana leaves by fusing Ebola virus glycoprotein (GP1) to the C-terminus of heavy chain of 6D8 monoclonal antibody (mAb), which is specific to the 6D8 epitope of GP1, and co-expressing the fusion with the light chain of 6D8 mAb. EIC was purified by ammonium sulfate precipitation and protein A or protein G affinity chromatography. EIC was shown to be immunogenic in mice, but the level of antibody against Ebola virus was not sufficient to protect the mice from lethal the Ebola challenge. Hence, different adjuvants were tested in order to improve the immunogenicity of the EIC. Among several adjuvants that we used, Poly(I:C), which is a synthetic analog of double-stranded ribonucleic acid that can interact with a Toll-like receptor 3, strongly increased the efficacy of our Ebola vaccine. The mice immunized with EIC co-administered with Poly(I:C) produced high levels of neutralizing anti-Ebola IgG, and 80% of the mice were protected from the lethal Ebola virus challenge. Moreover, the EIC induced a predominant T-helper type 1 (Th1) response, whereas Poly(I:C) co-delivered with the EIC stimulated a mixed Th1/Th2 response. This result suggests that the protection against lethal Ebola challenge requires both Th1 and Th2 responses. In conclusion, this study demonstrated that the plant-produced EIC co-delivered with Poly(I:C) induced strong and protective immune responses to the Ebola virus in mice. These results support plant-produced EIC as a good vaccine candidate against the Ebola virus. It should be pursued further in primate studies, and eventually in clinical trials.
ContributorsPhoolcharoen, Waranyoo (Author) / Mason, Hugh S (Thesis advisor) / Chen, Qiang (Thesis advisor) / Arntzen, Charles J. (Committee member) / Change, Yung (Committee member) / Ma, Julian (Committee member) / Arizona State University (Publisher)
Created2010
Description
Plant viral vectors have previously been used to produce high expression levels of antibodies and other proteins of interest. By utilizing a transformed Agrobacterium with the vector containing the protein of interest for infiltration, viral vectors can easily reach the plant cells making it an effective form of transient protein expression. For this project two different plant viral vectors were compared; the geminiviral vector derived from Bean yellow dwarf virus (BeYDV) and the MagnICON vector system derived from Tobacco Mosaic Virus(TMV) and Potato Virus X(PVX). E16, an antibody against West Nile virus, has previously been expressed using both systems but expression levels between the systems were not directly compared. Agrobacterium tumefaciens EHA105 cells were transformed with both systems and expression levels of E16 were quantified using ELISAs. Results showed very low expression levels of E16 using the geminiviral vector indicating a need for further investigation into the clone used as previous studies reported much higher expression levels with the system.
ContributorsMurphy, Skylar (Author) / Chen, Qiang (Thesis director) / Jugler, Collin (Committee member) / College of Health Solutions (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Virus-Like Particles (VLPs) are self-assembling structures that lack the viral genetic material. Therefore they are safer and more immunogenic than other forms of vaccines. The Hepatitis B core (HBc) VLPs are a novel mechanism through which delivery of DNA-based human vaccines are plausible. Production of VLPs require recombinant, rapidly replicating, plant-based systems such as the geminiviral replicon system. This project entails the cloning process of HBc-DIII fusion protein, a VLP that should form Domain III of the Envelope protein on West Nile Virus, into deconstructed geminiviral vector. The cloning process includes the HBc-DIII fusion protein DNA isolation, restriction enzyme digestion with NcoI and SacI, PCR changing the NcoI site on the HBc-DIII insert to XbaI, sequencing, ligation into geminiviral vector and transformation into an agrobacterium strain. The major impediment to the cloning process was the presence of multiple bands instead of the expected two bands while doing restriction enzyme digests. The troubleshooting process enabled speculating that due to the excess of restriction enzymes in the digestion volume, some of the DNA was not digested completely. Hence, multiple bands were observed. However, sequencing analysis and further cloning process ensured the presence of HBc-DIII insert band (approximately 800bp) in the Gemini vector. Lastly, the construct HBc-DIII in Gemini vector was ensured to be in agrobacterium for further experiments such as agro-infiltration.
ContributorsSuresh Kumar, Reshma (Author) / Chen, Qiang (Thesis director) / Zhang, Peiming (Committee member) / School of Molecular Sciences (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Vaccines are one of the most effective ways of combating infectious diseases and developing vaccine platforms that can be used to produce vaccines can greatly assist in combating global public health threats. This dissertation focuses on the development and pre-clinical testing of vaccine platforms that are highly immunogenic, easily modifiable, economically viable to produce, and stable. These criteria are met by the recombinant immune complex (RIC) universal vaccine platform when produced in plants. The RIC platform is modeled after naturally occurring immune complexes that form when an antibody, a component of the immune system that recognizes protein structures or sequences, binds to its specific antigen, a molecule that causes an immune response. In the RIC platform, a well-characterized antibody is linked via its heavy chain, to an antigen tagged with the antibody-specific epitope. The RIC antibody binds to the epitope tags on other RIC molecules and forms highly immunogenic complexes. My research has primarily focused on the optimization of the RIC platform. First, I altered the RIC platform to enable an N-terminal antigenic fusion instead of the previous C-terminal fusion strategy. This allowed the platform to be used with antigens that require an accessible N-terminus. A mouse immunization study with a model antigen showed that the fusion location, either N-terminal or C-terminal, did not impact the immune response. Next, I studied a synergistic response that was seen upon co-delivery of RIC with virus-like particles (VLP) and showed that the synergistic response could be produced with either N-terminal or C-terminal RIC co-delivered with VLP. Since RICs are inherently insoluble due to their ability to form complexes, I also examined ways to increase RIC solubility by characterizing a panel of modified RICs and antibody-fusions. The outcome was the identification of a modified RIC that had increased solubility while retaining high immunogenicity. Finally, I modified the RIC platform to contain multiple antigenic insertion sites and explored the use of bioinformatic tools to guide the design of a broadly protective vaccine.
ContributorsPardhe, Mary (Author) / Mason, Hugh S (Thesis advisor) / Chen, Qiang (Committee member) / Mor, Tsafrir (Committee member) / Wilson, Melissa (Committee member) / Arizona State University (Publisher)
Created2021
Description
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19) that emerged from a zoonotic host at the end of 2019 and caused a public health crisis. In this collection of studies, Nicotiana benthamiana plants are used to set the foundation for producing monoclonal antibodies (mAbs) with homogeneous glycosylation to neutralize SARS-CoV-2 and potentially address the immunopathology often observed with severe COVID-19. Specifically, a mAb against the human interleukin (IL)-6 receptor (sarilumab) was generated and evaluated in vitro for its potential to reduce IL-6 signaling that has been shown to be associated with more severe cases of COVID-19. Furthermore, multiple mAbs that bind to the receptor-binding domain (RBD) of SARS-CoV-2 and efficiently neutralize the virus were developed using plant-based expression. Several of these mAbs are from different classes of RBD-binding mAbs that have distinct binding sites from one another. Several mAbs from different classes showed synergy in neutralizing the ancestral strain of SARS-CoV-2 and a smaller subset showed synergy when tested against the highly mutated Omicron (B.1.1.529) variant. Of interest, a novel RBD-binding mAb, termed 11D7, that was raised against the ancestral strain and derived from a hybridoma, appears to have an epitope on the RBD that contributes more synergy to a mAb combination that efficiently neutralizes the B.1.1.529 variant of SARS-CoV-2. This epitope was partially mapped by competitive binding and shows that it overlaps with another known antibody that binds a cryptic, distal epitope, away from the receptor binding site, giving insight into the potential mechanism by which 11D7 neutralizes SARS-CoV-2, as well as potentially allowing it to resist SARS-CoV-2 immune evasion more efficiently. Furthermore, this mAb carries a highly homogeneous glycan pattern when expressed in N. benthamiana, that may contribute to enhanced effector function and provides a tool to elucidate the precise role of crystallizable fragment (Fc)-mediated protection in SARS-CoV-2 infection. Ultimately, these studies provide evidence of the utility of plant-made mAbs to be used as cocktail members, giving clarity to the use of less potent mAbs as valuable cocktail components which will spur further investigations into how mAbs with unique epitopes work together to efficiently neutralize SARS-CoV-2.
ContributorsJugler, Collin (Author) / Chen, Qiang (Thesis advisor) / Lake, Douglas (Committee member) / Steele, Kelly (Committee member) / Mason, Hugh (Committee member) / Arizona State University (Publisher)
Created2022
Description
Scientists are entrusted with developing novel molecular strategies for effective prophylactic and therapeutic interventions. Antivirals are indispensable tools that can be targeted at viral domains directly or at cellular domains indirectly to obstruct viral infections and reduce pathogenicity. Despite their transformative potential in healthcare, to date, antivirals have been clinically approved to treat only 10 out of the greater than 200 known pathogenic human viruses. Additionally, as obligate intracellular parasites, many virus functions are intimately coupled with host cellular processes. As such, the development of a clinically relevant antiviral is challenged by the limited number of clear targets per virus and necessitates an extensive insight into these molecular processes. Compounding this challenge, many viral pathogens have evolved to evade effective antivirals. Therefore, a means to develop virus- or strain-specific antivirals without detailed insight into each idiosyncratic biochemical mechanism may aid in the development of antivirals against a larger swath of pathogens. Such an approach will tremendously benefit from having the specific molecular recognition of viral species as the lowest barrier. Here, I modify a nanobody (anti-green fluorescent protein) that specifically recognizes non-essential epitopes (glycoprotein M-pHluorin chimera) presented on the extra virion surface of a virus (Pseudorabies virus strain 486). The nanobody switches from having no inhibitory properties (tested up to 50 μM) to ∼3 nM IC50 in in vitro infectivity assays using porcine kidney (PK15) cells. The nanobody modifications use highly reliable bioconjugation to a three-dimensional wireframe deoxyribonucleic acid (DNA) origami scaffold. Mechanistic studies suggest that inhibition is mediated by the DNA origami scaffold bound to the virus particle, which obstructs the internalization of the viruses into cells, and that inhibition is enhanced by avidity resulting from multivalent virus and scaffold interactions. The assembled nanostructures demonstrate negligible cytotoxicity (<10 nM) and sufficient stability, further supporting their therapeutic potential. If translatable to other viral species and epitopes, this approach may open a new strategy that leverages existing infrastructures – monoclonal antibody development, phage display, and in vitro evolution - for rapidly developing novel antivirals in vivo.
ContributorsPradhan, Swechchha (Author) / Hariadi, Rizal (Thesis advisor) / Hogue, Ian (Committee member) / Varsani, Arvind (Committee member) / Chen, Qiang (Committee member) / Arizona State University (Publisher)
Created2022