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- All Subjects: Molecular Biology
- All Subjects: Flaviviruses
- Creators: Lake, Douglas
- Creators: Chandler, Douglas
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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
The F1Fo ATP synthase is required for energy conversion in almost all living organisms. The F1 complex is a molecular motor that uses ATP hydrolysis to drive rotation of the γ–subunit. It has not been previously possible to resolve the speed and position of the γ–subunit of the F1–ATPase as it rotates during a power stroke. The single molecule experiments presented here measured light scattered from 45X91 nm gold nanorods attached to the γ–subunit that provide an unprecedented 5 μs resolution of rotational position as a function of time. The product of velocity and drag, which were both measured directly, resulted in an average torque of 63±8 pN nm for the Escherichia coli F1-ATPase that was determined to be independent of the load. The rotational velocity had an initial (I) acceleration phase 15° from the end of the catalytic dwell, a slow (S) acceleration phase during ATP binding/ADP release (15°–60°), and a fast (F) acceleration phase (60°–90°) containing an interim deceleration (ID) phase (75°–82°). High ADP concentrations decreased the velocity of the S phase proportional to 'ADP-release' dwells, and the F phase proportional to the free energy derived from the [ADP][Pi]/[ATP] chemical equilibrium. The decreased affinity for ITP increased ITP-binding dwells by 10%, but decreased velocity by 40% during the S phase. This is the first direct evidence that nucleotide binding contributes to F1–ATPase torque. Mutations that affect specific phases of rotation were identified, some in regions of F1 previously considered not to contribute to rotation. Mutations βD372V and γK9I increased the F phase velocity, and γK9I increased the depth of the ID phase. The conversion between S and F phases was specifically affected by γQ269L. While βT273D, βD305E, and αR283Q decreased the velocity of all phases, decreases in velocity due to βD302T, γR268L and γT82A were confined to the I and S phases. The correlations between the structural locations of these mutations and the phases of rotation they affect provide new insight into the molecular basis for F1–ATPase γ-subunit rotation.
ContributorsMartin, James (Author) / Frasch, Wayne D (Thesis advisor) / Chandler, Douglas (Committee member) / Gaxiola, Roberto (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2012
Description
Infertility has become an increasing problem in developed countries and in many cases can be attributed to compromised sperm quality. Assessment of male fertility typically utilizes semen analysis which mainly examines sperm morphology, however many males whose sperm appear normal are sub- or infertile, suggesting that sperm from these males may be deficient in a protein or suite of proteins. To date, very little is known about the composition of sperm or the complex maturation process that confers motility and fertilization competency to sperm. Chapter 1 discusses the use of whole cell mass spectrometry to identify 1247 proteins comprising the Rhesus macaque (Macaca mulatta) sperm proteome, a commonly used model of human reproduction. This study provides a more robust proxy of human sperm composition than was previously available and facilitates studies of sperm using the rhesus macaque as a model. Chapters 2 & 3 provide a systems level overview of changes in sperm proteome composition that occurs during epididymal transit. Chapter 2 reports the proteomes of sperm collected from the caput, corpus and cauda segments of the mouse epididymis, identifying 1536, 1720 and 1234 proteins respectively. Chapter 3 reports the sperm proteome from four distinct segments of the Rhesus macaque epididymis, including the caput, proximal corpus, distal corpus and cauda, identifying 1951, 2014, 1764 and 1423 proteins respectively. These studies identify a number of proteins that are added and removed from sperm during epididymal transit which likely play an important role in the sperm maturation process. To date no comparative evolutionary studies of sperm proteomes have been undertaken. Chapter 4 compares four mammalian sperm proteomes including the human, macaque, mouse and rat. This study identified 98 proteins common to all four sperm proteomes, 82 primate and 90 rodent lineage-specific proteins and 494, 467, 566, and 193 species specific proteins in the human, macaque, mouse and rat sperm proteomes respectively and discusses how differences in sperm composition may ultimately lead to functional differences across species. Finally, chapter 5 uses sperm proteome data to inform the preliminary design of a rodent contraceptive vaccine delivered orally using recombinant attenuated Salmonella vaccine vectors.
ContributorsSkerget, Sheri Jo (Author) / Karr, Timothy L. (Thesis advisor) / Lake, Douglas (Committee member) / Petritis, Konstantinos (Committee member) / Arizona State University (Publisher)
Created2013
Description
Synechocystis sp PCC 6803 is a photosynthetic cyanobacterium that can be easily transformed to produce molecules of interest; this has increased Synechocystis’ popularity as a clean energy platform. Synechocystis has been shown to produce and excrete molecules such as fatty acids, isoprene, etc. after appropriate genetic modification. Challenges faced for large–scale growth of modified Synechocystis include abiotic stress, microbial contamination and high processing costs of product and cell material. Research reported in this dissertation contributes to solutions to these challenges. First, abiotic stress was addressed by overexpression of the heat shock protein ClpB1. In contrast to the wild type, the ClpB1 overexpression mutant (Slr1641+) tolerated rapid temperature changes, but no difference was found between the strains when temperature shifts were slower. Combination of ClpB1 overexpression with DnaK2 overexpression (Slr1641+/Sll0170+) further increased thermotolerance. Next, we used a Synechocystis strain that carries an introduced isoprene synthase gene (IspS+) and that therefore produces isoprene. We attempted to increase isoprene yields by overexpression of key enzymes in the methyl erythritol phosphate (MEP) pathway that leads to synthesis of the isoprene precursor. Isoprene production was not increased greatly by MEP pathway induction, likely because of limitations in the affinity of the isoprene synthase for the substrate. Finally, two extraction principles, two–phase liquid extraction (e.g., with an organic and aqueous phase) and solid–liquid extraction (e.g., with a resin) were tested. Two–phase liquid extraction is suitable for separating isoprene but not fatty acids from the culture medium. Fatty acid removal required acidification or surfactant addition, which affected biocompatibility. Therefore, improvements of both the organism and product–harvesting methods can contribute to enhancing the potential of cyanobacteria as solar–powered biocatalysts for the production of petroleum substitutes.
ContributorsGonzalez Esquer, Cesar Raul (Author) / Vermaas, Willem (Thesis advisor) / Chandler, Douglas (Committee member) / Bingham, Scott (Committee member) / Nielsen, David (Committee member) / Arizona State University (Publisher)
Created2013
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
While the entire human genome has been sequenced, the understanding of its functional elements remains unclear. The Encyclopedia of DNA Elements (ENCODE) project analyzed 1% of the human genome and found that the majority of the human genome is transcribed, including non-protein coding regions. The hypothesis is that some of the "non-coding" sequences are translated into peptides and small proteins. Using mass spectrometry numerous peptides derived from the ENCODE transcriptome were identified. Peptides and small proteins were also found from non-coding regions of the 1% of the human genome that the ENCODE did not find transcripts for. A large portion of these peptides mapped to the intronic regions of known genes, thus it is suspected that they may be undiscovered exons present in alternative spliceoforms of certain genes. Further studies proved the existence of polyadenylated RNAs coding for these peptides. Although their functional significance has not been determined, I anticipate the findings will lead to the discovery of new splice variants of known genes and possibly new transcriptional and translational mechanisms.
ContributorsWang, Lulu (Author) / Lake, Douglas (Thesis advisor) / Chang, Yung (Committee member) / Touchman, Jeffery (Committee member) / Arizona State University (Publisher)
Created2010
Description
Small cell carcinoma of the ovary (SCCOHT) is a rare ovarian cancer affecting young women and characterized by mutation in SMARCA4 and silencing of SMARCA2, two tumor suppressors that function as ATPases in the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complex. SCCOHT patients face a 5-year survival rate of only 26%, but recently we have identified sensitivity of SCCOHT models to a natural product, triptolide. This study aims to ascertain the mechanism of action of triptolide. Previous SCCOHT epigenetic drug research has shown that some drugs reverse SMARCA2 epigenetic silencing to inhibit tumor growth, therefore it is hypothesized that triptolide acts the same and restores SWI/SNF function. Cells treated with triptolide have no change in SMARCA2 expression, suggesting that re-expression of epigenetically silenced tumor suppressor gene does not underlie its mechanism of action. Growth rates following triptolide treatment were observed in the presence and absence of SMARCA4, but no difference in sensitivity was observed. Thus, it is not likely that triptolide acts by restoring SWI/SNF. Others have observed that triptolide acts on xeroderma pigmentosa type B protein (XPB), a component of super-enhancers, which are DNA regions with high levels of transcription that regulate genes responsible for cell identity and oncogenes driving tumorigenesis. Both SCCOHT-1 and BIN67 cell lines treated with triptolide displayed lower expression of the super-enhancer associated MYC oncogene compared to untreated cells, supporting the theory that triptolide could be inhibiting super-enhancers regulating oncogenes.. A western blot confirmed reduced protein levels of RNA polymerase II and bromodomain 4 (BRD4), two essential components found at high levels at super-enhancers, in BIN67 cells treated with triptolide. ChIP-sequencing of Histone H3 Lysine-27 Acetylation (H3K27ac) marks in BIN67 and SCCOHT-1 cell lines identified super-enhancers in SCCOHT using tools CREAM and ROSE, which were mapped to neighboring genes associated genes and compared with the COSMIC database to identify oncogenes, of which the top 11 were examined by qRT-PCR to ascertain whether triptolide reduces their expression. It has been found that 6 out of 11 of the oncogenes examined (SALL4, MYC, SGK1, HIST1H3B, HMGA2, and CALR) decreased in expression when treated with triptolide. Thus, there is reason to believe that triptolide’s mechanism of action is via inhibition of super-enhancers that regulate oncogene expression.
ContributorsViloria, Nicolle Angela (Author) / Lake, Douglas (Thesis director) / Hendricks, William (Committee member) / Lang, Jessica (Committee member) / School of Life Sciences (Contributor) / School of Human Evolution & Social Change (Contributor) / School of International Letters and Cultures (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Macrophage fusion resulting multinucleated giant cells (MGCs) formation is associated with numerous chronic inflammatory diseases including the foreign body reaction to implanted
biomaterials. Despite long-standing predictions, there have been attempts to use live-cell
imaging to investigate the morphological features initiating macrophage fusion because
macrophages do not fuse on clean glass required for most imaging techniques. Consequently,
the mechanisms of macrophage fusion remain poorly understood. The goal of this research
project was to characterize the early and late stages of macrophage multinucleation using
fusogenic optical quality substrate. Live-cell imaging with phase-contrast and lattice-light
sheet microscopy revealed that an actin-based protrusion initiates macrophage fusion. WASpdeficient macrophages and macrophages isolated from myeloid cell-specific Cdc42-/- mice
fused at very low rates. In addition, inhibiting the Arp2/3 complex impaired both the formation
of podosomes and macrophage fusion.
Analyses of the late stages of macrophage multinucleation on biomaterials implanted into
mice revealed novel actin-based zipper-like structures (ZLSs) formed at contact sites between
MGCs. The model system that was developed for the induction of ZLSs in vitro allowed for
the characterization of protein composition using confocal and super-resolution microscopy.
Live-cell imaging demonstrated that ZLSs are dynamic formations undergoing continuous
assembly and disassembly and that podosomes are precursors of these structures. It was further
found that E-cadherin and nectin-2 are involved in ZLS formation by bridging the plasma
membranes together. ii
Macrophage fusion on implanted biomaterials inherently involves their adhesion to the
implant surface. While biomaterials rapidly acquire a layer of host proteins, a biological
substrate that is required for macrophage fusion is unknown. It was shown that mice with
fibrinogen deficiency as well as mice expressing fibrinogen incapable of fibrin polymerization
displayed a dramatic reduction of macrophage fusion on biomaterials. Furthermore, these mice
were protected from the formation of the dense collagenous capsule enveloping the implant. It
was also found that the main cell type responsible for the deposition of collagen in the capsule
were mononuclear macrophages but not myofibroblasts. Together, these findings reveal a
critical role of the actin cytoskeleton in macrophage fusion and identify potential targets to
reduce the drawbacks of macrophage fusion on implanted biomaterials.
ContributorsBalabiyev, Arnat (Author) / Ugarova, Tatiana (Thesis advisor) / Roberson, Robert (Committee member) / Chandler, Douglas (Committee member) / Baluch, Page (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
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative pathogen of Coronavirus Disease 2019 (COVID-19). Successful vaccination aims to elicit neutralizing antibodies (NAbs) which inhibit viral infection. Traditional NAb quantification methods (neutralization assays) are labor-intensive and expensive, with limited practicality for routine use (e.g. monitoring vaccination response). Thus, a rapid (10-minute) lateral flow assay (LFA) for quantification of SARS-CoV-2 NAbs was developed. Using the NAb LFA, an 18-month longitudinal study assessing monthly NAb titers was conducted in a cohort of over 500 COVID-19 mRNA vaccine recipients. Three NAb response groups were identified: vaccine strong responders (VSRs), moderate responders (VMRs), and poor responders (VPRs). VSRs generated high and durable NAb titers. VMRs initially generated high NAb titers but showed more rapid waning with time post-vaccination. Finally, VPRs rarely generated NAb titers ≥1:160, even after 3rd dose. Although strong humoral responses correlate with vaccine effectiveness, viral-specific CD4+ and CD8+ T cells are critical for long-term protection. Discordant phenotypes of viral-specific CD8+ and CD4+CXCR5+ T follicular helper (cTfh) cells have recently been associated with differential NAb responses. The second portion of this dissertation was to investigate whether/how SARS-CoV-2 T cell responses differ in individuals with impaired NAb titers following mRNA vaccination. Thus, phenotypic and functional characterization of T cell activation across NAb response groups was conducted. It was hypothesized that VPRs would exhibit discordant SARS-CoV-2 T cell activation and altered cTfh phenotypes. Peripheral blood mononuclear cells were isolated from VPRs, VMRs, VSRs, naturally infected, and normal donors. SARS-CoV-2 responsive T cells were characterized using in vitro activation induced marker assays, multicolor flow cytometry, and multiplex cytokine analysis. Further, CXCR5+ cTfh were examined for chemokine receptor expression (CCR6 and CXCR3). Results demonstrated that despite differential NAb responses, activation of SARS-CoV-2 responsive CD4+ and CD8+ T cells was comparable across NAb groups. However, double-positive CD4+CD8+, CD8low, and activated CD4+CXCR5+CCR6-CXCR3+ (Tfh1-like) T cells were expanded in VPRs compared to VMR and VSRs. Interestingly, a unique population of CD8+CXCR5+ T cells was also expanded in VPRs. These novel findings may aid in identification of individuals with impaired or altered immune responses to COVID-19 mRNA vaccination.
ContributorsRoeder, Alexa Jordan (Author) / Lake, Douglas (Thesis advisor) / McFadden, Grant (Committee member) / Borges Florsheim, Esther (Committee member) / Chang, Yung (Committee member) / Rahman, Masmudur (Committee member) / Arizona State University (Publisher)
Created2023