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- Title
- HELICAL PACKING REGULATES STRUCTURAL TRANSITIONS IN BAX.
- Creator
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Tschammer, Nuska, Khaled, Annette, University of Central Florida
- Abstract / Description
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Apoptosis is essential for development and the maintenance of cellular homeostasis and is frequently dysregulated in disease states. Proteins of the BCL-2 family are key modulators of this process and are thus ideal therapeutic targets. In response to diverse apoptotic stimuli, the pro-apoptotic member of BCL-2 family, BAX, redistributes from the cytosol to the mitochondria or endoplasmic reticulum and primes cells for death. The structural changes that enable this lethal protein to...
Show moreApoptosis is essential for development and the maintenance of cellular homeostasis and is frequently dysregulated in disease states. Proteins of the BCL-2 family are key modulators of this process and are thus ideal therapeutic targets. In response to diverse apoptotic stimuli, the pro-apoptotic member of BCL-2 family, BAX, redistributes from the cytosol to the mitochondria or endoplasmic reticulum and primes cells for death. The structural changes that enable this lethal protein to transition from a cytosolic form to a membrane-bound form remain poorly understood. Elucidating this process is a necessary step in the development of BAX as a novel therapeutic target for the treatment of cancer, as well as autoimmune and neurodegenerative disorders. A three-part study, utilizing computational modeling and biological assays, was used to examine how BAX, and similar proteins, transition to membranes. The first part tested the hypothesis that the C-terminal α9 helix regulates the distribution and activity of BAX by functioning as a "molecular switch" to trigger conformational changes that enable the protein to redistribute from the cytosol to mitochondrial membrane. Computational analysis, tested in biological assays, revealed a new finding: that the α9 helix can dock into a hydrophobic groove of BAX in two opposite directions in a self-associated, forward orientation and a previously, unknown reverse orientation that enables dimerization and apoptosis. Peptides, made to mimic the α9-helix, were able to induce the mitochondrial translocation of BAX, but not when key residues in the hydrophobic groove were mutated. Such findings indicate that the α9 helix of BAX can function as a "molecular switch" to mediate occupancy of the hydrophobic groove and regulate the membrane-binding activity of BAX. This new discovery contributes to the understanding of how BAX functions during apoptosis and can lead to the design of new therapeutic approaches based on manipulating the occupancy of the hydrophobic groove. The second and third parts of the study used computational modeling to examine how the helical stability of proteins relates to their ability to functionally transition. Analysis of BAX, as a prototypical transitioning protein, revealed that it has a broad variation in the distribution of its helical interaction energy. This observation led to the hypothesis tested, that proteins which undergo 3D structural transitions during execution of their function have broad variations in the distribution of their helical interaction energies. The result of this study, after examination of a large group of all-alpha proteins, was the development of a novel, predictive computational method, based on measuring helical interactions energies, which can be used to identify new proteins that undergo structural transitioning in the execution of their function. When this method was used to examine transitioning in other members the BCL-2 family, a strong agreement with the published experimental findings resulted. Further, it was revealed that the binding of a ligand, such as a small peptide, to a protein can have significant stabilizing or destabilizing influences that impact upon the activation and function of the protein. This computational analysis thus contributes to a better understanding of the function and regulation of the BCL-2 family members and also offers the means by which peptide mimics that modulate protein activity can be designed for testing in therapeutic endeavors.
Show less - Date Issued
- 2007
- Identifier
- CFE0001865, ucf:47392
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0001865
- Title
- REGULATION OF APOPTOTIC ALKALINIZATION THROUGH PHOSPHORYLATION OF SODIUM HYDROGEN EXCHANGER VIA P38 MITOGEN ACTIVATED PROTEIN KINASE.
- Creator
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GRENIER, AMY, KHALED, ANNETTE, University of Central Florida
- Abstract / Description
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Regulation of intracellular pH is responsible for many cellular processes, such as metabolism, cell cycle progression, and apoptosis. Many chemotherapeutic agents work by inducing target cells to undergo apoptosis, a cell death process still poorly understood. Previous studies demonstrated that a rise in intracellular pH activated apoptotic proteins leading to cytochrome C release. This "apoptotic alkalinization" occurred upon activation of the plasma membrane protein, sodium hydrogen...
Show moreRegulation of intracellular pH is responsible for many cellular processes, such as metabolism, cell cycle progression, and apoptosis. Many chemotherapeutic agents work by inducing target cells to undergo apoptosis, a cell death process still poorly understood. Previous studies demonstrated that a rise in intracellular pH activated apoptotic proteins leading to cytochrome C release. This "apoptotic alkalinization" occurred upon activation of the plasma membrane protein, sodium hydrogen exchanger-1 (NHE1), whose activity is regulated by the stress kinase p38 MAPK. In previous studies, upon cytokine withdrawal from cytokine-dependent lymphocytes induced the activity of the p38 MAP kinase which then phosphorylated the C-terminus of NHE1. To identify the p38 MAPK phosphorylation sites on NHE1, in vitro p38 MAP kinase assays coupled to deletion analysis of NHE1 and mass spectrometry, identified four possible p38 MAPK phosphorylation sites. To establish that NHE1 causes apoptotic alkalinization and determine whether the identified phosphorylation sites on NHE1 are functionally significant, we used PCR site directed mutagenesis to mutate T717, S722, S725, and S728 on the C-terminus of NHE1. Stable NHE1 deficient cell lines, expressing wild type (WT) NHE or the four mutated sites (F4MUTNHE), were assessed for apoptotic alkalinization using the pH-sensitive fluorescent protein, destabilized YFP. Our results show that NHE1 is required for apoptotic alkalinization, since expression of WT NHE restored alkalinization in an NHE deficient cell line, and that this process requires the phosphorylation of the p38 MAPK target sites, since mutation of all four sites prevented the apoptotic alkalinization response.
Show less - Date Issued
- 2006
- Identifier
- CFE0001078, ucf:46768
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0001078
- Title
- INTERLEUKIN-7 DIFFERENTIALLY REGULATES THE ACTIVATION, PROLIFERATION, AND HOMING OF T-CELLS: IMPLICATIONS FOR IMMUNOTHERAPY.
- Creator
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Kittipatarin, Christina, Khaled, Annette, University of Central Florida
- Abstract / Description
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Interleukin-7 (IL-7) is an essential lymphocyte growth factor required for the survival and proliferation of mature T-cells. As a therapeutic agent, IL-7 has the potential to restore T-cell numbers following immune depletion and to promote immunity against cancers. While the survival function of IL-7 is well established, less is known about how it supports T-cell expansion, a critical feature of the immune response. To study the biological effects of IL-7 on T-cell growth, we developed an in...
Show moreInterleukin-7 (IL-7) is an essential lymphocyte growth factor required for the survival and proliferation of mature T-cells. As a therapeutic agent, IL-7 has the potential to restore T-cell numbers following immune depletion and to promote immunity against cancers. While the survival function of IL-7 is well established, less is known about how it supports T-cell expansion, a critical feature of the immune response. To study the biological effects of IL-7 on T-cell growth, we developed an in vitro culture technique to expand T-cells ex vivo. A significant finding from our studies is that IL-7 did not induce the expansion of all T-cells, indicating that there are inherent differences in the response of individual T-cell subsets to IL-7. Culture with high doses of IL-7 (>150 ng/ml) preferentially expanded CD8 T-cells, but lead to the dramatic loss of CD4 T-cells which favored growth in lower dosages of IL-7 (<10 ng/ml). This effect was due to the regulation of LCK, a kinase predominantly associated with the CD4 co-receptor. We found that transgenic expression of the CD4 co-receptor onto CD8 T-cells promoted their growth in lower concentrations of IL-7. Conversely, inhibition of LCK activity in CD4 T-cells restored their responsiveness to high doses of IL-7 as indicated by the activation of the transcription factor STAT5, in a manner similar to CD8 T-cells. Interestingly, not all CD8 T-cells expanded in high doses of IL-7 and this effect was specific to CD8 T-cells that expressed an activated memory phenotype. We found that IL-7 promoted the proliferation of CD8 T-cells through Cdc25A, a phosphatase required for cell cycle progression. Expression of a constitutively active Cdc25A could maintain T-cell survival and proliferation in the absence of IL-7, demonstrating that Cdc25A is a crucial transducer of IL-7 growth signals. Inhibition of Cdc25A was sufficient to decrease proliferation and down-regulate the expression of activation/ memory markers on CD8 T-cells in the presence of IL-7. Upon further study, we identified a novel role for IL-7 through Cdc25A in the regulation of CD62L, an adhesion molecule required for lymph node entry. Culture with high doses of IL-7 down-regulated the expression of CD62L, suggesting that high doses of IL-7 could affect the ability of T-cells to enter or re-enter the lymph nodes. Collectively, our findings demonstrate that IL-7 administration at the supraphysiological doses currently used in the clinical trials could have a negative impact on the growth of CD4 T-cells and the homing of CD8 T-cells to the lymph nodes, effects which can impede the generation of an effective immune response.
Show less - Date Issued
- 2010
- Identifier
- CFE0003372, ucf:48449
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0003372
- Title
- THE CYTOKINE, INTERLEUKIN-7, TRANSCRIPTIONALLY REGULATES THE GENE EXPRESSION OF THE HEXOKINASE II TO MEDIATE GLUCOSE UTILIZATION IN T-LYMPHOCYTES.
- Creator
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Chehtane, Mounir, Khaled, Annette, University of Central Florida
- Abstract / Description
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The cytokine, interleukin-7 (IL-7), has essential growth activities that maintain the homeostatic balance of the immune system. Little is known of the mechanism by which IL-7 signaling regulates metabolic activity in support of its vital function in lymphocytes. We observed that IL-7 deprivation caused a rapid decline in ATP levels that were attributable to loss of intracellular glucose retention. To identify the transducer of the IL-7 metabolic signal, we examined the expression of three...
Show moreThe cytokine, interleukin-7 (IL-7), has essential growth activities that maintain the homeostatic balance of the immune system. Little is known of the mechanism by which IL-7 signaling regulates metabolic activity in support of its vital function in lymphocytes. We observed that IL-7 deprivation caused a rapid decline in ATP levels that were attributable to loss of intracellular glucose retention. To identify the transducer of the IL-7 metabolic signal, we examined the expression of three important regulators of glucose metabolism, the glucose transporter, GLUT-1, and two glycolytic enzymes, Hexokinase II (HXKII) and phosphofructokinase-1 (PFK1), using an IL-7-dependent T-cell line and primary lymphocytes. We found that in lymphocytes deprived of IL-7 loss of glucose uptake correlated with decreased expression of HXKII. Re-addition of IL-7 to cytokine deprived lymphocytes restored the transcription of the HXKII gene within 2 hours, but not that of GLUT-1 or PFK1. IL-7-mediated increases in HXKII, but not GLUT-1 or PFK-1, were also observed at the protein level. Inhibition of HXKII with 3-Bromopyruvate or specific siRNA decreased glucose utilization, as well as ATP levels, in the presence of IL-7, while over-expression of HXKII, but not GLUT-1, restored glucose retention and increased ATP levels in the absence of IL-7. This IL-7 mediated HXKII gene expression was abrogated with inhibition of JNK pathway. IL-7 also increased activation of AP-1 complex and DNA binding of JunD, a transcriptional complex thought to be negative regulator of proliferation. We found that over expression of HXKII caused cell cycle arrest and cell death, indicating that a potent IL-7 signal could produce negative growth signals. We conclude that IL-7 controls glucose utilization by regulating the gene expression of HXKII through activation of JNK-JunD pathway, suggesting a mechanism by which IL-7 supports bioenergetics that control cell fate decisions in lymphocytes.
Show less - Date Issued
- 2010
- Identifier
- CFE0003354, ucf:48430
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0003354
- Title
- DISTINCT DOMAINS OF BAX ARE INVOLVED IN MITOCHONDRIAL BIOENERGETICS AND APOPTOSIS.
- Creator
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Zhang, Ge, Khaled, Annette, University of Central Florida
- Abstract / Description
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Apoptosis is essential for cellular homeostasis and is also a pathologic feature of various diseases. The balance between Bcl-2 family proteins determines whether a cell will live or die. Bax, a member of the BCL-2 family proteins, is a pro-apoptotic protein that exists in both a soluble, cytoplasmic form and a membrane-bound form. Upon apoptotic stimuli, Bax undergoes a conformational change and translocates to the mitochondria, initiating apoptotic events. However, little is known about...
Show moreApoptosis is essential for cellular homeostasis and is also a pathologic feature of various diseases. The balance between Bcl-2 family proteins determines whether a cell will live or die. Bax, a member of the BCL-2 family proteins, is a pro-apoptotic protein that exists in both a soluble, cytoplasmic form and a membrane-bound form. Upon apoptotic stimuli, Bax undergoes a conformational change and translocates to the mitochondria, initiating apoptotic events. However, little is known about whether Bax is involved in the regulation of mitochondrial function under non-apoptotic conditions, and how Bax binds to mitochondria to exert its activity. Here, we investigate the role of Bax in the regulation of mitochondrial function under non-apoptotic conditions and explore the molecular mechanisms for Bax binding mitochondria under apoptotic stimuli. Using Bax-containing and Bax-deficient (Bax-/-) HCT-116 cells, we examined Bax cellular localization and its effects on mitochondria bioenergetics, and also tested whether over-expression of full-length Bax in Bax-/- cells would recover mitochondrial metabolic activity. To determine the effects of Bax localization upon mitochondrial function, we measured citrate synthase activity and ATP generation. We showed that Bax localized to the outer and inner mitochondrial membranes in non-apoptotic cells, enabling the activity of citrate synthase and the generation of ATP. Loss of Bax led to impairment of respiring mitochondria morphology and reduced oxidative capacity, all of which was restored by expression of full-length or C-terminal-deleted Bax. These findings indicate that under non-apoptotic conditions, the constitutive expression of Bax is necessary for mitochondrial bioenergetics. To determine the molecular mechanisms for Bax binding mitochondria under apoptotic stimuli, we previously performed in silico-mutagenesis and predicted that Lysines 189/190, in the C-terminal [alpha]9 helix, could regulate Bax binding to mitochondria. We demonstrated here that these lysines are the structural elements responsible for controlling how Bax interacts with the mitochondrial membrane. Expression of full-length Bax led to mitochondrial translocation and apoptosis, whereas deletion of the [alpha]9 helix resulted in cytosolic retention and dramatically reduced cell death. Mutation of the two lysine residues changed how Bax bound to mitochondrial membranes. We replicated the results achieved with full-length Bax by attaching the [alpha]9 helix of Bax to GFP or to a regulatory element, the degradation domain (DD), and induced apoptosis upon expression in cells. We demonstrated that the [alpha]9 helix alone promoted the mitochondrial translocation of Bax and increased apoptosis. These results indicate that the C-terminal [alpha]9 helix could be further studied for use in cancer therapies. Overall, we have demonstrated that the constitutive expression of the inactive form of Bax in non-apoptotic cells is necessary for mitochondrial bioenergetics, and have identified the C-terminal [alpha]9 helix of Bax as the effector domain of apoptotic function.
Show less - Date Issued
- 2011
- Identifier
- CFE0003986, ucf:48656
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0003986
- Title
- Influence of Chitosan-Alginate Scaffold Stiffness on Bone Marrow Stromal Cell Differentiation.
- Creator
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Arias Ponce, Isabel, Florczyk, Stephen, Fang, Jiyu, Khaled, Annette, Leon, Lorraine, University of Central Florida
- Abstract / Description
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Tissue grafts are the gold standard for replacing large volume tissue defects. Yet, they present several risks, including infection, low functional outcomes, and reduced graft integrity. Tissue engineering (TE) combines cells and biomaterial scaffolds to foster tissue growth and remodeling. Bone marrow stromal cells (BMSCs) have been shown to respond to the stiffness of their microenvironment, resulting in differentiation into different lineages. 3D porous chitosan-alginate (CA) scaffolds...
Show moreTissue grafts are the gold standard for replacing large volume tissue defects. Yet, they present several risks, including infection, low functional outcomes, and reduced graft integrity. Tissue engineering (TE) combines cells and biomaterial scaffolds to foster tissue growth and remodeling. Bone marrow stromal cells (BMSCs) have been shown to respond to the stiffness of their microenvironment, resulting in differentiation into different lineages. 3D porous chitosan-alginate (CA) scaffolds have been previously demonstrated for bone TE with osteoblasts and BMSCs; however, only a single scaffold composition (4 wt%) was studied. Three CA scaffold compositions (2, 4, 6 wt% CA) were produced. Scanning electron microscopy images were obtained to determine average pore sizes for 2, 4, and 6 wt% CA scaffolds, which were 233, 208, and 146 ?m. Compression testing was performed on CA scaffolds in dry and wet conditions, where higher concentrations yielded higher stiffnesses ranging from 0.22 to 5.34 kPa and 21.1 to 47.3 Pa, respectively. Fourier transform infrared spectroscopy performed on the CA scaffolds confirmed polyelectrolyte complex formation for all compositions. Human BMSCs from three donors were seeded on CA scaffolds, cultured in growth media for 14 days, then cultured in adipogenic or osteogenic differentiation media for 28 days to promote differentiation. Our hypothesis was that scaffold stiffness would influence BMSC differentiation, with softer scaffolds promoting adipogenesis and stiffer scaffolds promoting osteogenesis. BMSCs formed multicellular spheroids in all CA scaffold concentrations, while the 2 wt% CA scaffolds had smaller spheroids compared to the 4 wt% and 6 wt% CA scaffolds. Osteogenic and adipogenic differentiation were evaluated with Alizarin Red and Oil Red O staining, respectively. While positive staining was observed in all scaffold compositions, more robust differentiation was expected, thereby disproving our hypothesis. The polysaccharide composition of the CA scaffolds likely contributed to the spheroid formation and limited differentiation.
Show less - Date Issued
- 2018
- Identifier
- CFE0007389, ucf:52061
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007389
- Title
- Involvement of miRNAs in the Development of Androgen Independent Prostate Cancer.
- Creator
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Ottman, Richard, Chakrabarti, Ratna, Cole, Alexander, Khaled, Annette, Zervos, Antonis, University of Central Florida
- Abstract / Description
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Development of resistance to androgen deprivation therapy (ADT) is a major obstacle for the management of advanced prostate cancer. Therapies with androgen receptor (AR) antagonists and androgen withdrawal initially result in tumor regression but development of compensatory mechanisms including AR bypass signaling leads to tumor re-growth, independent of circulating androgens. The result is the emergence of castration resistant prostate cancer (CRPC), a highly morbid disease exhibiting...
Show moreDevelopment of resistance to androgen deprivation therapy (ADT) is a major obstacle for the management of advanced prostate cancer. Therapies with androgen receptor (AR) antagonists and androgen withdrawal initially result in tumor regression but development of compensatory mechanisms including AR bypass signaling leads to tumor re-growth, independent of circulating androgens. The result is the emergence of castration resistant prostate cancer (CRPC), a highly morbid disease exhibiting aberrant expression of many protein-coding and non-coding genes. Under the umbrella of non-coding RNAs is a class of small regulatory RNAs referred to as microRNAs (miRNAs). MicroRNAs are believed to function in the maintenance of cell homeostasis but are often differentially expressed in many different types of cancer including CRPC.In this study, the association of genome wide miRNA expression (1113 unique miRNAs) with development of resistance to ADT was determined. Androgen sensitive prostate cancer cells that progressed to ADT and AR antagonist Casodex (CDX) resistance upon androgen withdrawal and treatment with CDX were used. Validation of expression of a subset of 100 miRNAs led to identification of 43 miRNAs that are significantly altered during progression of cells to treatment resistance. A correlation of altered expression of 10 proteins targeted by some of these miRNAs in these cells was shown.Additionally, profiles of miRNA expressions in cancerous prostate tissues were created and compared with profiles of paired adjacent uninvolved areas of prostate tissue. Among the miRNAs identified from these analyses, a cluster of miRNAs, miR-17-92a, that is under-expressed in prostate tumors and in androgen independent prostate cancer cells was highlighted. The miR-17-92a cluster miRNAs are transcribed from a polycistronic transcription unit C13orf25 that generates six mature miRNAs: miR-17, miR-18a, miR-19a, miR-19b, miR-20a and miR-92a, and is commonly de-regulated in many cancers. In this research, the expression of miR-17-92a miRNAs was found to be reduced in cancerous prostate tissues when compared to uninvolved areas and also in aggressive prostate cancer cells. Restoration of expression of all members of miR-17-92a cluster showed decreased expression of cell cycle regulatory proteins cyclin D1 and SSH1; as well as LIMK1 and FGD4 of the RhoGTPase signaling pathway. Expression of miR-17-92a miRNAs caused decreased cell proliferation, reduced activation of AKT and MAP kinases, delayed tumorigenicity and reduced tumor growth in animals. Additionally, miR-17-92a miRNA expression inhibited EMT via reduced cell migration and expression of mesenchymal markers while elevating expression and surface localization of the epithelial marker e-cadherin. Expression of miR-17-92a miRNAs improved sensitivity of androgen dependent LNCaP104-S prostate cancer cells to the Androgen Receptor antagonist bicalutamide (CDX), AKT inhibitor MK-2206 2HCl, and docetaxel. Androgen refractory PC-3 cells also showed increased sensitivity to docetaxel, MK-2206 2HCl, and Aurora kinase inhibitor VX680 upon ectopic expression of miR-17-92a cluster miRNAs. In conclusion, dynamic alterations in miRNA expression occur early on during androgen deprivation therapy and androgen receptor blockade. The cumulative effect of these altered miRNA expression profiles is the temporal modulation of multiple signaling pathways promoting survival and acquisition of resistance. These early events are driving the transition to castration resistance and cannot be studied in already developed CRPC cell lines or tissues. Notably, these data demonstrate a tumor suppressor effect of miR-17-92a cluster miRNAs in prostate cancer cells and restoration of expression of these miRNAs has a therapeutic benefit for both androgen-dependent and -independent prostate cancer cells. Furthermore, these results can be used as a prognostic marker of cancers with a potential to be resistant to ADT.
Show less - Date Issued
- 2016
- Identifier
- CFE0006697, ucf:52866
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006697
- Title
- A Cytoplasmic-Replicating RNA Virus Sensitizes Cancer Cells to DNA Modifying Agents.
- Creator
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Fox, Candace, Parks, Griffith, Copik, Alicja, Khaled, Annette, Zervos, Antonis, University of Central Florida
- Abstract / Description
-
The Parainfluenza virus 5 (PIV5) mutant P/V-CPI- is restricted for spread in normal cells but not in cancer cells in vitro and is effective at reducing tumor burden in mouse model systems. Here we show that P/V-CPI- infection of human laryngeal cancer HEp-2 cells resulted in the majority of the cells dying, but unexpectedly, a population of cells emerged as P/V-CPI- persistently infected (PI) cells. P/V-CPI- PI cells had elevated levels of basal caspase activation, and viability was highly...
Show moreThe Parainfluenza virus 5 (PIV5) mutant P/V-CPI- is restricted for spread in normal cells but not in cancer cells in vitro and is effective at reducing tumor burden in mouse model systems. Here we show that P/V-CPI- infection of human laryngeal cancer HEp-2 cells resulted in the majority of the cells dying, but unexpectedly, a population of cells emerged as P/V-CPI- persistently infected (PI) cells. P/V-CPI- PI cells had elevated levels of basal caspase activation, and viability was highly dependent on activity of cellular inhibitors of apoptosis, such as Survivin. In challenge experiments with external inducers of apoptosis, the PI cells were highly sensitive to cisplatin-induced DNA damage and cell death. This increased cisplatin sensitivity correlated with defects in the phosphorylation cascade controlling DNA damage signaling pathways, as well as translocation of damage-specific DNA binding protein 1 (DDB1) to the nucleus. Similar sensitivity to cisplatin was seen with cells during acute infection with P/V-CPI-, as well as acute infections with WT PIV5. Based on this finding, we tested the hypothesis that histone deacetylase (HDAC) inhibitors would also act with P/V-CPI- infection to enhance cancer cell killing. Using human lung and laryngeal cancer cell lines, 10 HDAC inhibitors were tested for their effect on viability of P/V-CPI- infected cells. HDAC inhibitors such as scriptaid enhanced caspase-3/7, -8 and -9 activity induced by P/V-CPI- and overall cell toxicity. Scriptaid treatment also enhanced the spread of P/V-CPI- through a population of cancer cells and suppressed interferon-beta induction through blocking phosphorylation and nuclear translocation of Interferon Regulatory Factor 3 (IRF-3). These results support a therapeutic approach of combining parainfluenza infection and chemotherapy, but also raise questions on the mechanism by which a cytoplasmic-replicating RNA virus can alter cellular DNA damage responses.
Show less - Date Issued
- 2019
- Identifier
- CFE0007803, ucf:52355
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007803
- Title
- The Dynamic Functions of Bax are Dependent on Key Structural and Regulatory Features.
- Creator
-
Boohaker, Rebecca, Khaled, Annette, Cole, Alexander, Zervos, Antonis, Tatulian, Suren, University of Central Florida
- Abstract / Description
-
Bax is an essential mediator of cell fate. Since its discovery in 1985 as a protein that interacts with the anti-apoptotic protein, Bcl-2, key elements related to its function, structure and regulation remains to be determined. To this end, mitochondrial metabolism was examined in non-apoptotic Bax-deficient HCT-116 cells as well as primary hepatocytes from Bax-deficient mice. Although mitochondrial density and mitochondrial DNA content was the same in Bax-containing and Bax -deficient cells,...
Show moreBax is an essential mediator of cell fate. Since its discovery in 1985 as a protein that interacts with the anti-apoptotic protein, Bcl-2, key elements related to its function, structure and regulation remains to be determined. To this end, mitochondrial metabolism was examined in non-apoptotic Bax-deficient HCT-116 cells as well as primary hepatocytes from Bax-deficient mice. Although mitochondrial density and mitochondrial DNA content was the same in Bax-containing and Bax -deficient cells, MitoTracker staining patterns differed, suggesting the existence of Bax -dependent functional differences in mitochondrial physiology. Oxygen consumption and cellular ATP levels were reduced in Bax -deficient cells, while glycolysis was increased. These results suggest that cells lacking Bax have a deficiency in the ability to generate ATP through cellular respiration, supported by detection of reduced citrate synthase activity in Bax -deficient cells. Expression of either full length or C-terminal truncated Bax in Bax -deficient cells rescued ATP synthesis and oxygen consumption and reduced glycolytic activity, suggesting that this metabolic function of Bax was not dependent upon its C-terminal helix. Expression of BCL-2 in Bax-containing cells resulted in a subsequent loss of ATP measured, implying that, even under non-apoptotic conditions, an antagonistic interaction exists between the two proteins. Bax is composed of nine alpha-helices. While three of these helices have features of a trans-membrane region, the contribution of each domain to the apoptotic or non-apoptotic functions of Bax remains unknown. To examine this, we focused on the C-terminal alpha-9 helix, an amphipathic domain with putative membrane binding properties and discovered that it has an inherent membrane-binding and cytotoxic capacity. A peptide based on the last twenty amino acids (CT20p) of the alpha-9 helix was synthesized and proved a potent inducer of cell death independent of any apoptotic stimuli. The solubility of CT20p allowed it to be encapsulated in polymeric nanoparticles (NPs), and these CT20p-NPs caused the death of colon and breast cancer cells in vitro and induced tumor regression in vivo, using a murine breast cancer tumor model. CT20p caused increased mitochondrial membrane potential followed by cell death via membrane rupture, without the characteristic membrane asymmetry associated with apoptosis. Hence, while CT20p is based on Bax, its innate cytotoxic activity is unlike the parent protein and could be a powerful anti-cancer agent that bypasses drug resistance, can be encapsulated in tumor-targeted nanoparticles and has potential application in combination therapies to activate multiple death pathways in cancer cells. While previous work revealed novel aspects of the biology of Bax that were unrecognized, new structural information is needed to fully elucidate the complexity of Bax's function. One approach is to use computational modeling to assess the solved structure of Bax and provide insight into the structural components involved in the activity of the protein. Use of molecular dynamics simulators such as GROMACS, as well as other computational tools provides a powerful means by which to test the feasibility of certain modifications in defined parameters. Such work revealed that the removal of the C-terminal alpha-9 helix of Bax, which normally resides within a hydrophobic pocket, significantly destabilized the protein, perhaps explaining how the protein transitions from soluble to membrane-bound form and maintain energy production via aerobic respiration or, conversely, how the C-terminus helix conveys cytotoxicity. Collectively, this work reveals that Bax is more than an inducer of cell death but has complex activities yet to be determined.
Show less - Date Issued
- 2012
- Identifier
- CFE0004521, ucf:49285
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004521
- Title
- Dissertation Title: Development of molecular and cellular imaging tools to evaluate gene and cell based therapeutic strategies in vivo.
- Creator
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Xia, Jixiang, Ebert, Steven, Khaled, Annette, Cheng, Zixi, Daniell, Henry, University of Central Florida
- Abstract / Description
-
Molecular imaging modalities are important tools to evaluate the efficacy of gene delivery systems and cell-based therapies. Development and application of these modalities will advance our understanding of the mechanism of transgene expression and cell fate and functions. Physical gene transfer methods hold many advantages over viral vectors among gene therapeutic strategies. Here, we evaluated the efficacy of biolistic ((")gene gun(")) gene targeting to tissues with non-invasive...
Show moreMolecular imaging modalities are important tools to evaluate the efficacy of gene delivery systems and cell-based therapies. Development and application of these modalities will advance our understanding of the mechanism of transgene expression and cell fate and functions. Physical gene transfer methods hold many advantages over viral vectors among gene therapeutic strategies. Here, we evaluated the efficacy of biolistic ((")gene gun(")) gene targeting to tissues with non-invasive bioluminescence imaging (BLI) methods. Plasmids carrying the firefly luciferase reporter gene were transfected into mouse skin and liver using biolistics, and BLI was measured at various time points after transfer. With optimized DNA loading ratio (DLRs), reporter gene expression reached to peak 1day after transfer to mouse skin, and the maximum depth of tissue penetration was between 200-300?m. Similar peak expression of reporter gene was found in mouse liver but the expression was relatively stable 4-8 days post-biolistic gene transfer and remained for up to two weeks afterward. Our results demonstrated BLI was an efficient strategy for evaluation of reporter gene expression in the same animals over a period of up to two weeks in vivo. Different tissues showed different expression kinetics, suggesting that this is an important parameter to consider when developing gene therapy strategies for different target tissues. We also employed BLI to measure differentiation of mouse embryonic stem (ES) cells into beating cardiomyocytes in vitro and in vivo. A subset of these cardiomyocytes appears to be derived from an adrenergic lineage that ultimately contribute to substantial numbers of cardiomyocytes primarily on the left side of the heart. At present, it is unclear what the precise role of these cardiac adrenergic cells is with respect to heart development, though it is known that adrenergic hormones (adrenaline and noradrenaline) are essential for embryonic development since mice lacking them die from apparent heart failure during the prenatal period. To identify and characterize cardiac adrenergic cells, we developed a novel mouse genetic model in which the nuclear-localized enhanced green fluorescent protein (nEGFP) reporter gene was targeted to the first exon of the Phenylethanoamine N-transferase (Pnmt) gene, which encodes for the enzyme that converts noradrenaline to adrenaline, and hence serves as a marker for adrenergic cells. Our results demonstrate this knock-in strategy effectively marked adrenergic cells in both fetal and adult mice. Expression of nEGFP was found in Pnmt-positive cells of the adult adrenal medulla, as expected. Pnmt-nEGFP expression also recapitulated endogenous Pnmt expression in the embryonic mouse heart. In addition, nEGFP and Pnmt expression were induced in parallel during differentiation of pluripotent mouse ES cells into beating cardiomyocytes. This new mouse genetic model provides a useful new tool for studying the properties of adrenergic cells in different tissues. We also identified two limitations of the Pnmt-nEGFP model. One is that the amount of nEGFP expressed within individual adrenergic cells was highly variable. Secondly, expression of nEGFP in the embryonic heart was of low abundance and difficult to distinguish from background autofluorescence. To overcome these limitations, we developed two alternative genetic models to investigate adrenergic cells: (1) Mouse embryonic stem cells, which have been previously targeted with Pnmt-Cre recombinase gene, were additionally targeted with a dual reporter plasmid which covered both a loxP-flanked cDNA of red fluorescence protein (HcRed) and also EGFP. Under the undifferentiated status, cells emit red fluorescence as transcription stops before EGFP coding sequence. After differentiation into beating cardiomyoctyes, some cells switch fluorescence from red to green, indicating that excision of loxP-flanked sequences by Cre since Pnmt had been activated. (2) A surface marker, truncated low-affinity nerve growth factor receptor (?LNGFR) was used as the reporter gene as cells expressing this marker can be enriched by magnetic-activated cell sorting (MACS), a potentially efficient way to yield highly purified positive cells at low input abundance in a population. Through a series of subcloning steps, the targeting construct, Pnmt-?LNGFR-Neo-DTA was created and electroporated into 7AC5EYFP embryonic stem cells. Correctly targeted cells were selected by positive and negative screening. These cells provide a new tool with which to identify, isolate, and characterize the function of adrenergic cells in the developing heart, adrenal gland, and other tissues where adrenergic cells make important contributions.
Show less - Date Issued
- 2011
- Identifier
- CFE0004491, ucf:49287
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004491
- Title
- Stable expression of tuberculosis vaccine antigen in lettuce chloroplasts.
- Creator
-
Saikumar Lakshmi, Priya, Daniell, Henry, Khaled, Annette, Naser, Saleh, University of Central Florida
- Abstract / Description
-
Tuberculosis (TB) is caused by Mycobacterium tuberculosis and is one of the leading reasons of death by an infectious bacterial pathogen. The development of TB vaccines has been recognized as a major public health priority by the World Health Organization. In this study, a potential candidate antigen, ESAT-6 (6 kDa early secretory antigenic target) was fused with cholera toxin B subunit (CTB). Transplastomic lettuce plants were generated expressing these fusion proteins. Site-specific...
Show moreTuberculosis (TB) is caused by Mycobacterium tuberculosis and is one of the leading reasons of death by an infectious bacterial pathogen. The development of TB vaccines has been recognized as a major public health priority by the World Health Organization. In this study, a potential candidate antigen, ESAT-6 (6 kDa early secretory antigenic target) was fused with cholera toxin B subunit (CTB). Transplastomic lettuce plants were generated expressing these fusion proteins. Site-specific transgene integration into the chloroplast genome was confirmed by polymerase chain reaction and Southern blot analysis. In transplastomic leaves, expression levels of fusion protein (CTB-ESAT6) varied depending upon the developmental stage and time of leaf harvest with highest-level of accumulation in mature leaves harvested at 6PM. Transplastomic CTB-ESAT6 lettuce plants accumulated up to 0.75% of total leaf protein. Lyophilization increased CTB-ESAT6 protein content per gram of leaf material by 22 fold. Western blot analysis of lyophilized lettuce leaves showed that the CTB-ESAT6 fusion protein was stable and can be stored for prolonged period at RT. Hemolysis assay with purified CTB-ESAT6 protein showed partial hemolysis of red blood cells and confirmed functionality of ESAT-6 antigen. GM-1 binding assay demonstrated that the CTB-ESAT6 fusion protein formed pentamers to interact with GM1 ganglioside receptor. The expression of functional Mycobacterium tuberculosis antigens fused to CTB in transplastomic plants should facilitate development of a cost-effective and orally deliverable TB vaccine with potential for long term storage at room temperature.
Show less - Date Issued
- 2011
- Identifier
- CFE0004487, ucf:49303
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004487
- Title
- Signals Delivered By Interleukin-7 Regulate The Activities Of Bim And JunD In T Lymphocytes.
- Creator
-
Ruppert, Shannon, Khaled, Annette, Self, William, Zervos, Antonis, Teter, Kenneth, University of Central Florida
- Abstract / Description
-
Interleukin-7 (IL-7) is an essential cytokine for lymphocyte growth that has the potential for promoting proliferation and survival. While the survival and proliferative functions of IL-7 are well established, the identities of IL-7 signaling components in pathways other than JAK/STAT, that accomplish these tasks remain poorly defined. To this end, we used IL-7 dependent T-cells to examine those components necessary for cell growth and survival. Our studies revealed two novel signal...
Show moreInterleukin-7 (IL-7) is an essential cytokine for lymphocyte growth that has the potential for promoting proliferation and survival. While the survival and proliferative functions of IL-7 are well established, the identities of IL-7 signaling components in pathways other than JAK/STAT, that accomplish these tasks remain poorly defined. To this end, we used IL-7 dependent T-cells to examine those components necessary for cell growth and survival. Our studies revealed two novel signal transducers of the IL-7 growth signal: BimL and JunD. IL-7 promoted the activity of JNK (Jun N-terminal Kinase), and that JNK, in turn, drove the expression of JunD, a component of the Activating Protein 1 (AP-1) transcription factors. Inhibition of JNK/JunD blocked glucose uptake and HXKII gene expression, indicating that this pathway was responsible for promoting HXKII expression. After a bioinformatics survey to reveal possible JunD-regulated genes activated early in the IL-7 signaling cascade, our search revealed that JunD could control the expression of proteins involved in signal transduction, cell survival and metabolism, including Pim-1. Pim-1, an IL-7 induced protein, was inhibited upon JNK or JunD inhibition. Our hypothesis that JunD positively regulated proliferation was confirmed when the proliferation of primary CD8+ T-cells cultured with IL-7 was impaired upon treatment with JunD siRNA. These results show that the IL-7 signal is more complex than the JAK/STAT pathway, activating JNK and JunD to induce rapid growth through the expression of metabolic factors like HXKII and Pim-1. When metabolic activities are inhibited, cells undergo autophagy, or cell scavenging, to provide essential nutrients. Pro-apoptotic Bim was evaluated for its involvement in autophagy. Bim is a BH3-only member of the Bcl-2 family that contributes to T-cell death. Partial rescue of T-cells occurs when Bim and the interleukin-7 receptor are deleted, implicating Bim in IL-7-deprived T-cell apoptosis. Alternative splicing results in three different isoforms: BimEL, BimL, and BimS. To study the effect of Bim deficiency and define the function of the major isoforms, Bim-containing and Bim-deficient T-cells, dependent on IL-7 for growth, were used. Loss of Bim in IL-7-deprived T-cells delayed apoptosis, but blocked the degradative phase of autophagy. The conversion of LC3-I to LC3-II was observed in Bim-deficient T-cells, but p62, which is degraded in autolysosomes, accumulated. To explain this, BimL, was found to support acidification of lysosomes associated with autophagic vesicles. Key findings showed that inhibition of lysosomal acidification accelerated death upon IL-7 withdrawal only in Bim-containing T-cells, indicating that in these cells autophagy was protective. IL-7 dependent T-cells lacking Bim were insensitive to inhibition of autophagy or lysosomal acidification. BimL co-immunoprecipitated with dynein and Lamp1-containing vesicles, indicating BimL could be an adaptor for dynein to facilitate loading of lysosomes. In Bim deficient T-cells, lysosome-tracking probes revealed vesicles of less acidic pH. Over-expression of BimL restored acidic vesicles in Bim deficient T-cells, while other isoforms, BimEL and BimS, associated with intrinsic cell death. These results reveal a novel role for BimL in lysosomal positioning that may be required for the formation of functional autolysosomes during autophagy.
Show less - Date Issued
- 2012
- Identifier
- CFE0004435, ucf:49331
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004435
- Title
- Diabetes Phenotypes in Transgenic Pancreatic Cancer Mouse Models.
- Creator
-
Albury, Toya, Altomare, Deborah, Zhao, Jihe, Masternak, Michal, Khaled, Annette, University of Central Florida
- Abstract / Description
-
Protein Kinase B/AKT, a serine/threonine kinase with three isoforms (AKT1-3), is downstream of phosphatidylinositol 3-kinase (PI3K), and signals through the phosphorylation and subsequent activation or inhibition of downstream substrates, such as mammalian target of rapamycin complex 1 (mTORC1) or glycogen synthase kinase 3 beta (GSK-3?), respectively. The AKT1 isoform is predominantly recognized for regulation of cell survival, growth, and proliferation, due to its constitutive activation in...
Show moreProtein Kinase B/AKT, a serine/threonine kinase with three isoforms (AKT1-3), is downstream of phosphatidylinositol 3-kinase (PI3K), and signals through the phosphorylation and subsequent activation or inhibition of downstream substrates, such as mammalian target of rapamycin complex 1 (mTORC1) or glycogen synthase kinase 3 beta (GSK-3?), respectively. The AKT1 isoform is predominantly recognized for regulation of cell survival, growth, and proliferation, due to its constitutive activation in pancreatic cancers (e.g., islet cell carcinoma and pancreatic adenocarcinoma). The progression of pancreatic ductal adenocarcinoma (PDAC), the most lethal common cancer, is initiated by activation mutations of the KRas oncogene. This leads to additional molecular changes, such as activation of the AKT1 oncogene, which drives PDAC progression and tumor formation. By mating transgenic mice with activation of KRas (Pdx- Cre;LSL-KRasG12D) and mice with activation of AKT1 (Pdx- Tta;TetO-MyrAKT1) we were able to produce mice with two activated oncogenes (AKT1Myr/KRasG12D) for comparative studies. Kaplan-Meier survival curves, histology, and genomic/proteomic analysis were used to characterize the incidence and frequency of histological (e.g. presence of mucin-4 in pancreatic intraepithelial neoplasms) and genetic (e.g. loss of tumor suppressors p16Ink4a and p19Arf) alterations known to commonly occur in human pancreatic cancer, as well as delineate the role of AKT1 in accelerating pancreatic tumor progression and metastasis. We determined that AKT1Myr/KRasG12D mice, unlike other PDAC mouse models, accurately mimic the human PDAC progression molecularly, structurally, and temporally. Interestingly, the AKT1Myr and AKT1Myr/KRasG12D models both exhibit a pre-tumor, diabetic phenotype. While, AKT1 hyperactivation in various cancers has been thoroughly studied, its role in glucose metabolism has been noted, but comparatively overlooked. As early as the 1900s a relationship between diabetes and pancreatic cancer has been proposed. With 80% of PDAC patients suffering from hyperglycemia or diabetes prior to diagnosis, one prevailing theory is that new onset diabetes is an early marker for pancreatic cancer. This is also supported by experimental and clinical studies, such as the resolution of diabetes with tumor removal and the induction of hyperglycemia with the implantation of cancer cell lines. To better understand the role of AKT1 and its hyperactivation in glucose metabolism, AKT1Myr mice were characterized via metabolic (e.g. glucose/insulin tolerance test) and histological (e.g. immunohistochemistry) studies. Beginning at weaning, 3 weeks of age, the glucose intolerant AKT1Myr mice exhibited non-fasted hyperglycemia, which progressed to fasted hyperglycemia by 5 months of age. The glucose intolerance was attributed to a fasted hyperglucagonemia, and hepatic insulin resistance detectable by reduced phosphorylation of the insulin receptor following insulin injection into the inferior vena cava. Additionally, AKT1Myr/KRasG12D mice currently being studied, appear to display a more severe diabetic phenotype, with fasted hyperglycemia noticeable at an earlier age, fasted hyperglucagonemia, polyuria, muscle wasting, and bloating. Treatment of both models with doxycycline diet, to turn-off the transgene, caused attenuation of the non-fasted and fasted hyperglycemia, thus affirming AKT1 hyperactivation as the trigger. These newly revealed roles of AKT1, along with future studies of these mouse models, will better delineate the molecular mechanisms responsible for the individual and joint roles of AKT1 and KRas in pancreatic cancer oncogenesis, the initiation of cancer associated diabetes, and the association of these two diseases.
Show less - Date Issued
- 2015
- Identifier
- CFE0006245, ucf:51081
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006245
- Title
- Development of Cytotoxic Natural Killer Cells for Ovarian Cancer Treatment.
- Creator
-
Pandey, Veethika, Altomare, Deborah, Zhao, Jihe, Khaled, Annette, Estevez, Alvaro, University of Central Florida
- Abstract / Description
-
Ovarian cancer is a leading cause of gynecological malignancy. Cytoreductive surgery and frontline platinum/taxane-based chemotherapy provides good initial efficacy in the treatment, but poor long-term patient survival. This is mainly caused by tumor relapse due to intraperitoneal spreading and ineffective alternate therapies to treat these resistant tumors. The challenge in the field is to develop strategies that would prove effective in these patients and extend overall survival.Over the...
Show moreOvarian cancer is a leading cause of gynecological malignancy. Cytoreductive surgery and frontline platinum/taxane-based chemotherapy provides good initial efficacy in the treatment, but poor long-term patient survival. This is mainly caused by tumor relapse due to intraperitoneal spreading and ineffective alternate therapies to treat these resistant tumors. The challenge in the field is to develop strategies that would prove effective in these patients and extend overall survival.Over the years, various treatments have been developed for the treatment of cancer amongst which, adoptive cell immunotherapy has shown promising results. But despite the efficacy seen in the clinic, there are concerns with the complexity of treatment and associated side effects. Therefore, there is still a need for better understanding of how different components of the immune system react to the presence of tumor. In this study, healthy human peripheral blood mononuclear cells (PBMCs) were used to examine the immune response in a mouse model with residual human ovarian tumor, where natural killer (NK) cells were found to be the effector cells that elicited an anti-tumor response. Presence of tumor was found to stimulate NK cell expansion and cytotoxicity in mice treated intraperitoneally (IP) with PBMCs+Interleukin-2 (IL- 2). Intravenous (IV) adoptive transfer of isolated NK cells has been attempted in ovarian cancer patients before, but showed lack of persistence in patients resulting in lack of anti-tumor efficacy. Experiments in this study highlight the significance of NK cell-cytotoxic response to tumor, which may be attributed to interacting immune cell types in the PBMC population (when treated IP), as opposed to clinically used isolated NK cells showing lack of anti-tumor efficacy in ovarian cancer patients (when treated IV).iiiNK cell immunotherapy is mainly limited by insufficient numbers generated for adoptive transfer, limited in vivo life span after adoptive transfer, lack of cytotoxicity and some logistical concerns that impede its widespread implementation. Therefore there is a need to develop methods of NK cell expansion that provide stimulation similar to other immune cell types in the PBMC population. The second part of this study utilizes a method of in vivo NK cell expansion using a particle-based approach in which plasma membranes of K562-MB21-41BBL cells (K562 cells expressing membrane-bound IL-21 and 41BB ligand) are used for specific NK cell expansion from PBMCs. NK cells expanded with this method were cytotoxic, showed in vivo persistence and biodistribution in different organs.Collectively, these studies show that NK cells are a major innate immune component that can recognize and kill the tumor. Their cytotoxic ability, using particle-based stimulation, can be enhanced for a second-line treatment of relapsed tumors such as in ovarian cancer as well as other cancer types.
Show less - Date Issued
- 2015
- Identifier
- CFE0006369, ucf:51531
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006369
- Title
- Structural and functional characterization of enzymes of a novel group of tryptophylquinone cofactor containing oxidases.
- Creator
-
Sehanobish, Esha, Davidson, Victor, Khaled, Annette, Self, William, Tatulian, Suren, University of Central Florida
- Abstract / Description
-
Protein-derived cofactors are redox and catalytic centers that are generally formed by the posttranslational modifications of one or more amino acids. An important class of these cofactors are the quinones derived from tyrosine and tryptophan. Amongst redox proteins, it has been known till now that oxidases either contain a flavin or a tyrosylquinone cofactor, whereas tryptophylquinone ones are present within the dehydrogenases. In recent times, oxidases from a marine bacterium, Marinomonas...
Show moreProtein-derived cofactors are redox and catalytic centers that are generally formed by the posttranslational modifications of one or more amino acids. An important class of these cofactors are the quinones derived from tyrosine and tryptophan. Amongst redox proteins, it has been known till now that oxidases either contain a flavin or a tyrosylquinone cofactor, whereas tryptophylquinone ones are present within the dehydrogenases. In recent times, oxidases from a marine bacterium, Marinomonas mediterranea, have been shown to possess the latter. This study involves the characterization of two such proteins, lysine-?-oxidase (LodA) and glycine oxidase (GoxA). They have been reported to contain the same cysteine tryptophylquinone (CTQ) cofactor. Both require the co-expression of a second protein, LodB and GoxB respectively to generate matured CTQ containing active protein. Kinetic analysis of the reaction catalyzed by LodA showed that it followed the usual Michaelis-menten mode of interaction with its substrates. GoxA on the other hand exhibited allosteric cooperativity for its substrate glycine. This was attributed to the dimeric conformation of the wildtype GoxA based on size exclusion chromatographic studies. Mutagenesis study of amino acid residues based on the crystal structure of LodA and a homology model of GoxA, have given a detailed idea about their structure-function relationship. Kinetic studies on mutants of Tyr211 of LodA along with Lys530 present at the substrate channel, showed effects on both Km for the substrates and kcat for the reaction. As a result these residues have shown their involvement in forming a gate-like structure to control the to and fro movement of the substrate and products. Corresponding to this residue, the Phe237 of GoxA has proved to be important in maintaining the allostericity, by mediating the stable dimer formation. From the kinetic parameters, Cys448 of LodA was found to be responsible for substrate specificity and affinity. Whereas, mutants of His466 of GoxA that correspond to the Cys448 residue, were unable to yield CTQ containing active GoxA. On the other hand, Asp512 of LodA and Asp547 of GoxA that correspond to each other, have been implicated for their involvement in CTQ biogenesis. This study therefore highlights how even though this new pool of enzymes have great degrees of similarity in terms of the cofactors and conserved active site residues, there are major differences in the mechanism of the reaction that they catalyze which on a broader sense could influence the overall physiological importance of the enzyme in the biological system.
Show less - Date Issued
- 2016
- Identifier
- CFE0006390, ucf:51494
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006390
- Title
- Role of KLF8-CXCR4 signaling in Breast Cancer Metastasis.
- Creator
-
Mukherjee, Debarati, Zhao, Jihe, Khaled, Annette, Altomare, Deborah, Siddiqi, Shadab, University of Central Florida
- Abstract / Description
-
Kr(&)#252;ppel-like factor 8 (KLF8) has been strongly implicated in breast cancer metastasis. However, the underlying mechanisms remain largely unknown. In this study we report a novel signaling from KLF8 to C-X-C cytokine receptor type 4 (CXCR4) in breast cancer. Overexpression of KLF8 in MCF-10A cells induced CXCR4 expression at both mRNA and protein levels. This induction was well correlated with increased Boyden chamber migration, matrigel invasion and transendothelial migration (TEM) of...
Show moreKr(&)#252;ppel-like factor 8 (KLF8) has been strongly implicated in breast cancer metastasis. However, the underlying mechanisms remain largely unknown. In this study we report a novel signaling from KLF8 to C-X-C cytokine receptor type 4 (CXCR4) in breast cancer. Overexpression of KLF8 in MCF-10A cells induced CXCR4 expression at both mRNA and protein levels. This induction was well correlated with increased Boyden chamber migration, matrigel invasion and transendothelial migration (TEM) of the cells towards the ligand CXCL12. On the other hand, knockdown of KLF8 in MDA-MB-231 cells reduced CXCR4 expression associated with decreased cell migration, invasion and TEM towards CXCL12. Histological and database mining analyses of independent cohorts of patient tissue microarrays revealed a correlation of aberrant co-elevation of KLF8 and CXCR4 with metastatic potential. Promoter analysis indicated that KLF8 directly binds and activates the human CXCR4 gene promoter. Furthermore, CXCR4-CXCL12 engagement downstream of KLF8 leads to the feed-forward activation of FAK. Interestingly, KLF8 expression, through CXCR4 engagement, triggered the formation of filopodium-like protrusions (FLP) and thereby enhanced the proliferation rate of breast cancer cells in 3D Matrigel-on-Top culture, under prolonged treatment with CXCL12. This indicates that KLF8 plays a major role in promoting aggressive colonization of tumor cells in a CXCL12-enriched foreign tissue microenvironment, thereby aiding in secondary macrometastasis formation. Xenograft studies showed that overexpression of CXCR4, but not a dominant-negative mutant of it, in the MDA-MB-231 cells prevented the invasive growth of primary tumor and lung metastasis from inhibition by knockdown of KLF8. Apart from lung, KLF8 overexpression also induced spontaneous secondary metastasis to other CXCL12-rich organs through CXCR4 signaling. These results collectively suggest a critical role for KLF8 and the CXCR4-CXCL12 pathway in promoting breast cancer metastasis and shed new light on potentially more effective anti-cancer strategies.
Show less - Date Issued
- 2016
- Identifier
- CFE0006149, ucf:51127
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006149
- Title
- The CT20 peptide as an agent for cancer treatment.
- Creator
-
Bassiouni, Rania, Khaled, Annette, Altomare, Deborah, Zhao, Jihe, Estevez, Alvaro, University of Central Florida
- Abstract / Description
-
Due to cancer recurrence and the development of drug resistance, metastatic breast cancer is a leading cause of death in women. In the search for a new therapeutic to treat metastatic disease, we discovered CT20p, an amphipathic peptide based on the C-terminus of Bax. Due to inherent properties of its sequence and similarity to antimicrobial peptides, CT20p is a promising cytotoxic agent whose activity is distinct from the parent protein (e.g. does not cause apoptosis). CT20p is not membrane...
Show moreDue to cancer recurrence and the development of drug resistance, metastatic breast cancer is a leading cause of death in women. In the search for a new therapeutic to treat metastatic disease, we discovered CT20p, an amphipathic peptide based on the C-terminus of Bax. Due to inherent properties of its sequence and similarity to antimicrobial peptides, CT20p is a promising cytotoxic agent whose activity is distinct from the parent protein (e.g. does not cause apoptosis). CT20p is not membrane permeable but can be introduced to cells using polymeric nanoparticles, a method that promotes efficient delivery of the peptide into the intracellular environment.We demonstrated that CT20p was cytotoxic using triple negative breast cancer (TNBC) cell lines, primary breast tumor tissue, and breast tumor murine xenografts. Importantly, normal breast epithelial cells and normal primary breast cells were resistant to the lethal effects of the peptide. Examination of multiple cellular processes showed that CT20p causes cell death by promoting cytoskeletal disruption, cell detachment, and loss of substrate-mediated survival signals.In order to identify the intracellular target of CT20p, we performed pull-down experiments using a biotinylated peptide and found that CT20p binds directly to a type II chaperonin called chaperonin containing T-complex (CCT), which is essential for the folding of actin and tubulin into their native forms. The resulting effect of CT20p upon the cytoskeleton of cancer cells is disruption of vital cellular processes such as migration and adhesion. CCT gene expression and protein levels were examined across several breast cancer cell lines, and we found that susceptibility to CT20p correlated with higher CCT levels. Using human cancer tissue microarrays, we determined that CCT was present in significantly higher amounts in tumor tissues compared to normal tissues and that expression often increased with advanced cancer stage. These results indicate that CCT is a promising therapeutic target for the treatment of metastatic breast cancer and suggest that the use of cancer-targeted nanoparticles loaded with CT20p is a novel and effective therapeutic strategy for cancers, such as TNBC, that recur and are refractory to current treatments.
Show less - Date Issued
- 2015
- Identifier
- CFE0006207, ucf:51095
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006207
- Title
- Development of human and rodent based in vitro systems toward better translation of bench to bedside in vivo results.
- Creator
-
Berry, Bonnie, Hickman, James, Khaled, Annette, Lambert, Stephen, Sugaya, Kiminobu, University of Central Florida
- Abstract / Description
-
Prospective medicinal compounds progress through multiple testing phases before becoming licensed drugs. Testing of novel compounds includes a preclinical phase where the potential therapeutic is tested in vitro and/or in animal models in vivo to predict its potential efficacy and/or toxicity in humans. The failure of preclinical models to accurately predict human drug responses can lead to potentially dangerous compounds being administered to humans, or potentially beneficial compounds being...
Show moreProspective medicinal compounds progress through multiple testing phases before becoming licensed drugs. Testing of novel compounds includes a preclinical phase where the potential therapeutic is tested in vitro and/or in animal models in vivo to predict its potential efficacy and/or toxicity in humans. The failure of preclinical models to accurately predict human drug responses can lead to potentially dangerous compounds being administered to humans, or potentially beneficial compounds being kept in development abeyance. Moreover, inappropriate choice in model organism for studying disease states may result in pushing forward inappropriate drug targets and/or compounds and wasting valuable time and resources in producing much-needed medications. In this dissertation, models for basic science research and drug testing are investigated with the intention of improving current preclinical models in order to drive drugs to market faster and more efficiently. We found that embryonic rat hippocampal neurons, commonly used to study neurodegenerative disease mechanisms in vitro, take 3-4 weeks to achieve similar, critical ion-channel expression profiles as seen in adult rat hippocampal cultures. We also characterized a newly-available commercial cell line of human induced pluripotent stem cell-derived neurons for their applicability in long-term studies, and used them to develop a more pathologically relevant model of early Alzheimer's Disease in vitro. Finally, we attempted to create an engineered, layered neural network of human neurons to study drug responses and synaptic mechanisms. Utilization of the results and methods described herein will help push forward the development of better model systems for translation of laboratory research to successful clinical human drug trials.
Show less - Date Issued
- 2015
- Identifier
- CFE0006261, ucf:51031
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006261
- Title
- The contribution of visceral fat to positive insulin signaling in Ames dwarf mice.
- Creator
-
Menon, Vinal, Masternak, Michal, Khaled, Annette, Altomare, Deborah, Estevez, Alvaro, University of Central Florida
- Abstract / Description
-
Ames dwarf (df/df) mice are homozygous for a spontaneous mutation in the prop1 gene due to which there is no development of anterior pituitary cells (-) somatotrophs, lactotrophs and thyrotrophs, leading to a deficiency of growth hormone (GH), prolactin (PRL) and thyrotropin (TSH). They tend to become obese as they age, but still live longer and healthier lives compared to their wild-type littermates, being very insulin sensitive, showing no signs of diabetes and cancer. These mutant mice...
Show moreAmes dwarf (df/df) mice are homozygous for a spontaneous mutation in the prop1 gene due to which there is no development of anterior pituitary cells (-) somatotrophs, lactotrophs and thyrotrophs, leading to a deficiency of growth hormone (GH), prolactin (PRL) and thyrotropin (TSH). They tend to become obese as they age, but still live longer and healthier lives compared to their wild-type littermates, being very insulin sensitive, showing no signs of diabetes and cancer. These mutant mice also have high circulating levels of anti-inflammatory and anti-diabetic adiponectin. Plasma levels of this adipokine usually decrease with an increase in accumulation of visceral fat (VF). We thus believe that VF in df/df mice, developed in the absence of GH signaling, may be functionally different from the same fat depots in normal (N) mice and may be beneficial, rather than detrimental, to the overall health of the animal. We performed surgeries involving removal of VF depots (epididymal and perirenal fat) in both groups of mice and hypothesize that the beneficial effects of visceral fat removal (VFR) will be present exclusively in N mice as VF in df/df mice contributes to enhanced insulin sensitivity by producing decreased levels of pro-inflammatory adipokines like TNF? and IL-6. We found that VFR improved insulin sensitivity only in N mice but not in the df/df mice. This intervention led to an upregulation of certain players of the insulin signaling pathway in the skeletal muscle of N mice only, with no alteration in df/df mice. The subcutaneous fat of df/df mice showed a downregulation of these insulin signaling genes upon VFR. Compared to N mice, epididymal fat of df/df mice (sham-operated) had increased gene expression of some of the players involved in insulin signaling and a decrease in transcript levels of TNFa. Ames dwarf mice had decreased levels of IL-6 protein in EF and in circulation. High circulating levels of adiponectin and decreased levels of IL-6 in circulation could contribute to the high insulin sensitivity observed in the Ames dwarf mice. Understanding the mechanisms responsible for VF having positive effects on insulin signaling in df/df mice would be important for future treatment of obese diabetic patients.
Show less - Date Issued
- 2013
- Identifier
- CFE0004890, ucf:49654
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004890
- Title
- FMF assay for assessing vaccine generated antibodies in a biomimetic manner.
- Creator
-
Dhir, Vipra, Khaled, Annette, Self, William, Warren, William, University of Central Florida
- Abstract / Description
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Traditional functional assays such as hemagglutination inhibition (HAI) and micro-neutralization (MN) assays have been routinely used for assessing the vaccine response, since influenza vaccine has been administered in people (1940). Such assays are not always predictive regarding the protection conferred by the influenza vaccine and are not able to monitor neutralization related to stem region of influenza hemagglutinin responsible for virus membrane fusion in the endosomes. In order to...
Show moreTraditional functional assays such as hemagglutination inhibition (HAI) and micro-neutralization (MN) assays have been routinely used for assessing the vaccine response, since influenza vaccine has been administered in people (1940). Such assays are not always predictive regarding the protection conferred by the influenza vaccine and are not able to monitor neutralization related to stem region of influenza hemagglutinin responsible for virus membrane fusion in the endosomes. In order to study Influenza vaccine response in a more biomimetic manner and overcome the deficiencies of the traditional functional assays, we developed a fluorescent membrane fusion assay (fMF). The assay uses viruses labeled with Octadecyl Rhodmaine B Chloride (R18) to monitor two major neutralization pathways: blocking the attachment of virus to the target cells and blocking of virus membrane fusion in the endosomes. The latter was tested using endosomal acidification inhibitor Bafilomycin a1 which blocked membrane fusion by 85%. Specificity of the assay was tested using two different subtypes of viruses H1N1 (A/Puerto Rico/8/1934 and A/Brisbane/59/2007), and H3N2 virus (A/Aichi/68) with their respective subtype specific stem specific monoclonal antibodies: M145, Aca-1, Aca-2 (H1N1 specific) and Aca-3 (H3N2 specific). Subtype specific mAbs blocked membrane fusion, while a mismatch in virus subtype and the mAb resulted in lack of blocking. We also studied the effect of H1N1 head specific mAb Aca-4, which not only blocked attachment of the virus, but also demonstrated blocking of membrane fusion. Results were validated by testing pre- and post- sera from 2009 seasonal Influenza vaccination and to show that at higher Ab concentration the majority of virus (85%) was blocked from attaching cells, but at lower Ab concentration, where attachment could not be prevented, blocking of membrane fusion was still in effect - up to 50%. Sera screening experiments showed that sera antibodies work beyond just blocking attachment. They also may neutralize the already attached virus by blocking fusion of the viral membrane in the endosomes. The assay has the capacity to monitor blocking of attachment and fusion in a single run. Therefore, it is more representative regarding the natural process of infection and the corresponding neutralization pathways. The assay is unique in terms of assessing stem specific antibodies; stem specific response and its measurement are relevant for the advancement of a universal influenza vaccine.
Show less - Date Issued
- 2015
- Identifier
- CFE0005604, ucf:50255
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0005604