Browsing by Author "Finkielstein, Carla V."

Now showing 1 - 20 of 56
  • Thumbnail Image
    Association of the circadian factor Period 2 to p53 influences p53’s function in DNA-damage signaling
    Gotoh, Testuya; Vila-Caballer, Marian; Liu, Jingjing; Schifhauer, Samuel; Finkielstein, Carla V. (American Society for Cell Biology, 2014-11-19)
    Circadian period proteins influence cell division and death by associating with checkpoint components, although their mode of regulation has not been firmly established. hPer2 forms a trimeric complex with hp53 and its negative regulator Mdm2. In unstressed cells, this association leads to increased hp53 stability by blocking Mdm2-dependent ubiquitination and transcription of hp53 target genes. Because of the relevance of hp53 in checkpoint signaling, we hypothesize that hPer2 association with hp53 acts as a regulatory module that influences hp53’s downstream response to genotoxic stress. Unlike the trimeric complex, whose distribution was confined to the nuclear compartment, hPer2/hp53 was identified in both cytosol and nucleus. At the transcriptional level, a reporter containing the hp21WAF1/CIP1 promoter, a target of hp53, remained inactive in cells expressing a stable form of the hPer2/hp53 complex even when treated with γ-radiation. Finally, we established that hPer2 directly acts on the hp53 node, as checkpoint components upstream of hp53 remained active in response to DNA damage. Quantitative transcriptional analyses of hp53 target genes demonstrated that unbound hp53 was absolutely required for activation of the DNA-damage response. Our results provide evidence of the mode by which the circadian tumor suppressor hPer2 modulates hp53 signaling in response to genotoxic stress.
  • Thumbnail Image
    Biochemical studies of enzymes in insect cuticle hardening
    Liu, Pingyang (Virginia Tech, 2013-03-28)
    In insects, the cuticle provides protection against physical injury and water loss, rigidness for muscle attachment and mechanical support, and flexibility in inter-segmental and joint areas for mobility. As most insects undergo metamorphosis, they need to shred off old cuticle and synthesize new cuticle to fit the body shape and size throughout their life cycles. The newly formed cuticle, mainly composed of cuticular proteins, chitin, and sclerotizing reagents, needs to be hardened through the crosslinks between cuticular proteins and sclerotizing reagents. This dissertation concerns the biochemical activities of several pyridoxal 5-phosphate (PLP)-dependent decarboxylases with most of them involved in insect cuticle hardening. Herein, we first present a detailed overview of topics in reactions and enzymes involved in insect cuticle hardening. Aspartate 1-decarboxylase (ADC) is at the center of this dissertation. beta-alanine, the product of ADC-catalyzed reaction from aspartate, is the component of an important sclerotizing reagent, N-beta-alanyldopamine; the levels of beta-alanine in insects regulate the concentrations of dopamine, therefore affecting insect sclerotization and tanning (collectively referred as cuticle hardening in this dissertation). Biochemical characterization of insect ADC has revealed that this enzyme has typical mammalian cysteine sulfinic acid decarboxylase (CSADC) activity, able to generate hypotaurine and taurine. The result throws lights on research in the physiological roles of insect ADC and the pathway of insect taurine biosynthesis. Cysteine was found to be  an inactivator of several PLP-dependent decarboxylases, such as ADC, glutamate decarboxylase (GAD) and CSADC. This study helps to understand symptoms associated with the abnormal cysteine concentrations in several neurodegenerative diseases. A mammalian enzyme, glutamate decarboxylase like-1 (GADL1), has been shown to have the same substrate usage as insect ADC does, potentially contributing to the biosynthesis of taurine and/or beta-alanine in mammalian species. Finally, the metabolic engineering work of L-3, 4-dihydroxyphenylalanine decarboxylase (DDC) and 3, 4-dihydroxylphenylacetaldehyde (DHPAA) synthase has revealed that the reactions of these enzymes could be determined by a few conserved residues at their active site. As both enzymes have been implicated in the biosynthesis of sclerotizing reagents, it is of great scientific and practical importance to understand the similarity and difference in their reaction mechanisms. The results of this dissertation provide valuable biochemical information of ADC, DDC, DHPAA synthase, and GADL1, all of which are PLP-dependent decarboxylases. ADC, DDC, DHPAA synthase are important enzymes in insect cuticle hardening by contributing to the biosynthesis of sclerotizing reagents. Knowledge towards understanding of these enzymes will promote the comprehension of insect cuticle hardening and help scientists to search for ideal insecticide targets. The characterization of GADL1 lays groundwork for future research of its potential role in taurine and beta-alanine metabolism.
  • Thumbnail Image
    Circadian Control of Cell Cycle Progression
    Santos, Carlo Steven (Virginia Tech, 2009-03-31)
    Tumorigenesis is the result of uncontrolled cell growth due to the deregulation of cell cycle checkpoints 1. Period 2 (Per2) is a tumor suppressor that oscillate in expression in a 24-hour cycle 2, 3. Here, we show that Per2 interacts with the tumor suppressor protein p53. Both G1 and G2 checkpoint pathways involve a p53 dependent pathway which can trigger the cell to go through cell arrest or programmed cell death4. Understanding all the mitigating factors involved in regulating cell cycle progression under DNA damage can offer a better idea in how cells become immortal. Initially discovered through screening of a human liver cDNA library, the novel interaction between p53-Per2 was further documented using co-precipitation. Interestingly, under genotoxic stress conditions, p53 and Per2 were not found to bind which leads us to suspect that Per2 does not affect active p53 which may possibly be due to post translational modifications of its active state. Furthermore we investigated p53's ability to act as a transcription factor in the presence of Per2, showing that the Per2-p53 complex prevents p53 from binding to DNA. This implies that the tetramerization of p53 may also be another factor in Per2's ability to bind to p53. A truncated p53 lacking the last 30 amino acids that theoretically increase p53's ability to form a tetramer showed a drastic reduction in binding to Per2 5, 6. On the other hand, p53 lacking the tetramerization domain showed binding similar to wildtype. Consequently we speculate that the ability of Per2 to modulate p53 and act as a tumor suppressor protein may be dependent on either the post translational modifications of p53 or its oligomeric state.
  • Thumbnail Image
    Circadian disruption promotes tumor-immune microenvironment remodeling favoring tumor cell proliferation
    Aiello, I.; Fedele, M. L. Mul; Román, F.; Marpegan, L.; Caldart, C.; Chiesa, J. J.; Golombek, D. A.; Finkielstein, Carla V.; Paladino, N. (American Association for the Advancement of Science, 2020-10-14)
    Circadian disruption negatively affects physiology, posing a global health threat that manifests in proliferative, metabolic, and immune diseases, among others. Because outputs of the circadian clock regulate daily fluctuations in the immune response, we determined whether circadian disruption results in tumor-associated immune cell remodeling, facilitating tumor growth. Our findings show that tumor growth rate increased and latency decreased under circadian disruption conditions compared to normal light-dark (LD) schedules in a murine melanoma model. Circadian disruption induced the loss or inversion of daily patterns of M1 (proinflammatory) and M2 (anti-inflammatory) macrophages and cytokine levels in spleen and tumor tissues. Circadian disruption also induced (i) deregulation of rhythmic expression of clock genes and (ii) of cyclin genes in the liver, (iii) increased CcnA2 levels in the tumor, and (iv) dampened expression of the cell cycle inhibitor p21WAF/CIP1, all of which contribute to a proliferative phenotype.
  • Thumbnail Image
    The circadian factor Period 2 modulates p53 stability and transcriptional activity in unstressed cells
    Gotoh, Testuya; Vila-Caballer, Marian; Santos, Carlos S.; Liu, Jingjing; Yang, Jianhua; Finkielstein, Carla V. (American Society for Cell Biology, 2014-10-01)
    Human Period 2 (hPer2) is a transcriptional regulator at the core of the circadian clock mechanism that is responsible for generating the negative feedback loop that sustains the clock. Its relevance to human disease is underlined by alterations in its function that affect numerous biochemical and physiological processes. When absent, it results in the development of various cancers and an increase in the cell’s susceptibility to genotoxic stress. Thus we sought to define a yet-uncharacterized checkpoint node in which circadian components integrate environmental stress signals to the DNA-damage response. We found that hPer2 binds the C-terminal half of human p53 (hp53) and forms a stable trimeric complex with hp53’s negative regulator, Mdm2. We determined that hPer2 binding to hp53 prevents Mdm2 from being ubiquitinated and targeting hp53 by the proteasome. Down-regulation of hPer2 expression directly affects hp53 levels, whereas its overexpression influences both hp53 protein stability and transcription of targeted genes. Overall our findings place hPer2 directly at the heart of the hp53-mediated response by ensuring that basal levels of hp53 are available to precondition the cell when a rapid, hp53-mediated, transcriptional response is needed.
  • Thumbnail Image
    Circadian modulation of the estrogen receptor alpha transcription
    Villa, Linda Monique (Virginia Tech, 2012-07-12)
    The circadian clock is a molecular mechanism that synchronizes physiological changes with environmental variations. Disruption of the circadian clock has been linked to increased risk in diseases and a number of disorders (e.g. jet lag, insomnia, and cancer). Period 2 (Per2), a circadian protein, is at the center of the clock's function. The loss or deregulation of per2 has been shown to be common in several types of cancer including breast and ovarian [1, 2]. Epidemiological studies established a correlation between circadian disruption and the development of estrogen dependent tumors. The expression of estrogen receptor alpha (ERα) mRNA oscillates in a 24-hour period and, unlike Per2, ERα peaks during the light phase of the day. Because up regulation of ERα relates to tumor development, defining the mechanisms of ERα expression will contribute to our comprehension of cellular proliferation and regulation of normal developmental processes. The overall goal of this project is to investigate the molecular basis for circadian control of ERα transcription. Transcriptional activation of ERα was measured using a reporter system in Chinese hamster ovary (CHO) cell lines. Data show that Per2 influences ERα transcription through a non-canonical mechanism independent of its circadian counterparts. Breast cancer susceptibility protein 1 (BRCA1) was confirmed to be an interactor of Per2 via bacterial two-hybrid assays, in accordance with previous studies [2]. BRCA1 is a transcriptional activator of ERα promoter in the presence of octamer transcription factor-1 (OCT-1) [3]. Our results indicate that the DNA binding domain of OCT-1, POU, to directly interact with Per2 and BRCA1, in vitro. Pull-down assays were used to map direct interaction of various Per2 and BRCA1 recombinant proteins and POU. Chromatin immunoprecipitation assays confirmed the recruitment of PER2 and BRCA1 to the estrogen promoter by OCT-1 and the recruitment of Per2 to the ERα promoter decreases ERα mRNA expression levels in MCF-7 cells. Our work supports a circadian regulation of ERα through the repression of esr1 by Per2 in MCF-7 cells.
  • Thumbnail Image
    A comparative analysis exposes an amplification delay distinctive to SARS-CoV-2 Omicron variants of clinical and public health relevance
    Brown, Katherine L.; Ceci, Alessandro; Roby, Clinton; Briggs, Russell B.; Ziolo, D.; Korba, R.; Mejia, R.; Kelly, S. T.; Toney, D.; Friedlander, Michael J.; Finkielstein, Carla V. (Taylor & Francis, 2023)
    Mutations in the SARS-CoV-2 genome may negatively impact a diagnostic test, have no effect, or turn into an opportunity for rapid molecular screening of variants. Using an in-house Emergency Use Authorized RT-qPCR-based COVID-19 diagnostic assay, we combined sequence surveillance of viral variants and computed PCR efficiencies for mismatched templates. We found no significant mismatches for the N, E, and S set of assay primers until the Omicron variant emerged in late November 2021. We found a single mismatch between the Omicron sequence and one of our assay's primers caused a > 4 cycle delay during amplification without impacting overall assay performance. Starting in December 2021, clinical specimens received for COVID-19 diagnostic testing that generated a Cq delay greater than 4 cycles were sequenced and confirmed as Omicron. Clinical samples without a Cq delay were largely confirmed as the Delta variant. The primer-template mismatch was then used as a rapid surrogate marker for Omicron. Primers that correctly identified Omicron were designed and tested, which prepared us for the emergence of future variants with novel mismatches to our diagnostic assay's primers. Our experience demonstrates the importance of monitoring sequences, the need for predicting the impact of mismatches, their value as a surrogate marker, and the relevance of adapting one's molecular diagnostic test for evolving pathogens.
  • Thumbnail Image
    Creating a Positive Departmental Climate at Virginia Tech: A Compendium of Successful Strategies
    Finney, Jack W.; Finkielstein, Carla V.; Merola, Joseph S.; Puri, Ishwar; Taylor, G. Don; Van Aken, Eileen M.; Hyer, Patricia B.; Savelyeva, Tamara (Virginia Tech, 2008-05-05)
    “Creating a Positive Departmental Climate at Virginia Tech: A Compendium of Successful Strategies” was created as part of the AdvanceVT Departmental Climate Initiative (DCI). The Department Climate Committee collected policies and practices from a variety of sources to provide department chairs and heads with opportunities to learn about departmental issues at Virginia Tech, to understand more fully the ways in which these issues manifest themselves within departments, and to share both successful and unsuccessful strategies illustrative of the different approaches departments have taken towards promoting effective, efficient, and pleasant work environments.
  • Thumbnail Image
    Crosstalk Signaling Between Circadian Clock Components and Iron Metabolism
    Schiffhauer, Samuel Peter (Virginia Tech, 2017-04-25)
    Circadian rhythms are daily molecular oscillations within cells ranging from prokaryotes to humans. This rhythm is self sustaining, and receives external cues in order to synchronize an organism's behavior and physiology with the environment. Many metabolites utilized in metabolic processes seem to follow a pattern of circadian oscillation. Iron, an essential component in cellular processes such as respiration and DNA synthesis, is obtained almost exclusively through diet, yet little is known about how the clock governs iron metabolism. The regulation of iron within the cell is very tightly controlled, as iron is highly reactive in the generation of oxidative stress and the excretion of excess iron is very limited. There are limited findings indicating that there are molecular ties between the circadian clock and the regulation of iron metabolism. The first half of my dissertation focuses on the role of the circadian clock in modulating expression of iron metabolic components. We found that key components of iron import, in TFRC, and export, in SLC40A1, show altered expression in response to changes in the expression of clock transcription components. Furthermore, in circadian synchronized HepG2 hepatocytes TFRC and SLC40A1 showed rhythms in their mRNA expression, although expression of these genes was highly altered in conditions of high iron availability. We also examined IREB2, which expresses a master regulator of iron concentration in IRP2. IRP2 showed rhythms in phase with circadian component PER2, and IRP2's rhythmicity was lost under iron overload conditions. We observed that the ability of these three critical iron metabolic components to respond to sudden increases in available iron was mitigated in cells with clock impairment. Whole cistrome and transcriptome analysis was used to determine that rhythmicity in TFRC and SLC40A1 are not equal in their recruitment of circadian protein binding or in the stage of transcription in which circadian rhythms are generated. The cumulative effect of all of this regulation is that rhythmic variation in intracellular hepatic ferrous iron is clock controlled. The second half of my dissertation focuses on understanding how iron uptake influences clock resetting. Initially, iron was added to the cells in the form of ferrous sulfate, or chelated out of the cells using 2-2'-dipyridyl and clock gene expression was monitored. Altered rhythmicity of these components was seen at both the mRNA and protein level in cells with disrupted iron homeostasis. Then, we measured changes in period, phase, and amplitude of these rhythms, ultimately using a luciferase reporter cell line to demonstrate that even slight changes in cellular iron produce an effect on rhythmic period. We find that the circadian clock and iron metabolism pathway are intimately related, and that the intracellular iron concentration plays a role in circadian clock behavior. Overall, our research illustrates the importance of the circadian clock in liver metabolism and physiology. Improper iron metabolism due to genetic or dietary shortcomings is common in humans, and our work builds on the importance of chronotherapy in treatment of these conditions. Conversely, our research into the effect intracellular iron has on the clock contributes to the growing body of research into how circadian clocks, especially the peripheral clock of the liver, receive input from a range of metabolites in conjunction with signals from the master oscillator of the suprachiasmatic nucleus.
  • Thumbnail Image
    Development and implementation of a scalable and versatile test for COVID-19 diagnostics in rural communities
    Ceci, Alessandro; Muñoz-Ballester, Carmen; Tegge, Allison N.; Brown, Katherine L.; Umans, Robyn A.; Michel, F. Marc; Patel, Dipankumar; Tewari, Bhanu P.; Martin, James E.; Alcoreza, Oscar Jr.; Maynard, Thomas M.; Martinez-Martinez, Daniel; Bordwine, Paige; Bissell, Noelle; Friedlander, Michael J.; Sontheimer, Harald; Finkielstein, Carla V. (Nature Publishing Group, 2021-07-20)
    Rapid and widespread testing of severe acute respiratory coronavirus 2 (SARS-CoV-2) is essential for an effective public health response aimed at containing and mitigating the coronavirus disease 2019 (COVID-19) pandemic. Successful health policy implementation relies on early identification of infected individuals and extensive contact tracing. However, rural communities, where resources for testing are sparse or simply absent, face distinctive challenges to achieving this success. Accordingly, we report the development of an academic, public land grant University laboratory-based detection assay for the identification of SARS-CoV-2 in samples from various clinical specimens that can be readily deployed in areas where access to testing is limited. The test, which is a quantitative reverse transcription polymerase chain reaction (RT-qPCR)-based procedure, was validated on samples provided by the state laboratory and submitted for FDA Emergency Use Authorization. Our test exhibits comparable sensitivity and exceeds specificity and inclusivity values compared to other molecular assays. Additionally, this test can be re-configured to meet supply chain shortages, modified for scale up demands, and is amenable to several clinical specimens. Test development also involved 3D engineering critical supplies and formulating a stable collection media that allowed samples to be transported for hours over a dispersed rural region without the need for a cold-chain. These two elements that were critical when shortages impacted testing and when personnel needed to reach areas that were geographically isolated from the testing center. Overall, using a robust, easy-to-adapt methodology, we show that an academic laboratory can supplement COVID-19 testing needs and help local health departments assess and manage outbreaks. This additional testing capacity is particularly germane for smaller cities and rural regions that would otherwise be unable to meet the testing demand.
  • Thumbnail Image
    Differential Impact of VEGF and FGF2 Signaling Mechanisms on Flt1 Pre-mRNA Splicing
    Payne, Laura Beth (Virginia Tech, 2016-06-19)
    The human proteome is exponentially derived from a limited number of genes via alternative splicing, where one gene gives rise to multiple proteins. Alternatively spliced gene products, although crucial for normal physiology, are also linked to an increasing number of pathologies. Consequently, a growing focus is currently being placed on elucidating the extrinsic cues and ensuing signaling mechanisms which direct changes in gene splicing to yield functionally distinct proteins. Of note is the dysregulation of the vascular endothelial growth factor (VEGF) receptor, Flt1 and its soluble splice variants, sFlt1_v1 and sFlt1_v2, in the pregnancy-related disorder, preeclampsia. Preeclampsia is characterized by proteinuria and hypertension and is responsible for almost 600,000 maternal and fetal yearly deaths, worldwide. Here, we examined the impact of endothelial mitogens VEGF and FGF2 (fibroblast growth factor 2), both of which are upregulated in preeclampsia, on Flt1 transcript variants in umbilical vein endothelial cells. We tested the hypothesis that VEGF modulates the expression of Flt1 variants via the signaling kinase Akt and its impact on SR proteins. VEGF was observed to induce expression of overall Flt1 mRNA, principally as variants Flt1 and sFlt1_v1. Conversely, FGF2 induced a shift in splicing toward sFlt1_v2 without significant increase in overall Flt1. Based on inhibitor studies, the VEGF and FGF2 signals were transduced via ERK, but with the involvement of different upstream components. We mapped predicted SR protein binding to Flt1 pre-mRNA and identified two candidate proteins, SRSF2 and SRSF3, that may be involved in VEGF- or FGF2-induced Flt1 pre-mRNA splicing. Examination of SRSF2 and SRSF3 relative mRNA expression levels, following inhibition of VEGF- and FGF2-activated kinases, indicates that FGF2 significantly downregulates SRSF3 mRNA levels via PKC-independent activation of ERK. Additionally, our data suggest that FGF2 may impact Flt1 and sFlt1_v1 via SR protein kinases Akt and SRPK, while conversely regulating sFlt1_v2 levels via Clk. We did not find evidence of VEGF-induced Flt1 variant splicing via SR protein kinase activation or SRSF2 and SRSF3 mRNA levels. Thus, VEGF and FGF2 signals were tranduced via related but distinct mechanisms to differentially influence Flt1 pre-mRNA splicing. These findings implicate VEGF and FGF2 and their related intracellular signaling mechanisms in soluble Flt1 regulation.
  • Thumbnail Image
    Disabled-2 regulates platelet heterotypic and homotypic aggregation through sulfatide binding
    Welsh, John Douglas (Virginia Tech, 2010-03-29)
    At the site of vascular injury platelet aggregation serves to stem blood flow, initiate the inflammatory response, and stimulate wound healing. Platelets become stimulated, release their granule contents, and become adherent to one another. Platelet granules contain important clotting factors and regulators of aggregation. Disabled-2 (Dab2) is a negative regulator of platelet aggregation released from platelet α-granules. Dab2 binds to the αIIbβ3 integrin, through the PTB domain, and blocks fibrin binding to the integrin which serves as the major cause of platelet-platelet interactions. Dab2 is also capable of binding to sulfatides, through the N-PTB region, expressed on the outer leaflet of adjacent cells. Dab2-sulfatide binding decreases Dab2's ability to interact with the αIIbβ3 integrin, however sulfatides activate and stimulate platelet-platelet and platelet-leukocyte aggregation. Sulfatide addition to platelets stimulates increased αIIbβ3 integrin and P-selectin expression through stimulation of continued platelet degranulation, and these surface receptors mediate platelet heterotypic and homotypic aggregation. Here, we show that Dab2 N-PTB binding of sulfatides serves to increase the inhibitory affect of Dab2. Sulfatide stimulation of platelet degranulation can be blocked by the addition of N-PTB. Inhibition of sulfatide induced αIIbβ3 integrin and P-selectin expression result in decreased platelet-platelet aggregation under flow. N-PTB also blocks sulfatide induced platelet-leukocyte interactions and aggregation. Experimental data supports the hypothesis that Dab2-sulfatide binding serves to increase the inhibition of platelet aggregation.
  • Thumbnail Image
    Dynamics of Cell Fate Decisions Mediated by the Interplay of Autophagy and Apoptosis in Cancer Cells:  Mathematical Modeling and Experimental Observations    
    Tavassoly, Iman (Virginia Tech, 2013-08-21)
    Autophagy is a conserved biological stress response in mammalian cells that is responsible for clearing damaged proteins and organelles from the cytoplasm and recycling their contents via the lysosomal pathway. In cases where the stress is not too severe, autophagy acts as a survival mechanism. In cases of severe stress, it may lead to programmed cell death. Autophagy is abnormally regulated in a wide-range of diseases, including cancer. To integrate the existing knowledge about this decision process into a rigorous, analytical framework, we built a mathematical model of cell fate decision mediated by autophagy. The model treats autophagy as a gradual response to stress that delays the initiation of apoptosis to give the cell an opportunity to survive. We show that our dynamical model is consistent with existing quantitative measurements of time courses of autophagic responses to cisplatin treatment. To understand the function of this response in cancer cells we have provided a systems biology experimental framework to study dynamical aspects of autophagy in single cancer cells using live-cell imaging and quantitative uorescence microscopy. This framework can provide new insights on function of autophagic response in cancer cells.
  • Thumbnail Image
    The Dynamics of the Unreplicated DNA Checkpoint in Xenopus laevis Embryos and Extracts
    Adjerid, Nassiba (Virginia Tech, 2008-03-25)
    When unreplicated or damaged DNA is present, cell cycle checkpoint pathways cause cell cycle arrest by inhibiting cyclin-dependent kinases (Cdks). In Xenopus laevis, early embryonic development consists of twelve rapid cleavage cycles between DNA replication (S) and mitosis (M) without checkpoints or gap phases. However, checkpoints are engaged in Xenopus once the embryo reaches the midblastula transition (MBT). At this point, the embryo initiates transcription, acquires gap phases between S and M phases, and establishes a functional apoptotic program. During the cell cycle, there are two main checkpoints that regulate entrance into S and M phases. The focus of this study is the role of protein kinase Chk1 and the phosphatase Cdc25A in the DNA replication checkpoint. In the absence of active Chk1, Cdc25A activates cyclin dependent kinases (Cdks) allowing the cell to progress into S or M phase. Chk1 regulates cell cycle arrest in the presence of unreplicated DNA in somatic cells by phosphorylating Cdc25A and leading to its degradation. Chk1 is also transiently activated at the MBT in Xenopus laevis embryos, even when there is no block to DNA replication or damaged DNA. One goal of this work is to understand the developmental role and regulation of checkpoint signaling pathways due to its monitoring of DNA integrity within the cell. Chk1 plays a critical but not fully understood role in cell cycle remodeling and early embryonic development. In order to understand the function and regulation of Chk1 in checkpoints, the features of the MBT that activate Chk1 must be identified. The activation of Chk1 by two time-dependent events in the cell cycle, the critical nuclear to cytoplasmic (N/C) ratio and the cyclin E/Cdk2 maternal timer are explored in this study. Embryos treated with aphidicolin, resulting in a halted replication fork and therefore a reduced DNA concentration, were tested for Chk1 activation and Cdc25A degradation. Chk1 and Cdc25A were observed to undergo activation and degradation, respectively, in embryos with a reduced DNA concentration. In addition, embryos were injected with Δ34Xic cyclin E/Cdk2 inhibitor, in order to disturb the maternal timer and tested for Chk1 activation and Cdc25A degradation. Both Chk1 and Cdc25A were unaffected by the disruption of the cyclin E/Cdk2 maternal time in the embryo. Therefore, the N/C ratio and the cyclin E/Cdk2 maternal timer do not affect Chk1 activation and therefore Cdc25A degradation. Another means of characterizing the unreplicated DNA checkpoint is through the use of mathematical modeling of the checkpoint-signaling cascade of the cell cycle. Mathematical modeling is the translating of biological pathways into mathematical equations that can simulate interactions without performing laboratory experiments. The Novák-Tyson checkpoint model made important predictions of hysteresis and bistability in the frog egg checkpoint model, predictions that were later confirmed experimentally. The model was updated with additional interactions, such as those including Myt1, a second inhibitor kinase, and lamin proteins, which become phosphorylated at the onset of nuclear envelope breakdown (NEB) at entry into mitosis. Also, experimental data was fit into the model while maintaining hysteresis and bistability. Therefore, the unreplicated DNA checkpoint model was updated with new interactions and experimental data while still preserving previously identified dynamic characteristics of the system. As described, Cdc25A regulation is dynamic in the embryo. The checkpoint original model represents the activity of Cdc25 phosphatase on the mitosis promoting factor (MPF) that leads the cell into mitosis. In the checkpoint model, Cdc25C is the phosphatase activating MPF. However, the model does not include Cdc25A, which is an integral part of the checkpoint-signaling pathway due to its role in activating the cyclin/Cdk complex allowing entry into S and possibly M phase. Experimental studies were performed in which Cdc25A levels were reduced in embryos and extracts using Cdc25A morpholinos. Embryos and extracts showed delayed cell cycle and mitotic entry, demonstrating the importance of Cdc25A plays in the cell cycle. Based upon experimental data, the mathematical model of the DNA replication checkpoint was expanded to include Cdc25A. The expanded model should more accurately demonstrate how checkpoints affect the core cell cycle machinery. Cdc25A was incorporated into the model by gathering experimental data and designing a signaling cascade, which was translated into differential equations. The updated model was then used to simulate the effect of synthesis and degradation rates of Cdc25A on the entry into mitosis dynamics. Therefore, using mathematical modeling and experimental design, we can further understand the role that Cdc25A plays in cell cycle progression during development. Understanding the regulation of Chk1 activity at the MBT and the role of Cdc25A in checkpoint signaling will help us further characterize the dynamics of early embryonic development. The use of mathematical modeling and experimental tools both contribute to further our understanding of controls of the checkpoint signaling pathway and therefore leading us one step closer to truly being able to model a pathway and make predictions as to the behavior of the cell during early embryonic development.
  • Thumbnail Image
    Engineered microsystems and their application in the culture and characterization of three-dimensional (3D) breast tumor models
    Menon, Nidhi (Virginia Tech, 2021-05-26)
    Microsystems are a broad category of engineered technologies in the micro and nano scale that have a diverse range of applications. They are emerging as a powerful tool in the field of biomedical research, drug discovery, as well as clinical diagnostics and prognostics, especially with regards to cancer. One of the major challenges in precision and personalized medicine in cancer lies in the technical difficulties of ex-vivo cell culture and propagation of the limited number of primary cells derived from patients. Therefore, our aims are to 1. Develop a biologically relevant platform for culturing cancer cells and characterize how it influences the cell growth and phenotype compared to conventional 2-dimensional(2D) cell culturing techniques, 2. Isolate secondary metabolites from endophytic fungi and screen them on the platform for potential anticancer properties in a preliminary drug discovery pipeline, 3. Design and develop biosensors for quantifying cell responses in real-time within these systems. Several biomaterial scaffolds with microscale architectures have been utilized for engineering the tumor extracellular matrix, but very few studies have thoroughly characterized the phenotypic changes in their cell models, which is critical for translational applications of biomaterial systems. The overall objective of these studies is to engineer a biomimetic platform for the culture of breast cancer cells in vitro and to quantify and profile their phenotypic changes. In order to do this, we first evaluated a blank-slate matrix consisting of thiolated collagen, hyaluronic acid and heparin, cross-linked chemically via Michael addition reaction using diacrylate functionalized poly (ethylene glycol). The hydrogel network was used with triple-negative breast cancer cells and showed significant changes in characteristics, with cells self-assembling to form a 3D spheroid morphology, with higher viability, and exhibiting significantly lower cell death upon chemotherapy treatment, as well as had a decrease in proliferation. Furthemore, the transcriptomic changes quantified using RNA-Seq and Next-Gen Sequencing showed the dramatic changes in some of the commonly targeted pathways in cancer therapy. Furthermore, we were able to show the importance of our biomimetic platform in the process of drug discovery using fungal endophytes and their secondary metabolites as the source for potential anticancer molecules. Additionally, we developed gold nanoparticle and antibody-based (ICAM1 and CD11b) sensors to quantify cell responses spatiotemporally on our platform. We were able to show quenching of the green fluorescent fluorophores due to the Förster Resonance Energy Transfer mechanism between the fluorophore and the gold nanometal surface. We also observed antigen-dependent recovery of fluorescence and inhibition of energy transfer upon the antibody binding to the cell-surface receptors. Future efforts are directed towards incorporating the hydrogel system with antigen-dependent sensors in a conceptually-designed microfluidic platform to spatiotemporally quantify the expression of surface proteins in various cells of the tumor stroma. This includes the migration,infiltration, and polarization of specific immune cells. This approach will provide further insight into the heterogeneity of cells at the single-cell resolution in defined spaces within the 3D microfluidic platform.
  • Thumbnail Image
    Evaluation of potential photodynamic therapy agents and patient-relevant biomarker combinations for the selective targeting of cancer
    Rodriguez Corrales, Jose Angel (Virginia Tech, 2018-08-21)
    Cancer, the second leading cause of death worldwide, is characterized by uncontrolled and abnormal cell growth. Even though researchers have made significant progress in its treatment over the past several decades, innovative therapeutic approaches that both improve patient survival and lessen the many debilitating side effects of conventional cancer treatments are vital. Accordingly, we first investigated the mechanism of interaction of a bimetallic complex, Ru(II)-Rh(III), with DNA. Non-covalent binding of Ru(II)-Rh(III) is strong and involves electrostatic and, potentially, groove binding interactions. Ru(II)-Rh(III) photobinds and photocleaves DNA through an O2-independent, metal-center mediated mechanism that could be beneficial in hypoxic tumors. Furthermore, the extent of covalent binding and cleavage of DNA, which inhibit PCR amplification, is dependent upon the strength of the non-covalent interactions. These results suggest that the toxicity of Ru(II)-Rh(III) could be selectively generated in tissues irradiated with light (e.g., a tumor). Secondly, we identified protein combinations selectively present in melanoma, which could be utilized in heteromultivalency. Heteromultivalent scaffolds display higher affinity towards cells that express a protein combination in comparison to those with only one of the proteins, which facilitates cell discrimination. Using an empirically-optimized threshold-based screening method and expression profiles of melanoma patients and normal tissues, we identified surface proteins and protein combinations that are selectively found in melanoma patients and not in normal tissues. After a preliminary validation process using the scientific literature, we used immunofluorescence to confirm differential expression of some of these combinations in established melanoma cell lines in comparison to immortalized keratinocytes controls. Finally, we investigated the resazurin assay, a method used for the evaluation of proliferation and cytotoxicity in more than 2,000 publications. We found that only ~14% of these utilized validated assay conditions, while ~40% failed to report essential analytical parameters needed for their replication. We evaluated assay parameters needed for accurate estimation of cell number in eight cell lines, and found that these are highly variable and independent of tissue type, growth kinetics, and energetic parameters. Furthermore, we obtained some insights into the biochemical reduction of resazurin and proposed minimum reporting standards, along with a sample protocol for assay validation.
  • Thumbnail Image
    Identification and Regulatory Role of E3 Ligases in the Time-Dependent Degradation of the Circadian Factor Period 2
    Liu, Jingjing (Virginia Tech, 2016-06-20)
    Circadian rhythms are self-sustained, 24h, biological oscillatory processes that are present in organisms ranging from bacteria to human. Circadian rhythms, which can be synchronized by external cues, are important for organisms to adjust their behavior, physiological activity, and metabolic reactions to changes in environmental conditions. Another well-established oscillatory mechanism that shares common organizational and regulatory features with the circadian system, is the cell division cycle. Recent findings reveal that some essential regulators are common to both the cell cycle and the circadian clock. The first half of my thesis (Chapter 2-3) focuses on the function of Period 2 (Per2), a key regulatory component of the negative feedback arm of the clock and tumor suppressor protein, as a modulator of cell cycle response. We found that Per2 binds the C-terminus end of the tumor suppressor p53 thus forming a trimeric complex with p53's negative regulator Mdm2 and preventing Mdm2-mediated p53's ubiquitination and degradation. Thus, Per2 stabilizes p53 under unstressed conditions allowing for basal levels of the protein to exist and be available for a rapid response to take place in case of any stressed signals. Our experiments prove that Per2 plays an indispensible role in p53 signaling pathway. The second half of my thesis (Chapter 4-5) focuses on how Mdm2 and Per2 interplay regulate Per2 availability and its impact on circadian clock function. My research found that Mdm2 targets Per2 for ubiquitination as Mdm2 depletion stabilizes Per2 and, conversely, Mdm2 ectopic expression shorten Per2's half-life. Accordingly, association of Per2 to Mdm2 maps C-terminus of the p53 binding region in Mdm2 and thus, the RING domain remains accessible. Next, we tested the hypothesis that Mdm2-dependent ubiquitination of Per2 directly impacts circadian clock period length. Accordingly, addition of sempervirine nitrate (SN), a specific molecular inhibitor of Mdm2, to MEF cells abrogated Per2 ubiquitination leading to the accumulation of a stable pool of Per2. By recording the oscillatory behavior of the Per2:Luc reporter system in MEF cells treated with SN at different circadian times, we found that inhibition of Mdm2 E3 ligase activity promoted phase advance only when treatment took place during the degradation period. This is in agreement with our findings that radiation, but not light pulses, causes the same phase behavior. Considering the established role of both Mdm2 and p53 in the response of cells to genotoxic stress and Per2 in modulating the clock, the existence of the Mdm2-Per2-p53 complex opens the possibility of various stimuli triggering regulatory mechanisms converging in a critical node. Overall, our work provides a holistic view of how signals are integrated at multiple levels to ensure that environmental signals are sense and responses triggered timely.
  • Thumbnail Image
    Impact of mutations in non-structural proteins on SARS-CoV-2 replication
    Datsomor, Eugenia Afi (Virginia Tech, 2024-06-14)
    The late 2019 marked the onset of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that led to the unprecedented COVID-19 pandemic, with profound global health and socioeconomic impacts. This thesis offers a thorough examination of the molecular biology, evolution, and disease-causing mechanisms of SARS-CoV-2, as well as recent advancements in understanding the structural and functional implications of mutations in viral proteins. The prevailing belief is that SARS-CoV-2 originated from a zoonotic transmission involving bats as the natural reservoir hosts, with an unknown intermediate host facilitating transmission to humans. Genomic sequencing and phylogenetic analysis have identified similarities between SARS-CoV-2 and bat coronaviruses, particularly RaTG13, indicating a potential bat origin. However, the exact circumstances and intermediate hosts of the spillover event remain under investigation. In its structure, SARS-CoV-2 is an enveloped virus with a positive-sense single-stranded RNA genome. This genome encodes both structural and non-structural proteins crucial for viral replication and the development of the disease. The spike (S) protein facilitates viral entry by binding to the angiotensin-converting enzyme 2 (ACE2) receptor. Meanwhile, non-structural proteins are involved in viral RNA synthesis, immune evasion, and the assembly of virions. Alterations in the genetic makeup of the SARS-CoV-2 genome, notably within the spike protein, can impact transmission efficiency, viral load, and immune evasion. Notable mutations such as D614G, N501Y, and E484K have been associated with increased transmissibility and reduced neutralization by antibodies. Understanding the effects of these mutations on viral fitness and pathogenicity is crucial for informing public health interventions and vaccine development efforts. The impacts of Non-structural proteins (NSPs) on viral replication and transmission are however understudied. In this study, we focused on mutations in the several NSPs including NSP1, 2, 3, 13,14, and 15 of the early Omicron (BA.1) and XBB 1.5 variants and investigated their impact on structure and the functional implications using bioinformatics tools and protein structure prediction methods. Our analysis focused on potential alterations in NSP1's structure and hence its ability to suppress host gene expression and modulate immune responses, shedding light on the mechanisms by which SARS-CoV-2 evolves to evade host defenses. Overall, this thesis gives insights into the emergence, structure, replication cycle, evolution, and pathogenesis of SARS-CoV-2, highlighting the importance of ongoing research efforts in understanding and combatting this global health threat and provides a detailed structural analysis of mutations in NSPs.
  • Thumbnail Image
    Insights on the Regulation of the PERIOD 2 Gene in the Cellular Response to DNA Damage
    Jiang, Liang (Virginia Tech, 2019-05-24)
    Circadian rhythm is a ~24-h mechanism that keeps our physiology and behavior in synchrony with environmental changes. PERIOD2 (PER2) is a core component of the circadian clock and a candidate tumor suppressor as its knockout expression results in a cancer-prone animal. p53 is an effector in the DNA damage response and regulates downstream effectors by trans-activation. Recent studies in our lab show that PER2 can bind to p53, and regulates the trans-activation function. This project studied the subcellular distribution of PER2 in response to DNA damage, and explored the role of p53 in the regulation of PER2 subcellular distribution. We found that PER2 accumulates in the nucleus in response to DNA damage, and such accumulation is independent of p53. In addition, we analyzed Single Nucleotide Polymorphisms (SNP) of PER2 in the 1000 Genome project to gain insight onto how missense mutations in PER2 lay at the interface of p53:PER2 binding. In a separate project, we also performed bioinformatics analysis on the iron related genes to discuss the circadian regulation of iron genes in the liver. These findings shed light on the regulation of PER2 under genotoxic stress, genetic variations of Per2 in normal human population, and expression of circadian genes under iron controlled diets.
  • Thumbnail Image
    Interlocking mechanisms regulating the circadian clock response to DNA damage
    Zou, Xianlin (Virginia Tech, 2021-06-15)
    Almost all organisms have an endogenously generated and self-sustained time-keeping system that oscillates with a periodicity of about 24 h, namely the circadian clock, that help them adapt to daily environmental changes. Mammalian circadian rhythms are generated and maintained by transcription-translation feedback loops (TTFLs) and include post-translational modifications to help fine-tune the oscillation. Circadian rhythms control a broad range of cellular signaling pathways including those mechanisms involved in cell division and DNA damage response (DDR). We have previously established that the core clock component PERIOD2 (PER2) binds to the tumor suppressor protein p53, a key regulatory checkpoint component that modulates cell cycle progression and the cellular response to genotoxic stress. PER2 binding to p53 modulates p53's stability, cellular localization, and transcriptional activity. As described in Chapter 2, we now identified PER2 as a previously uncharacterized substrate for the ubiquitin E3 ligase mouse double minute 2 homolog (MDM2), an oncoprotein and negative regulator of p53. Our findings showed that the association between PER2 and MDM2 is independent of the presence of p53. In addition, MDM2 targets PER2 for ubiquitylation and degradation in a phosphorylation-independent fashion. Lastly, our studies showed that MDM2 collaborates with β-transducin repeat-containing proteins (β-TrCPs), an E3 ligase that targets PER2 for ubiquitylation in a phosphorylation-dependent manner, to control PER2 degradation and thus the length of circadian period. Because the p53:MDM2 pathway plays a critical role in the cellular response to genotoxic stress, the project described in Chapter 3 is based on the hypothesis that DNA damage caused by radiation shifts the circadian clock phase via the p53:PER2:MDM2 complex. Firstly, we generated Trp53KO (Trp53 gene encodes mouse p53) cell lines in NIH 3T3 Per2:dLuc reporter cells expressing luciferase driven by the Per2 promoter. Phase-response curves (PRCs) for Trp53WT and Trp53KO reporter cells were obtained in response to ionizing radiation (IR) treatments. Results indicated that Trp53 knockout did not affect radiation-induced circadian phase shifts, whereas increased p53 levels induced by transient inhibitor treatments prevented phase shifts when IR was performed at the trough of PER2 abundance. Additional mechanisms were unveiled that kinases ATM (Ataxia Telangiectasia Mutated), ATR (ATM- and Rad3-related) and CHK2 (Checkpoint Kinase 2) regulate radiation-induced phase shifts. Lastly, we found that CLOCK (Circadian Locomotor Output Cycles Kaput) and CRY1 (CRYPTOCHROME 1) were phosphorylated in response to radiation. Taken together, these results indicate that radiation-induced clock phase shifts involve the activity of kinases ATM, ATR and CHK2, and the modification in CLOCK and CRY1. Chapter 4 is a review of current findings about the interaction between circadian rhythms and the cell division cycle regulation pathway. The article highlights a multidisciplinary approach that combines mathematical modeling and experimental data to reveal how p53:PER2:MDM2 acts as a node controlling timely cell cycle progression. In summary, our work provided evidence that MDM2 targets PER2 for ubiquitylation and degradation in a phosphorylation-independent manner, and this influences circadian oscillation. Furthermore, the exploration of p53:PER2:MDM2 association shed light on how radiation-induced DNA damage shifts clock phase. These findings expose a crosstalk mechanism that senses DNA damage and shifts the clock system.