• p21B, a variant of p21WAFiycipl, is induced by the p53 family

      Nozell, Susan; Department of Biochemistry and Molecular Biology (2011-12)
      Alternative splicing or expression from an alternate promoter can produce variants of a gene. To determine whether the p21 W*r,/Cipl locus is regulated by these mechanisms, we searched for and found two transcripts, p21B and p21C, that are expressed from an alternate promoter in the first intron of the p21 gene. While p21C encodes the p21 cyclin-dependent kinase inhibitor, p21B encodes a novel protein and is ubiquitously expressed in sixteen human tissues tested. Both p21B and p21C are induced by DNA damage, p53, and p73, and are potentially regulated by a proximal p53 response element in their own promoter. p21B is localized to the Golgi apparatus, and overexpression of p21B inhibits cell proliferation and induces apoptosis. These findings indicate that the p21 locus expresses at least two structurally distinct, but functionally related, variants of the p21 gene from discrete promoters.
    • ParaDIME: Genome-wide differential DNA methylation analyses using next generation sequencing

      Pabla, Sarabjot; Institute of Molecular Medicine and Genetics (12/27/2016)
      Epigenetic modifications are key players in the regulation of a plethora of cellular and physiological processes. DNA methylation is one of the most widely studied epigenetic modification. Genomic abnormalities in DNA methylation have been implicated in various complex diseases including cancer and autoimmunity. With advent of next generation sequencing, investigating DNA methylation patterns at genome-wide scale has become increasingly feasible. However, the pace of developing appropriate statistical methods to analyze large scale DNA methylation data has been slower. This can be attributed to both statistical and computational challenges faced by current methods. In order to overcome these statistical and computational shortcomings, we developed ParaDIME, a web application for differential DNA methylation analysis. ParaDIME tests CpG dinucleotide sites or pre-defined regions of CpG sites for differential DNA methylation using Rao-Scott chi squared test. ParaDIME not only uses a nonparametric test that accounts for differential sequencing coverage but also uses permutation testing to compute exact p values. In order to overcome computation challenges of large amount of permutations, we use parallel computing to share the workload and decrease execution time significantly. To test ParaDIME in-silico, we initially simulated bisulfitesequencing data and tested it against two most widely used methods: MethylSig and MethylKit. It performed equal or better at accurately detecting differentially methylated regions than both the methods. Especially, at important, low differences of percent methylation, ParaDIME performed better than existing tools. In order to test ParaDIME’s ability to detect biologically relevant differentially methylation regions (DMRs), it was then tested on publically available methylation data from chronic lymphocytic leukemia patients. Our method was able to detect previously known and experimentally verified DMR in CLL, especially DMRs located in Nfatc1 and FOXA2 genes. Additionally, it was able to detect other DMRs in genes present in caner related pathways. Due to ParaDIME’s ability to detect biologically relevant DMRs, we employed it in an integrative analysis study to identify epigenetically regulated genes in Sjogren’s syndrome mouse model, B6.NOD aec1/aec2. We performed reduced representation bisulfite sequencing and RNA sequencing on salivary glands of four and eighteen weeks old B6.NOD aec1/aec2 compared to age and gender matched C57BL/6 mice. After removing age and mouse model effect, we discovered 89 differentially expressed as well as differentially methylated genes. Spearman rank order correlation analysis found a significant correlation between DNA methylation and gene expression. Autoimmunity related genes Klf9 and Nfkbid showed significant negative correlation whereas, other genes like Fgf12 and Coll11a2 genes showed significant positive correlation. Subnetwork enrichment using MATISSE showed three jointly active connected subnetworks that were highly enriched in Immune system related pathways, especially, T cell and B cell activation along with cytokine signaling and endocrine system development. Evidence presented in this report presents a novel and a robust differential DNA methylation analysis method with high accuracy to detect disease-relevant DMRs. ParaDIME is a user-friendly and scalable web application with appropriate test statistic to analyze large-scale DNA methylation studies.
    • Partial Elucidation of the Primary Structure of the Heavy Chain of a Myeloma Protein: igG GAR

      Apelgren, Lynn David; Department of Cell and Molecular Biology (1977-06)
    • Partnering With a Formal Program: Expanding the Boundaries of Family Caregiving for Frail Older Adults

      Poole, Deborah K.; Department of Biobehavioral Nursing (1999-12)
      Caring for frail older adults at home is an increasingly common lifestyle among American families. A growing array of community-based programs has been developed to assist family caregivers in this endeavor. Certain of these programs are comprehensive in nature and require a particularly close working relationship between the program’s health professionals and the lay caregiver at home. A paucity of literature exists that can act as a guide to formal and informal caregivers within such a context as they strive to develop an effective working relationship. This study used grounded theory methodology to develop a substantive theory of the process by which family caregivers of frail older adults establish and maintain a working relationship with a comprehensive formal caregiving system. The context of the study was a program belonging to the Program of All-inclusive Care for the Elderly (PACE) network. An initial sample of six primary caregivers of PACE participants was selected. The primary means of data collection was in-depth individual interviews with documents review also being used as a data source. An additional 13 primary caregivers were chosen via theoretical sampling for a total sample size of 19 informants. The method of constant analysis was employed to direct data acquisition and analysis until saturation was complete and the core variable was identified. The basic social-psychological problem identified by informants was termed Helplessness, defines by them as “needing additional help with caregiving.” Partnering with the Program was the basic social-psychological process informants used to relieve their helplessness in caregiving. Partnering with the Program was comprised of three phases: Connecting, Discovering Self, and Transcending Self. The first phase of Connecting represented “the honeymoon phase” of the relationship with the program and was made up of three stages: finding out, “joining up”, and adjusting. Discovering Self, the second phase, had three stages: communicating concerns, evaluating the program’s response, and expecting more. Informants in this phase related with the program in a conflicted manner, wanting to assert their autonomy but realizing their dependence on the program. The final phase, Transcending Self, was also made up of three stages. These stages were monitoring, advocating, and choosing to work it out. The hallmark of the final phase was that informants chose to have a positive, family-like personal relationship with the program staff rather than perpetuate conflict over unmet desires about service provision. This substantive theory provided information heretofore unavailable regarding the trajectory of close healthcare relationships from the perspective of the family caregiver. Implications of the theory related to health and social policy, clinical practice with older adults, and nursing knowledge are made explicit in the final chapter of the report.
    • Patterns of Compensation in Alcohol Dependent Women

      Ambrogne, Janet A.; Department of Physiological and Technological Nursing (1999-04)
      Over the past two decades there has been an increase in the number of research studies addressing alcohol and drug dependent women. Findings from these studies support a number of characteristics unique to women that have implications for treatment. Despite these findings, the majority of existing treatment modalities for substance dependence have continued to be based on traditional models of additions and treatment. Further, few studies have explored women's perceptions of their alcohol use, or the struggles that women encounter in their efforts to temper their alcohol use. The purpose of this qualitative study was to forward an interpretive theory about how alcohol dependent women experience temperance. This study was a focused ethnography, and was guided by a feminist perspective. The techniques of in-depth interviewing and participant observation were employed as a means of eliciting the perspective of a purposive sample of fourteen women. Concurrent data collection and analysis generated an interpretive theory that went beyond the women’s experiences with temperance. The theory: From Chaos to Connection: Patterns of Compensation in Alcohol Dependent Women, answered the questions, “What are women’s experiences with alcohol dependence?” A cultural theme of patterns of compensation was identified. The women used alcohol and other drugs as compensatory mechanisms to alleviate feelings of inadequacy and depression. Through participation in treatment and twelve-step self-help groups such as Alcoholics Anonymous, the women learned to replace their former compensatory mechanisms of drinking and using, with compensatory strategies designed to manage the disease of alcoholism. Even with successful abstinence, the women continued to struggle with feelings of depression and low self-worth. While they had learned purposeful strategies that facilitated abstinence, feelings of depression and inadequacy endured. These findings challenge the utility of dominant models of addiction such as the disease model, which focuses on substance dependence as the primary problem, in adequately meeting the needs of women who use alcohol and other substances to alleviate negative feelings such as depression. Findings from this study support the need to integrate other models of addiction, such as the Self-Medication Hypothesis into treatment programs for women. Implications for nursing, policy and future research directions are forwarded.
    • Penalized Least Squares and the Algebraic Statistical Model for Biochemical Reaction Networks

      Linder, Daniel F. II; Department of Biostatistics and Epidemiology (2013-07)
      Systems biology seeks to understand the formation of macro structures such as cellular processes and higher level cellular phenomena by investigating the interactions of systems’ individual components. For cellular biology, this goal is to understand the dynamic behavior of biological materials within the cell, a container consisting of smaller materials such as mRNA, proteins, enzymes and other intermediates necessary for regulating intracellular functions and chemical species levels. Understanding these cellular dynamics is needed to help develop new drug therapies, which can be targeted to specific molecules or specific genes, in order to perturb the system for a desired result. In this work we develop inferential procedures to estimate reaction rate coefficients in cellular systems of ordinary differential equations (ODEs) from noisy data arising from realizations of molecular trajectories. It is assumed that these systems obey the so called chemical mass action law of kinetics, with corresponding deterministic mass action limit as the system size becomes infinite. The estimation and inference is based on the penalized least squares estimates, where the covariance structure of these estimates corresponds to the solution of a system of coupled nonautonomuous ODEs. Another topic discussed here is that of network topology estimation. The algebraic statistical model (ASM) offers a means of performing this topological inference for the special class of conic networks. We prove that the ASM recovers the true network topology as the number of samples grows without bound, a property known in the literature as sparsistency. We propose a method to extend the ASM to a wider class of networks that are decomposable into multiple cones.
    • Perceived Conditions of Employment Among Hospital Registered in Georgia: A Comparative Analysis

      Futch, Joan W.; School of NursinG (1982-03)
      Employment conditions of registered nurses have to date not been studied in the State of Georgia. This investigation was conducted in order to compare ~urses' satisfaction or dissatisfaction with conditions of employment to retention, hospital setting, and hospital size, The present study identified specific factors in ~he work environment that contribute to the registered nurses' satisfaction or dissatisfaction with employment conditions.. This investigation, which is a partial replication of an earlier st ud.y by Wandel t ( 1980) , arose· out of concern for the shortage . of registered nurses in Georgia. A descriptive survey design was used to determine the relationship between perceived conditions of employment o~ register$d n~ses employed in 200- 400 bed acute medical-surgical hospitals. in the State of Georgia and their perceived job. satisfaction. It was hypothesi.zed thata (1) nurses employed in a hospital with a low turnover rate would express greater satisfaction with conditions of employment than nurses employed in a hospital with a high turnover rate; (2) nurses employed in a rural hospital would express greater satisfaction with conditions of employment than nurses employed in an urban hospital; and ( J) nurses employed in a 200 - 2?5 bed hospital would express great,er satisfaction with conditions of employment than nurses employed in a,J25 - 400 bed hospital. The results of ! test values, at the .05 level of significance, demonstrated statistically significant results for all three hypotheses posed.
    • Perceived effects on the family system when a wife/mother returns to school as reported by returnee

      Jackson, Jo Anne Christian; School of Nursing (1986-06)
      The foc~s of this study was the· female's ret~rn to school and her petception of its effect·on the family system. A tota~ of ·twenty females, selected through a snowball effect,· were interviewed ·in their homes. An interview technique· with demographic questionnaire 'Was utilized. The results of the st.udy indicated that the return to school had various similarities for the female. The suppoit 6f . . ·. spouse and f~mily was viewe~ as crucial. Di.~ision.of · labor d-id not change drastically nor did. t'ime spent with families. Quality of time with family.became more important and.the most positive effect of the return to school was seen as the increased interqction between husband and children. The study has implications for educators, counselors, health car~ providers and the families~ The greatest implication of the. study is the fact that the return to school. was viewed by most women in this study as being positive.
    • Perceived Professional Risk of School Nurses Associated with Delegation of Nursing Care Responsibilities to Unlicensed Personnel

      Hamilton, Bernita K; Department of Physiological and Technological Nursing (1997-05)
      The increasing numbers of children who require health care services while attending school have prompted the delegation of nursing care responsibilities to unlicensed personnel. School nurses have expressed legal and professional concerns regarding delegation. The purpose of this study was to describe current delegation practices of school nurses to unlicensed personnel, examine legal and professional standards which impact delegation decisions, and explore the perceived professional risk of school nurses associated with delegation and risk to the health and safety of students. A professional and legal regulation of practice model provided the conceptual framework. A descriptive design was used to investigate the delegation practices of school nurses in Georgia. Eighty-seven (N=193) school nurses returned completed questionnaires. Summary statistics were used to analyze the data. A Demographic Questionnaire provided information about sample characteristics. Analysis of data from the School Health Care Questionnaire determined the performance and delegation of nursing care responsibilities. Approximately 70% of the school nurses reported delegation to unlicensed personnel. Crosstabulation of performance and delegation revealed the most frequently delegated procedures as oral, inhalation, ophthalmic/otic, and topical medication administration; seizure procedures; gastrostomy feedings; vision and hearing screenings; and urinary catheterizations. The investigator-developed Professional Risk Related to Delegation Scale determined the importance of standards used in delegation decisions and the risk associated with delegation practices. The majority of participants rated the legal and professional standards as considerable to extreme importance in delegation decisions. Findings supported that items consistent with appropriate delegation practices had lower risk scores; whereas, items consistent with inappropriate delegation had higher risk scores. Overall, the school nurses reported moderate to very high professional risk and risk to the health and safety of students associated with delegation to unlicensed personnel. Findings show that school nurses in Georgia are concerned about professional risk associated with delegation to unlicensed personnel. These findings have implications for development of delegation practice models and refinement of legal and professional statutes and standards for the regulation of delegation.
    • Perceived Supportive Behaviours and Occupational Stress Among Nurses

      Allanach, Elaine J.; Department of Nursing (1988-04)
    • Peripheral Insulin Resistance Mediates Microvascular Dysfunction in Obese Mice via Increase NAD(P)H Oxidase Isoform one Mediated Superoxide Production

      Ali, Mohammed Irfan; Department of Physiology; Georgia Regents University (2010-07)
      The overall goal of the current study was to determine if improving the net glycemic load and lipid derangements associated with IR in obesity by genetically deleting PTP1B in a mouse model of obesity would improve microvascular function.
    • Person Variables, Psychosocial State Variables, and Reported Health Behaviors: Relationship to Preterm Delivery

      Kelley, Maureen; Department of Physiological and Technological Nursing (1993-12)
      The purpose of this study was to use a conceptual model of examine selected possible relationships among person variables, health behavior variables, and psychosocial state variables including anxiety, depression, life events, mastery, self-esteem, stress, and social support. Reported health behavior variables include smoking, drinking, drug use, prepregnant weight for height gain during pregnancy. The dependent variable was preterm delivery, which was defined as delivery before 37 weeks gestation. The dependent variable was preterm delivery, which was defined as delivery before 37 weeks gestation. The analyses used two subgroups of women. The subgroup consisted of 1163 women who delivered moderately preterm infants (32-37 weeks gestation). The second subgroup consisted of 1258 women who delivered both moderately preterm and very preterm infants (27-37 weeks gestation). Data were analyzed utilizing both univariate and multivariate statistics, with logistics regression as the principle multivariate technique. As a group, person variables and psychosocial state variables had direct relationships, as posited in the hypotheses and supported in the literature, to preterm delivery. Health behavior variables were directly related to preterm delivery in the variable set that contained both moderately preterm and very preterm infants. Indirect relationships were supported for the hypothesis that added psychosocial state to health behaviors. Individual variables that were associated with preterm delivery were self-esteem and mastery. Results of this study were significantly different than results of a parallel study using this same data set , but examining the association between psychosocial variables and intrauterine growth retardation
    • PHOTOBIOMODULATION AS A MITOCHONDRIAL TARGETED TREATMENT STRATEGY IN NEONATAL HYPOXIC ISCHEMIC ENCEPHALOPATHY

      Tucker, Donovan; Tucker, Lorelei; Department of Neuroscience and Regenerative Medicine (Augusta University, 2019-05)
      Neonatal hypoxic ischemic encephalopathy (HIE), initiated by hypoxic-ischemic (HI) injury to the brain in the perinatal period, is a leading cause of infant mortality and disability. HI damage to the developing brain triggers a complex pathology, initiating with mitochondrial insult, which culminates in neuronal cell death. Photobiomodulation (PBM), the application of near-infrared light, is an experimental neuroprotective strategy targeting the activity of mitochondrial cytochrome c oxidase (CCO), but its effect on HIE is unknown. This work was designed to shed light on the effect of PBM on a neonatal rat HI injury model. Postnatal day 10 mixed-sex pups underwent HI insult followed by 7 daily PBM treatment sessions via a continuous wave diode laser (808 nm). HI pups suffered significant ipsilateral hemispheric brain shrinkage and substantial cell death in the cortex and hippocampal CA1 and CA3 subregions. PBM treatment reduced neuronal cell death in the cortex and hippocampal subregions and reduced hemispheric brain shrinkage. HI pups displayed impaired motor function and spatial learning and memory which was ameliorated by PBM. Blood-brain barrier integrity was compromised in HI animals, as evidenced by reduced extravasation of Evans blue, but was reversed by PBM. PBM also mitigated microglial activation and upregulation of pro-inflammatory cytokines in HI pups. PBM treatment induced robust reduction in oxidative damage markers and protein carbonyl production in the cortex and hippocampus. Investigation of mitochondrial function revealed that PBM markedly attenuated mitochondrial dysfunction and preserved ATP production in neonatal HI rats. Furthermore, PBM treatment profoundly suppressed HI-induced mitochondrial fragmentation. PBM administration reduced activation of pro-apoptotic caspase 3/9 and TUNEL-positive neurons in HI pups. Finally, we demonstrated that the neuroprotective action of PBM could be reversed in a primary hippocampal neuronal OGD model by application of low-dose KCN, a CCO inhibitor. Taken together, our findings demonstrated that PBM treatment contributed to a robust neuroprotection via attenuation of mitochondrial dysfunction, oxidative stress, and neuronal apoptosis in the neonatal HI brain. Additionally, we demonstrated that these effects are, in part, mediated by modulation of CCO activity. This suggests that PBM may offer a promising role as a potential treatment strategy for HIE.
    • Physical Dissociation of G Protein Heterotrimers in Living Cells

      Digby, Gregory J.; Department of Neuroscience and Regenerative Medicine (2008-06)
      On the basis of numerous studies using cell membranes, it is commonly assumed that active G protein heterotrimers physically dissociate into GTPbound Gα and Gβγ subunits. However, due to inadequate evidence in vivo, several groups question this hypothesis. To explore this problem, we have developed an assay that measures G protein dissociation in living cells. We examined protein mobility using fluorescence recovery after photobleaching (FRAP) and found that Gβ1γ2 subunits formed heterotrimers with inactive immobile Gα subunits. When we activated heterotrimers with receptors, Gβγ subunits released from Gα, suggesting that G protein heterotrimers physically dissociate in living cells. To our knowledge this is the first definitive measure of this event in vivo. When different Gα isoforms were compared, we found that Gαi/o subunits released Gβγ dimers more readily than Gαs subunits, suggesting that heterotrimers differentially dissociate. To determine if differential release of Gβγ is a mechanism for Gα specific activation of Gated inwardly rectifying potassium (GIRK) channels, we activated GIRKs in cells expressing GαoA or Gαs subunits. We found that GαoA heterotrimers were more effective activators of GIRK channels than Gαs heterotrimers when comparable amounts of each were available. Thus, since GαoA subunits also released Gβγ dimers more efficiently than Gαs subunits, we propose that differential dissociation provides a mechanism for Gα specific activation of GIRK. In addition to providing a clear demonstration of G protein dissociation, we also propose a more complete model of the G protein cycle where active G proteins are in continuous association-dissociation equilibrium. Accordingly, at any given time during activation, G protein heterotrimers can cycle through several dissociation-association events until GTP is hydrolyzed. A model of the G protein cycle where GαGTP + Gβγ and GαGTPGβγ are both present during activation is included.
    • PKC and ATR Mediated Regulation of Cisplatin-Induced Renal Tubular Cell Apoptosis

      Pabla, Navjotsingh; Department of Cellular Biology and Anatomy (2009-03)
      Cisplatin is one of the most widely used anti-cancer drug. However, its use and efficacy is limited due to nephrotoxicity. One fourth of patients treated with cisplatin develop varying degree of renal impairment, frequently resulting in acute kidney injury. Due to high mortality associated with acute kidney injury, effort has been made to understand the molecular basis of cisplatin nephrotoxicity and develop effective renoprotective strategies. In kidneys, cisplatin is accumulated in tubular cells; however the uptake mechanism that is responsible for high accumulation of cisplatin in renal cells is unclear. In tubular cell, cisplatin accumulation induces cell death by apoptosis. Mechanistically, our laboratory has demonstrated a critical role of p53 in tubular cell apoptosis during cisplatin nephrotoxicity. However, the proximal events that contribute to p53 activation and related signaling are unknown. The focus of my work was to decipher these early events during cisplatin nephrotoxicity. Firstly, my results suggest that the copper transporter Ctr1 is highly expressed in renal tubular cells and is responsible for renal uptake of cisplatin. Secondly, I show that DNA damage response involving ATR-Chk2 is responsible for p53 activation and consequent apoptosis during cisplatin-induced kidney injury and nephrotoxicity. Thirdly, I have identified that PKCd is a novel regulator of cisplatin nephrotoxicity. During cisplatin treatment PKCd is activated in a Src dependent manner and is responsible for activation of MAPKs, contributing to renal cell death. Most importantly, my results suggest that pharmacological inhibition of PKCd ameliorates renal injury without affecting the anticancer efficacy of cisplatin. These results have not only provided new insights into the 3 molecular mechanism of cisplatin nephrotoxicity, but have also identified a novel strategy to mitigate the side effects of cisplatin in normal renal tissues.
    • Plasma Membrane Disruption in Orthodontic Tooth Movement

      Orellana, Maria F.; Department of Oral Biology (2002-04)
      (Introduction) One hundred years ago, in 1900, Dr. Edward H. Angle and a dozen colleagues came together to establish dentistry's first specialty, which is known today as orthodontics and dentofacial orthopedics. Orthodontics is a science and an art. It is the art o f creating healthy, beautiful smiles by moving teeth with precise, gradual force expertly applied, and the science concerned with the study o f the growth o f the craniofacial complex, the development o f occlusion and the treatment o f dentofacial abnormalities. Mechanical forces exerted on tooth roots and transmitted to the periodontal tissues initiate the remodeling activity that facilitates the movement o f teeth through bone. The specific changes in the bone surrounding the root o f an orthodontically moved tooth are characterized as resorption and deposition. Resorption o f bone is seen on the compression side o f the tooth. In contrast, bone is deposited in the tension side of the tooth that is being moved in the opposite direction. The biologic response to sustained force against the teeth is a function o f force magnitude; forces great enough to occlude blood vessels lead to pain, sterile necrosis and a process described as undermining resorption that inevitably leads to a delay in tooth movement. Lighter forces allow activation o f osteoclasts, and thus the removal of bone from the compression side by the painless process o ffrontal resorption. Clinicians face the challenge o f maintaining tissue vitality by avoiding undermining resorption, while applying forces heavy enough to produce frontal resorption. An understanding of the cellular and molecular mechanisms that enable bone to adapt to changes in its mechanical environment is important for solving the different challenges o f clinical orthodontics. Almost a century of research has been devoted to examining this phenomenon by morphologic methods. The histologic changes have consequently been well documented, but there are many unanswered questions that must be addressed in order to explain how mechanical deformation is transduced into a desirable biologic response. The aim o f the present investigation was to characterize a novel cellular mechanism for uptake and release of molecules important in bone remodeling by periodontal ligament cells. Specifically, the plasma membrane disruption theory was examined in light o f its role in mechanotransduction in orthodontic tooth movement. These are the first studies linking the placement o f mechanical loading, as occurs in orthodontic tooth movement, with plasma membrane disruption and resealing of periodontal ligament cells. The release o f bFGF and II-ip from the cells of the periodontal ligament was also examined following application o f in vivo strain.
    • Polycyclic Aromatic Hydrocarbons: The Nuclear Meabolizing System and Inability of Vitamen A Deficiency to Alter Metabolism, DNA Binding, And Subsequent DNA Repair

      Bornstein, William; Department of Cell and Molecular Biology (1978-09)
      Polycyclic aromatic hydrocarbons (PAR's) are ubiquitous environmental pollutants formed by incomplete combustion. Consequently, they are present in tobacco smo~e as well as in industrial effluents produced by the burning of fos~il fuels. Within this class of compounds,· many members are known carcinogens for animals and are, presumably, also carcinogenic for man. Since it is estimated that 80-90% of all human cancers have environmental factors as a component of their .etiology, the study of the mechanisms of PAH carcino'genesis is of paramount basic ·and clinical. importance. The carcinogenic PAH's require metabolic activation leading to the formation of "proxj_mate" and "ultimate" carcinogens. The latter activated electrophilic species bind to cellular macromolecules--DNA, RNA, anq protein--and.such . binding is postulated to be a critical event leading to neoplasia •. Repair of the lesion to macromolecules, e. g., DNA, may subsequently modify the results of~ such binding. ·In addition, a variety of these "post-initiation"· factors may ultimately determine whether or not such neoplastic transformation is clinically expressed. Metabolic activation had been believed to occur exclusively within the endoplasmic reticulum. Recently, however, the nucleus has been demonstrated to contain enzymes capable of activating PAH's. Nuclear activation is suspected t~ be of particular importance because of its proximity to the genetic apparatus of the cell and because of the.extreme lability of the putative ultimate metabolite of. the archetypal PAH-benzo[ a] pyrene (BP) . · The activation of BP .require.s. the concerted action of two enzymes--aryl hydrocarbon hydroxylase (AHH) and epoxide hydrase. Although the nuclear form of AHH had been studied prior to the present studies; nuc.le.ar. epoxide hydrase· has been poorly .characterized. In the studies herein reported, we have employed a "pincer" tactic in order to further characterize the metabolic activation of BP. Thus we· have, on·· the one hand, focused our attention on a specific nuclear enzyme activity--nuclear epoxide hydrase-.;..and have comp·ared and contrasted this activity with its microsomal counterpart. On the other hand, we have investigated the relationship between a known. ,modifier of BP tumorigenesis-- vitamin A def'iciency--and the metabolic activation of BP, its binding·to DNA, and the subsequent repc:tir of DNA.
    • Polyfunctional CD4+ T cells synergize with chemotherapy to reprogram tumor metabolism towards a curative outcome

      Habtetsion, Tsadik Ghebreamlak; Department of Biochemistry and Molecular Biology (8/3/2017)
      CD4+ T cells are critical mediators of anti-tumor immunity. Accumulating evidence from preclinical and clinical studies suggests that tumor-reactive CD4+ T cells in adoptive T cell therapy (ACT) have the potential to effectively control tumor growth. In most ACT clinical settings, chemotherapeutic agents are used to induce an immunostimulatory milieu which facilitates the effector function of donor T cells. Although the efficacy of ACT has been well-established, currently only a fraction of patients with certain types of malignancy have benefited, highlighting the need for improved ACT strategies. Recent studies have revealed that the metabolic reprogramming by cancer cells attenuate antitumor immune response by imposing nutrient restrictions in the tumor microenvironment, which leads to defective T cell responses. In the current study, we set out to explore how Cyclophosphamide (CTX) and tumor reactive CD4+ T cells alter the metabolic features of cancer cells. By comparing the global metabolic profiling of tumors pre and post-treatment, we found that CXT+CD4 ACT elicited a metabolic catastrophe affecting multiple pathways critical for cancer progression. Particularly, CTX+CD4 ACT led to marked reduction in glutathione (GSH) levels, increased accumulation of reactive oxygen species (ROS) and oxidative DNA damage product in tumors. Importantly, administration of N-acetyl-L-cysteine diminished the curative effect of CTX+CD4 ACT. Moreover, pharmacological inhibition of GSH using Buthionine Sulfoximine (BSO) following CTX significantly delayed tumor growth in mice. Mechanistically, we found that TNFα synergized with chemotherapy to reduce intracellular GSH levels and promote ROS induction. TNFα enhanced cell death in chemotherapy pre-treated tumor cells and the cytotoxic effect was reversed by adding GSH exogenously. Importantly, the curative effect of CTX+CD4 ACT was abrogated after TNFα neutralization. Additionally, we found that CTX+CD4 ACT led to tumor vascular disruption causing hemorrhagic necrosis of tumors. IFNγR-deficient mice failed to reject tumor after CTX+CD4 ACT and had intact tumor vasculature. Collectively, our data reveal that tumor reactive CD4+T cells disrupt the redox homeostasis of cancer cells in TNFα-dependent manner. Whereas CD4+T cells derived IFNγ targeted tumor endothelial cells to cause vascular disruption and tissue ischemia. The combined action of these two cytokines leads to eventual eradication of established tumor.