• 5' Flanking Sequence Mutations Associated With Low Expression of Fetal Y-Globin Genes

      Brinson, Eleanor Campbell; Department of Cell and Molecular Biology (1988-06)
    • 96 plays a role in the virulence of C. jejuni

      Rathbun, Kimberly M; Department of Medicine (2009-05)
      Campylobacter jejuni is a gastrointestinal pathogen of humans but part of the normal flora of poultry. C. jejuni therefore grows well at both 37°C and 42°C. Proteomic studies on temperature regulation in C. jejuni strain 81-176 revealed the upregulation at 37°C of CJ0596, a predicted periplasmic chaperone that is similar to proteins found to be involved in outer membrane protein (OMP) folding and virulence in other bacteria. The cj0596 gene was highly conserved in multiple strains and species of Campylobacter (24 in total), implying the importance of this gene. To study the role CJ0596 plays in Campylobacter pathogenesis, a mutant derivative of strain 81-176 was constructed in which the cj0596 gene was precisely deleted. This mutant was complemented by restoring the gene to its original chromosomal location. The mutant strain demonstrated a decreased growth rate and lower final growth yield, yet was more motile than wild-type. The cj0596 mutant also showed altered levels of several outer membrane proteins (OMPs), and changes in membrane-associated characteristics (antimicrobial sensitivity, autoagglutination, and biofilm formation). In either single or mixed infections, the mutant was less able to colonize mice than wild-type. Purified, recombinant CJ0596 had peptidyl-prolyl cistrans isomerase (PPIase) activitty, but did not functionally complement an E. coli surA mutant. These results suggest that C. jejuni CJ0596 is a PPIase and loss of CJ0596 alters phenotypes that have been shown to be related to the pathogenesis of the bacterium.
    • A HERMENEUTIC PHENOMENOLOGICAL APPROACH TO EXPLORING THE LIVED EXPERIENCES OF PARTICIPANTS IN GEORGIA’S P-20 COLLABORATIVES

      Maple, Carol Willyn; Gamble, Phyllis; McCoy, Felina; Department of Advanced Studies and Innovation (Augusta University, 2020-04)
      This research effort used a hermeneutic phenomenological approach to investigate 1) What are the lived experiences of participants in Georgia’s P-20 Collaboratives? and 2) How are regional P-20 Collaboratives using mutual resources and continuous professional development to meet the needs of all stakeholders? Data provided by representatives from school systems (P-12s), colleges and universities (IHEs), Regional Education Service Agencies (RESAs), and State Education Agencies (SEAs) via a qualitative questionnaire, online discussion boards, contextual documents, and convening observations were analyzed to explore the perceived benefits and challenges of participation in Georgia’s P-20 Collaboratives and to examine the effectiveness of the regional Collaboratives in meeting the mission statement. Three emergent themes derived from the data were: 1) Mutually beneficial outcomes are derived from networking and sharing resources, but are hindered by low and inconsistent attendance by participants, 2) Diverse perspectives needed for effective reciprocal learning are limited by issues with focus, commitment, regular communication, and consistent participation by the right stakeholders, and 3) The majority of the respondents (68%) perceived that the regional collaboratives were effective in meeting the mission. However (14%) suggested improvements and (14%) felt that the mission statement lacked clarity.
    • A HERMENEUTIC PHENOMENOLOGICAL APPROACH TO EXPLORING THE LIVED EXPERIENCES OF PARTICIPANTS IN GEORGIA’S P-20 COLLABORATIVES

      McCoy, Felina Rae; Department of Advanced Studies and Innovation (Augusta University, 2020-05)
      This research effort used a hermeneutic phenomenological approach to investigate 1) What are the lived experiences of participants in Georgia’s P-20 Collaboratives? and 2) How are regional P-20 Collaboratives using mutual resources and continuous professional development to meet the needs of all stakeholders? Data provided by representatives from school systems (P-12s), colleges and universities (IHEs), Regional Education Service Agencies (RESAs), and State Education Agencies (SEAs) via a qualitative questionnaire, online discussion boards, contextual documents, and convening observations were analyzed to explore the perceived benefits and challenges of participation in Georgia’s P-20 Collaboratives and to examine the effectiveness of the regional Collaboratives in meeting the mission statement. Three emergent themes derived from the data were: 1) Mutually beneficial outcomes are derived from networking and sharing resources, but are hindered by low and inconsistent attendance by participants, 2) Diverse perspectives needed for effective reciprocal learning are limited by issues with focus, commitment, regular communication, and consistent participation by the right stakeholders, and 3) The majority of the respondents (68%) perceived that the regional collaboratives were effective in meeting the mission. However (14%) suggested improvements and (14%) felt that the mission statement lacked clarity. Keywords: P-20 collaboratives, collaboration, hermeneutic phenomenology, teacher preparation, regional partnerships, professional development, teacher quality
    • A HERMENEUTIC PHENOMENOLOGICAL APPROACH TO EXPLORING THE LIVED EXPERIENCES OF PARTICIPANTS IN GEORGIA’S P-20 COLLABORATIVES

      Gamble, Phyllis J.; Department of Advanced Studies and Innovation (Augusta University, 2020-05)
      This research effort used a hermeneutic phenomenological approach to investigate 1) What are the lived experiences of participants in Georgia’s P-20 Collaboratives? and 2) How are regional P-20 Collaboratives using mutual resources and continuous professional development to meet the needs of all stakeholders? Data provided by representatives from school systems (P-12s), colleges and universities (IHEs), Regional Education Service Agencies (RESAs), and State Education Agencies (SEAs) via a qualitative questionnaire, online discussion boards, contextual documents, and convening observations were analyzed to explore the perceived benefits and challenges of participation in Georgia’s P-20 Collaboratives and to examine the effectiveness of the regional Collaboratives in meeting the mission statement. Three emergent themes derived from the data were: 1) Mutually beneficial outcomes are derived from networking and sharing resources, but are hindered by low and inconsistent attendance by participants, 2) Diverse perspectives needed for effective reciprocal learning are limited by issues with focus, commitment, regular communication, and consistent participation by the right stakeholders, and 3) The majority of the respondents (68%) perceived that the regional collaboratives were effective in meeting the mission. However (14%) suggested improvements and (14%) felt that the mission statement lacked clarity. Keywords: P-20 collaboratives, collaboration, hermeneutic phenomenology, teacher preparation, regional partnerships, professional development, teacher quality
    • Acquiring Situation Awareness through Hand-Off in a Critical Care Environment

      Holden, Tina; Nursing (Augusta University, 2019-12)
      Hand-off communication is associated with 80% of hospital errors. Situation awareness (SA) has been targeted as a strategy to reduce errors and enhance patient safety when providing hand-off communication. Few studies have focused on the influence of SA in hand-off communication in the intensive care unit where the risk of errors is high. The purpose of this study was to develop a substantive theory of critical care nursing hand-off. The study was guided by Endsley’s SA framework. A qualitative study design using Straussian grounded theory methods was used to develop a substantive theory related to critical care nursing hand-off. Data collection strategies included observation of 20 critical care nursing hand-offs followed by 34 semi-structured interviews and took place from 2017 to 2019 in a medical and surgical ICU at two academic tertiary care facilities. Data analysis was conducted using constant comparative analysis and was guided by Endsley’s model of SA. Results revealed that hand-off is a basic social process with a core category of handing-off awareness. The process contained four phases: interactive, reflective, maintenance, and preparatory. The interactive phase was characterized by communication between the giver and receiver of hand-off. During that phase, the 10 critical elements of hand-off were passed on to the receiver. These critical elements included the code status, past medical history, story, systems assessment, trends, changes, rationale, level of organ support, and anticipation. Handing off these elements in a way that flows with logical order affects awareness. Nursing behaviors of the giver associated with handing off awareness are linked to the critical elements. For the receiver, these behaviors include arriving prepared, reporting the critical elements, controlling flow, and making connections between the critical elements. Behaviors for the receiver include being an active listener, validating information, and asking questions within the flow of information. In the reflective phase, the resilient nurse bridges gaps in awareness. The maintenance phase is characterized by nursing actions that support hand-off information recall. In the maintenance phase, SA is maintained through artifacts. Artifacts are tools used by nurses to aid in the cognitive function of hand-off. The preparatory phase is characterized by information synthesis and organization. The four phases of hand-off are re-occurring and are influenced by individual and organizational factors. Individual factors include a nurse’s personal process, experience, socialization, and emotional intelligence. Organizational factors include unit policies, unit artifacts, and safety culture. Theory and research implications include the need for future research to further expand the framework of SA in hand-off, the use of qualitative methods to provide insight into complex areas of healthcare, and the need for educational interventions on SA hand-off. Practice implications include evaluation of current hand-off practices in the ICU and evaluation of organizational influences on hand-off. The study concludes that the theory of handing-off awareness in the ICU is a continuous process that occurs over four phases in a repetitive cycle that starts again with each shift change. The critical elements, flow, nursing behaviors, time, and external factors influence the ability of the nurse to achieve optimal SA.
    • Activation of Arginase and the Endothelin System in Models of Ischemic Retinopathy

      Patel, Chintan; Department of Biochemistry and Molecular Biology; Department of Biochemistry and Molecular Biology (2014-07)
      Ischemic retinopathies, such as diabetic retinopathy (DR) and retinopathy of prematurity (ROP) are characterized by microvascular degeneration, followed by an abnormal hypoxia-induced neovascularization (NV). Although the triggering insult varies among the diseases, they share a common end result of capillary loss due to increased oxidative stress, cellular inflammation and vascular injury and dysfunction. We have linked activation of the urea hydrolase enzyme arginase to the latter complications in models of DR. Both arginase and nitric-oxide synthase (NOS) enzymes utilize L-arginine as substrate. NOS dysfunction due to limitations in L-arginine availability has been implicated in the pathogenesis of diabetic complications. Our studies in streptozotocin-induced diabetic mice and high glucose treated retinal ECs have demonstrated signs of retinal vascular activation and injury. These were associated with increased arginase activity and expression, decreased bioavailable nitric oxide (NO), increased superoxide formation and increased leukostasis. Blockade of the arginase pathway prevented these alterations, suggesting a primary role of arginase in retinal vascular dysfunction and injury. Our studies have also shown that endothelium-dependent retinal vasorelaxation was impaired in diabetic mice, however, deletion of arginase improved retinal vessel function and improved blood flow. During ischemic retinopathies, disturbances in retinal blood flow can result in vasoconstriction, ischemia, tissue hypoxia and formation of neovascularization (NV). Such alterations have been linked to development of ROP, a blinding disease that adversely affects premature infants due to oxygen-induced damage of the immature retinal vasculature resulting in pathological NV. Our studies using a mouse model of ROP, the oxygen-induced retinopathy (OIR) model indicate that a potent vasoactive and angiogenic factor endothelin (EDN) is responsible for pathological NV. Our analysis revealed significant increases in EDN1, EDN2 and endothelin A receptor (EDNRA) mRNA and EDN2 protein expression during ischemia. EDN2 was localized to endothelial cells and retinal glia in OIR retinas. Treatment of OIR mice with EDNRA blocker, BQ-123, significantly increased vessel sprouting resulting in enhancement of physiological angiogenesis and decreased pathological NV. OIR triggered a significant increase in STAT3 activation and VEGFA production and increased mRNA expression of angiogenic and inflammatory mediators, which were all reduced by BQ-123 treatment. These studies suggest that EDNRA activation during OIR promotes vessel degeneration and pathological NV. Collectively, both arginase and endothelins are increased in models of ischemic retinopathies. These two pathways could be interrelated through an unknown cross-talk mechanism that needs to be elucidated.
    • Activities and Perceived Outcomes of Nurse Case Managers: Building a Case Management Model for Rural Hospitals

      Anderson-Loftin, Wanda; Department of Physiological and Technological Nursing (1996-12)
      The primary purpose of this study was to describe the activities and perceived outcomes of nurse case managers in a rural hospital setting and the relationship of nurse case managers' education, experience, and age to activities and perceived outcomes. Results of the study will be used to further develop and refine a portion of the investigator-developed model of Nursing Case Management for Rural Hospitals which served as the study framework. Nurse case managers in non-federal, rural hospitals listed in the American Hospital Association's (1995) guide to U. S. hospitals were surveyed using an investigatordeveloped instrument. Psychometric qualities of the instrument were determined as part of the study. The sample (N = 302) consisted primarily of white, middle-aged females. The majority were ADN or BSN nurses who averaged two to three years experience in case management and 16 years experience in nursing. Descriptive, correlational, and multivariate statistics were used to analyze the data. Results indicated that individual advocacy was the most frequent activity, and comments suggested that nurse case managers were becoming aware of the centrality of advocacy to their practice and job satisfaction. The second most frequent activity was teaching, with clinical practice third. The most frequent pattern of activities reflected assessment and coordination of community resources through an advocacy role for the client while performing managed care/quality assurance activities. The top ten perceived outcomes were: (a) increased patient satisfaction, (b) reduced fragmentation of care, (c) reduced length of stay, (d) increased job satisfaction, (e) increased job enjoyment, (f) increased quality of life, (g) increased functional health, (h) increased self-care, (i) increased autonomy, and (j) attainment of goals within the time-frame for reimbursement. Two-thirds of the nurse case managers thought their activities prevented delays in care and provided clients with a regular source of care, thus increasing access to care. Other significant (pi .05) findings indicated: (a) ADN, BSN, and MSN nurse case managers engaged in more teaching than nurse case managers with diplomas, (b) system advocacy was higher for MSN than for diploma, ADN, or BSN case managers, and (c) experienced nurse case managers engaged in more clinical practice than inexperienced nurse case managers.
    • Activity-dependent regulation of the dopamine transporter is mediated by Cam Kinase II signaling

      Padmanabhan, Shalini; School of Graduate Studies (2009-01)
      Dopamine signaling in the brain governs a variety of functions such as locomotor activity, reward, attention and working memory. The dopamine transporter (DAT) plays a crucial role in the clearance of extracellular dopamine and thus helps terminate dopamine neurotransmission. DAT is also the target for psychostimulant drugs of abuse and therapeutic agents. Changes in DAT expression occur in neuropsychiatric disorders such as attention deficit hyperactivity disorder (ADHD) and chro_nic psychostimulant use, and variability in DAT abundance is associated with differences in working memory. However, mechanisms regulating DAT expression are poorly understood. We tested the hypothesis that neuronal activity is one of the non-genetic determinants of DAT abundance. Chronic perturbations in neuronal firing, caused by pharmacological agents, significantly altered DAT expression and function in primary cultures of mesencephalic neurons. Pharmacological experiments showed that calcium entry through L-type voltage-gated calcium channels and calcium/calmodulindependent protein kinase II (CaMKII) activity played a role in activity-dependent changes in DAT expression. In order to further evaluate the role of CaMKII in DAT regulation, the effect of sustained depolarization, a stimulus often used to study activity-dependent changes in gene expression, on DAT expression was tested. Surprisingly, chronic KCl-induced depolarization decreased DAT expression and function. Measurement of CaMKII activity in dopam·ine neurons showed that chronic depolarization led to a decrease in CaMKI I activity, even in the presence of elevated intracellular calcium, due to activation of the serine/threonine protein phosphatase 2A. Moreover, increasing CaMKII activity in dopamine neurons by introducing a constitutively active CaMKII mutant caused a significant increase in DAT abundance while inhibiting CaMKII activity in dopamine neurons using a dominant-negative CaMKII mutant decreased DAT abundance suggesting that CaMKII activity is both sufficient and required to cause changes in DAT expression in a cell autonomous fashion. Taken together, our data demonstrate that CaMKI I activity can govern DAT expression and may play an important role in dopamine neurotransmission in the brain.
    • Activity-Dependent Regulation of the Dopamine Transporter is Mediated by Cam Kinase II Signaling

      Padmanabhan, Shalini; Department of Pharmacology (2009-01)
      Dopamine signaling in the brain governs a variety of functions such as locomotor activity, reward, attention and working memory. The dopamine transporter (DAT) plays a crucial role in the clearance of extracellular dopamine and thus helps terminate dopamine neurotransmission. DAT is also the target for psychostimulant drugs of abuse and therapeutic agents. Changes in DAT expression occur in neuropsychiatric disorders such as attention deficit hyperactivity disorder (ADHD) and chronic psychostimulant use, and variability in DAT abundance is associated with differences in working memory. However, mechanisms regulating DAT expression are poorly understood. We tested the hypothesis that neuronal activity is one of the non-genetic determinants of DAT abundance. Chronic perturbations in neuronal firing, caused by pharmacological agents, significantly altered DAT expression and function in primary cultures of mesencephalic neurons. Pharmacological experiments showed that calcium entry through L-type voltage-gated calcium channels and calcium/calmodulindependent protein kinase II (CaMKII) activity played a role in activity-dependent changes in DAT expression. In order to further evaluate the role of CaMKII in DAT regulation, the effect of sustained depolarization, a stimulus often used to study activity-dependent changes in gene expression, on DAT expression was tested. Surprisingly, chronic KCI-induced depolarization decreased DAT expression and function. Measurement of CaMKII activity in dopamine neurons showed that chronic depolarization led to a decrease in CaMKII activity, even in the presence of elevated intracellular calcium, due to activation of the serine/threonine protein phosphatase 2A. Moreover, increasing CaMKII activity in dopamine neurons by introducing a constitutively active CaMKII mutant caused a significant increase in DAT abundance while inhibiting CaMKII activity in dopamine neurons using a dominant-negative CaMKII mutant decreased DAT abundance suggesting that CaMKII activity is both sufficient and required to cause changes in DAT expression in a cell autonomous fashion. Taken together, our data demonstrate that CaMKII activity can govern DAT expression and may play an important role in dopamine neurotransmission in the brain.
    • AN ACTOR-NETWORK VIEW OF THE CYBER DOMAIN’S EFFECTS ON DEMOCRATIZATION THROUGH ELECTIONS

      Garrett, Eric; Department of Social Sciences (Augusta University, 2020-05)
      Cyberspace conceptualizations include combinations of Internet infrastructure, the devices used to access it, and applications used to encapsulate or communicate data. Other conceptualizations are more abstract. Whether it directly enables democracy as a public sphere, drives economics in the private sector, or securitizes as a domain for information operations, or cyber warfare. These conceptualizations obscure understanding cyberspace’s first order effects on events, and second order understanding related to intelligence and security studies. The research question, can actor-network theory supply a robust theoretical framework to understand and describe cyberspace’s core qualities as a democratizing medium, will be examined in Kenyan, Nigerian, and Zambian use of cyberspace related to elections. Examination of these data points through four elemental characteristics of cyberspace, proliferation, evolution, “spatial hereness,” and linkability, within an actor-network theory will lead to a determination if the totality of cyberspace is a democratizing medium. Lastly, this paper will make general recommendations that can lead to greater understanding of cyberspace that can influence policy and decision making as well as encourage democratic maturity in cyberspace by applying the considerations gained from an actor-network theory perspective.
    • ADAM17 AND AGING-RELATED VASCULAR DYSFUNCTION

      Dou, Huijuan; Department of Physiology (1/25/2018)
      A disintegrin and metalloproteinase ADAM17 (tumor necrosis factor–converting enzyme) regulates soluble TNF levels. We tested the hypothesis that aging-induced activation in adipose tissue (AT)-expressed ADAM17 contributes to the development of remote coronary microvascular dysfunction in obesity. We found that the increased activity of endothelial ADAM17 is mediated by a diminished inhibitory interaction with caveolin-1, due to age-related decline in caveolin-1 expression in obese patients and mice or to genetic deletion of caveolin-1. Coronary arterioles (CA) and AT were examined in patients who underwent heart surgery. Excess, ADAM17-shed TNF from AT arteries in older obese patients was sufficient to impair CA dilation in a bioassay in which the AT artery was serially connected to a CA. CA and AT were also studied in 6-month and 24-month lean and obese mice. We found that obesity elicited impaired endothelium-dependent CA dilations only in older patients and in aged obese mice. Transplantation of AT from aged obese, but not from young or aged, mice increased serum cytokine levels, including TNF, and impaired CA dilation in the young recipient mice. In patients and mice, obesity was accompanied by age-related activation of ADAM17, which was attributed to vascular endothelium–expressed ADAM17. Additionally, ADAM17 mediates shedding of JAM-A (junctional adhesion molecule-A). We hypothesized that ADAM17 activation, via increased JAM-A shedding impairs flow mechanosensing and induces abnormal artery remodeling in aging. We found a reduced lumen diameter and increased wall thickness in AT of aged patients. ECs using plasmid JAM-A were aligned to flow direction earlier than GFP treated control cells. Site-directed mutagenesis was employed to generate JAM-A cleavage resistant mutants, we detected soluble JAM-A in the supernatants from cells transfected with plasmid JAM-A, but not from cells expressing mutant JAM-A plasmids. Importantly, soluble JAM-A is significantly increased in the supernatant from cells with combined action of plasmid JAM-A and recombinant ADAM17, when compared to cells with plasmid JAM-A alone. Collectively, our data revealed that age-related reduction in Cav-1 expression and subsequently increased the activity of endothelial ADAM17 led to excess TNF production, which acts remotely to promote coronary arteriole dysfunction. Whereas activation of ADAM17 in vascular endothelium mediates increased JAM-A shedding and causes ECs misalignment. Our data suggest that the combined action of TNF and JAM-A lead to development of CMD and its related vascular remodeling in older obese patients.
    • Adoption of AACN verification of feeding tube placement practice alert by critical care nurses

      Bourgault, Annette M/; College of Graduate Studies (2012-04)
      The intent of clinical practice guidelines is to help bridge gaps between evidence and practice, yet there is no correlation between availability of guidelines and changes in practice. Little is known about how critical care nurses adopt guidelines, since few studies have sampled nurses exclusively. This descriptive, exploratory study examined factors influencing adoption of the American Association of Critical-Care Nurses (AACN) Verification of Feeding Tube Placement Practice Alert and four clinical practices recommended by this guideline. An online questionnaire, based on Rogers' Diffusion of Innovations conceptual framework was completed by 370 critical care nurses from 45 states. Fifty five percent were aware of the Practice Alert and 45% had adopted the guideline in practice. Only · 24% of nurses who adopted the guideline had also adopted/implemented all four clinical practices. Practice adoption ranged from 23-94%, although 10-73% of nurses indicated partial adoption, performing practices only some of the time. There was also a gap between nurses being aware of practices (60-98%) and adopting the practices (23-94%). Predictors of AACN Verification of Feeding Tube Placement Practice Alert adoption measured by logistic regression included BSN or higher nursing education (OR · = 2.49), and the guideline characteristics of observability (OR = 1.46) and trialability (OR= 1.37). Predictors of clinical practices included membership in a professional association (OR= 4.09), staff nurse/charge nurse role (OR= 4.01), traditional communication behavior (OR= 2.83), research/web-based communication behavior (OR = 2.11 ), academic medical center (2.11 ), and perception of a policy. Policy was the only significant predictor of all four practices (OR 7.08, 42.71, 17.09, 2.78 respectively). Only practice four, avoidance of auscultation by air bolus method, was associated with Practice Alert adoption, although this was not significant when other practice guidelines were controlled for. Factors influencing nurses' decisions to adopt guidelines within an organization are multifaceted. More research is necessary to obtain a better understanding of these factors to achieve the ultimate goal of increasing translation of evidence into clinical practice.
    • Adrenal zona glomerulosa targeting in transgenic mice

      Parmar, Jeniel; School of Graduate Studies (2009-12)
      The final step in the production of aldosterone is performed by the enzyme aldosterone synthase (CYP11 B2)-. CYP11 B2 is primarily expressed in the zona glomerulosa (ZG). of the adrenal cortex. Adrenocortical, expression of CYP11 B2 is primarily regulated by circulating levels of angiotensin II (Ang II) and K+, but the molecular mechanisms that control its ZG-specific expression are not clearly defined. Con$iderable in vitro analyses have been performed towards defining the mechanisms that control CYP11 B2 expression. Previous studies from our laboratory and others have identified · several cis-regulatory elements on the 5' · flanking promoter region (at -71/64, -129/114, -351/343 and -773/766) that regulate basal expression as well as maximal stimulation of CYP11 B2 gene transcription. Moreover, key· transcription factors that bind these cis-regulatory regions including NGFIB, NURR1, SF-1 and COUP-TF have also been identified. Hence, through several in vitro analyses, a considerable evidence exists supporting the contention that these regulatory elements found within the 5' flanking promoter region may control ZG-specific expression of CYP 11 B2 gene. However, thus far, all evidence is based on in. vitro analyses of transcriptional regulation, which does not always depict in vivo_occurrences. To initiate our in vivo assessment of CYP1182 promoter, we began by comparing the DNA sequences between human, mouse, and rat CYP1182 genes, which interestingly revealed high sequence similarity in the · 5' flanking promoter region of the CYP1182 gene. This result suggested that the cisregulatory regions identified by in vitro analyses likely plays an important role in CYP1182 ZG-specific gene expression. Therefore, we generated transgenic mouse lines by pronuclear injection of a Transgenic (Tg) DNA construct containing 985 base pairs (bp) of the mouse Cyp11 b2 promoter driving expression of a Lacz reporter gene.· Importantly, 4 founder Tg mouse lines revealed Lacz expression exclusively in the adrenal ZG. Mice fed a normal sodium diet (0.3 %) and a low sodium diet (0.03 %) showed Lacz mRNA expression exclusively in adrenal tissue. Furthermore, (3-galactosidase protein (the product of LacZ) was localized solely in the ZG of the Tg mice. Hence, the role of the proximal promoter region of the Cyp11 b2 gene was confirmed, in vivo, as this region allowed induction of Lacz exclusively in the adrenal ZG of Tg mice. Moreover, with the expression of Lacz properly restricted to adrenal ZG, we concluded that regions required for Cyp11 b2 gene repression in the adjacent inner two zones of the adrenal cortex were also confined within the 985 bp promoter. This regulatory fragment. will be an invaluable tool for adrenal ZG targeting of genes believed to play a role in adrenocortical diseases and aldosterone dysregulation. While developing Tg mice, we also focused on characterization and development of novel adrenocortical cell lines. As aforementioned, in vitro culture models have allowed a multitude of studies that have broadened our understanding of normal adrenocortical endocrine function. Primary cultures of adrenocortical cells have been an excellent source for in vitro studies. However, the eventual onset of senescence in primary cultures of cells creates a recurring need for the costly · and difficult isolations of fresh adrenocortical cells. Hence, the use of primary cultures has been increasingly supplemented by immortalized cell lines. We utilized an adrenocortical carcinoma to develop a human adrenocortical cell line. We entitled it the human adrenocortical carcinoma cell line clone 15 (HAC15). HAC15 represents only the second human adrenocortical cell line available that exhibits physiological hormonal responses, steroid.ogenesis, and expression of steroid-metabolizing enzymes. The ability of HAC15 to respond to Ang II, K+, and ACTH makes it the first adrenal cell line capable of responding to the three main physiologic regulators of the adrenal cortex.
    • Adrenal Zona Glomerulosa Targeting in Transgenic Mice

      Parmar, Jeniel; Department of Physiology (2009-12)
      The final step in the production of aldosterone is performed by the enzyme aldosterone synthase (CYP11B2). CYP11B2 is primarily expressed in the zona glomerulosa (ZG) of the adrenal cortex. Adrenocortical expression of CYP11B2 is primarily regulated by circulating levels of angiotensin II (Ang II) and K+, but the molecular mechanisms that control its ZG-specific expression are not clearly defined. Considerable in vitro analyses have been performed towards defining the mechanisms that control CYP11B2 expression. Previous studies from our laboratory and others have identified several c/s-regulatory elements on the 5' flanking promoter region (at -71/64, -129/114, -351/343 and -773/766) that regulate basal expression as well as maximal stimulation of CYP11B2 gene transcription. Moreover, key transcription factors that bind these c/s-regulatory regions including NGFIB, NURR1, SF-1 and COUP-TF have also been identified. Hence, through several in vitro analyses, a considerable evidence exists supporting the contention that these regulatory elements found within the 5' flanking promoter region may control ZG-specific expression of CYP11B2 gene. However, thus far, all evidence is based on in vitro analyses of transcriptional regulation, which does not always depict in vivo occurrences. To initiate our in vivo assessment of CYP11B2 promoter, we began by comparing the DNA sequences between human, mouse, and rat CYP11B2 genes, which interestingly revealed high sequence similarity in the 5' flanking promoter region of the CYP11B2 gene. This result suggested that the cisregulatory regions identified by in vitro analyses likely plays an important role in CYP11B2 ZG-specific gene expression. Therefore, we generated transgenic mouse lines by pronuclear injection of a Transgenic (Tg) DNA construct containing 985 base pairs (bp) of the mouse Cyp11b2 promoter driving expression of a LacZ reporter gene. Importantly, 4 founder Tg mouse lines revealed LacZ expression exclusively in the adrenal ZG. Mice fed a normal sodium diet (0.3 %) and a low sodium diet (0.03 %) showed LacZ mRNA expression exclusively in adrenal tissue. Furthermore, (3-galactosidase protein (the product of LacZ) was localized solely in the ZG of the Tg mice. Hence, the role of the proximal promoter region of the Cyp11 b2 gene was confirmed, in vivo, as this region allowed induction of LacZ exclusively in the adrenal ZG of Tg mice. Moreover, with the expression of LacZ properly restricted to adrenal ZG, we concluded that regions required for Cyp11b2 gene repression in the adjacent inner two zones of the adrenal cortex were also confined within the 985 bp promoter. This regulatory fragment will be an invaluable tool for adrenal ZG targeting of genes believed to play a role in adrenocortical diseases and aldosterone dysregulation. While developing Tg mice, we also focused on characterization and development of novel adrenocortical cell lines. As aforementioned, in vitro culture models have allowed a multitude of studies that have broadened our understanding of normal adrenocortical endocrine function. Primary cultures of adrenocortical cells have been an excellent source for in vitro studies. However, the eventual onset of senescence in primary cultures of cells creates a recurring need for the costly and difficult isolations of fresh adrenocortical cells. Hence, the use of primary cultures has been increasingly supplemented by immortalized cell lines. We utilized an adrenocortical carcinoma to develop a human adrenocortical cell line. We entitled it the human adrenocortical carcinoma cell line clone 15 (HAC15). HAC15 represents only the second human adrenocortical cell line available that exhibits physiological hormonal responses, steroidogenesis, and expression of steroid-metabolizing enzymes. The ability of HAC15 to respond to Ang II, K+, and ACTH makes it the first adrenal cell line capable of responding to the three main physiologic regulators of the adrenal cortex.
    • Adult patients' descriptions of postoperative pain : an exploratory study

      Rufo, Mary Kathryn Liu; School of Nursing (1970-12)
    • AKAP350 targets to the centrosome and scaffolds a novel transforming acidic coiled-coil protein, TACC4

      Steadman, Brent T.; School of Graduate Studies (2003-06)
      AKAP350 is a multiply-spliced scaffolding protein that localizes to the centrosome -and Golgi apparatus. Two important characteristics of AKAP350 are its ability to interact with multiple signaling partners and to target to specific cellular structures. Here, we have identi_fied a novel interacting partner of AKAP350 from the Transforming Acidic Coiled-Coil protein family, TACC4. Localization of fusion GFP-TACC4 proteins to the centrosome in interphase Jurkat cells required AKAP350 interaction. During mitosis, GFP-TACC4 expression targeted to the spindle apparatus and delayed cell cycle progression. Multiple GFP-TACC4 transfected HeLa cells displayed Mad2-positive kinetochore staining suggesting GFP-TACC4 expression prolonged activation of the spindle checkpoint. AKAP350 localizes to the centrosome by a specific targeting sequence in its carboxyl terminus known as the PACT domain. Expression of GFP-PACT altered structures of the mitotic apparatus in Jurkat • cells. In interphase cells, hypertrophied centrosomes, fragmented centrosomes, and supernumerary centrosomes were increased with GFP-PACT expression. Mitotic cells displayed increased tripolar and multipolar spindle apparatuses. Expression of GFP-PACT in rat intestinal epithelial (RIE-1) cells altered its morphologic growth characteristics in a manner indicative of cellular transformation. We propose that AKAP350 scaffolds a multivalent complex to the centrosome that may play role in signaling pathways important for cellular transformation. Therefore, AKAP350 may provide further evidence that centrosome alterations are important in the biology of cancer.