• Identification and Characterization o f CRIPlb: A Novel CBi Cannabinoid Receptor Interacting Protein

      Niehaus, Jason L.; Department of Biological Sciences (2006-07)
      G protein-coupled receptors (GPCRs) transduce extracellular stimuli to intracellular signals through their interaction with heterotrimeric G proteins. Signaling diversity and specificity is imparted primarily through variations o f G protein subunits. Protein-protein interactions between intracellular accessory proteins and GPCRs also modify signaling by altering receptor activity or signaling pathways. The ability of intracellular proteins to interact with the CBi cannabinoid receptor was investigated to determine whether particular signaling properties of CBi resulted from interaction with specific CBi interacting proteins. A novel protein named C RIPlb was discovered to interact with the C-terminal tail o f CB). The interaction between CRIPlb and CBi was characterized using the yeast two-hybrid assay. Functional consequences of the CRIPlb- CB| interaction were investigated by examining protein localization by confocal microscopy and measuring CBi mediated N-type Ca2+ channel activity in the presence of CRIPlb by whole-cell patch clamp recordings. The yeast two-hybrid assay indicated that the last nine amino acids of the CBi C-terminal tail were required for interaction with CRIPlb. Heterologous expression of C RIPlb and CBi in HEK 293 cells did not reveal evidence of colocalization, nor was CBi able to significantly traffic C RIPlb to the plasma membrane. However, CRIPlb and CBi were found to colocalize in superior cervical ganglion (SCG) neurons. Whole-cell voltage-clamp recordings of N-type Ca2+ channels in SCG neurons indicated that CRIPlb had no effect on agonist- or inverse agonist-induced modulation of Ca2+ current by CBi. Furthermore, the level of CBi constitutive activity was not significantly altered by CRIPlb. The high affinity of CBi for G proteins, as demonstrated by the ability of CBi to sequester G proteins from other Gi/0 coupled receptors, was unaffected by expression of CRIP lb. These results provide evidence that CRIPlb is a novel CBi accessory protein that interacts with the C-terminal tail of CBi. While CRIPlb and CBi can apparently interact in a neuronal expression system, the ability of CRIPlb to modify CBi signaling was not detected in any of the pathways investigated. Thus, the distinctive signaling properties of CB|, such as constitutive activity and G protein sequestration do not originate from nor are modified by CRIPlb.
    • Identification and Characterization of Two New Players in DNA Double-Strand Break Repair - PSF and p54(nrb)

      Udayakumar, Durga; Department of Molecular Medicine (2005-12)
      The stability and integrity of a genome depends on how accurately the genetic information is passed on to each daughter of a dividing cell. This accuracy is compromised when the genome is exposed to various stressful conditions, including ionizing radiation (IR), radiomimetic agents such as bleomycin, neocarzinostatin, and etoposide, free radicals generated from metabolic processes, and also errors during replication. The effect is DNA damage resulting in potentially lethal double-strand breaks (DSBs). The causes are as follows: DSBs are also created as intermediates during specialized recombination processes, such as V(D)J recombination, immunoglobulin class switching and somatic hypermutation.
    • Identification of Muscarinic Receptor Subtypes and Arachidonic Acid Metabolites Mediating the Effects of Actylcholine in The Pulmonary Circulation of Rabbits and Cats

      El-Kashef, Hassan; Department of Pharmacology (1987-05)
      Acetylch.oline (ACh) is known ·to reduce vascular pressure throughout the systemic. circulation; in. the·-- pulmonary circulatfon: ACh: can·- produce:either vasodi-1ationr or' ·vasoconstri-ction. For example,. ACh', produces: vasoconstrictfon · in· the, pulmonary ·circula~i on· of rabbits .. but dtlati on in· the· -feline. pu·lmof!ary-, vasculature·~-- The·· mechanism(s-) responsible·' for· the· actions of ACh in the pulmona.ry vasculature-- remain unc;lear. This study was designed to i·nve·st.igate poss·ible mechanisms. responsible for the actions. of ACh in the ra~bit ancf cat pulmonari_.circulation·~- Our hypothesi's is tbat . variations in· the· v.as.cular ·-response~ to: ACh may- be due to differe.nces in med-iators. (e.g. ·prostanoids)· released upon. activation .. of the muscarinic receptor~ These· di.ffe-rences cou 1 d be attributed,· either to. differences ... in muscarinic receptor subtypes.-. or to differences. in the·. metabolism of· arachidonic acid- tn rabbit. vs. cat. In anesthetized rabbits,_ ACh-induced pulmonary=- vasoconstriction-··. was,. totatly, inhibited::'·- by:: the: phosphotipasey A'2· inhibitor· qu-inacrine··,. the cycl o~oxygenase"' inhibitors:; indomethacin·,. and,.~ mec lofenamate·,, the- thtomboxane/ A:2:· synthetase=, i nh~i bi:tot· ]-;.;.{1'- Imidazolyl.)-· Heptano.fc. Acid .. (7;..IHA), and'- by,- the··' thromboxane·· A- 2 . receptor·· antagpntst. SQ:. 29-~.548:, .. but not·,. by·- the· 1 .ipoxygenase:-- inhibitor- nordihydrogua-iaret.ic: acid( NDGA}':. AGh·: s:igni-ftcantly; increased> plasma; thromboxane···B2.' (TXB'2J:· levels;. in·-. anesthetized:·,_ rabb_its:. A.Ch:~induced:. decrease'~ in, system.ic ar.teria:l pressure'" was, not· affected: by:·. any o.f'· the·· i nh'ibftors,. mentionedJ above--._ Small>. doses: of· the, select.i ve:· Mi- muscari nfc. receptor· subtype· antagonists:, pi renzepihe and_:. tr.ihexypheni:d~l,.. s:i gni-f.i cantly:- fnhi bjted:-: the· ACh.-i nduced:~- increase:'-·. i m pulmonary; vascuJa·r res:fstance- but. not: the~ ACh-induced=;· decrease Ht systemic. arterfal~: pressur.e··. The:.- s·ame" dose·· o.f secover.i ne:, .. a-~.- se·l ecti ve, M2-- muscar.i.nic: ii recept.or subtype. antagonist, dld ·not· change the · ACh ·effects on the pu 1 mona ry or· sys.temi c · v qSCu latu,re. Larger doses of pirenzepine, trihexyphen-idyl and\· secoverine,· totally. inhibited· the.- effects:: of ACh in· the .. pu:lmonary· and: systemic: circulatjons. Atrop.ine··. equally:- inh.i.bi ted:· the· effects. of ACn· on'" the pulmonary..- and.: systemic vascul atures- at.- any. do$es~ . . used': In .. fso_lated· rabbit·. lungs .. perfused·:·in:· situ~: w:ith · b]ood·-,_'free· medium;< . . . . . . . the ACh-fnduced· increases fn .. ·pulmonary v.ascular resistance, TXB2 level·s: a:nd ·6-Keto~PG·Fra were significa.nt.ly inhibited. by small concentrations.· of· pi.renzepine but .not· secove·rine. Larger concentrations. of· p.irenzepine~ and:'· · . . secover·ine totally ·inhibited· these· effects of ACh. In -anesthetized c~ts, the· ACh~inducec:l decre-ase in pulmonary vascular resfstance was partially~ · inhibited· by quinacrine and·' indomethacin and· significantly:· .potenti~ted by NOGA. The ACh-induced ·decrease in systemic arterial .pressure was not s·ignificantly affected by· any. of these· inhibitors. Small. doses. of secover-ine:'' but not . pfrenzepi ne;~" i nh.ibited·· the: pulmonary: but,. not .. the:· systemic vascular-. response·, tct.. ACh.,,_ i!!.=- v:ivo~ Howev-er,_. large··· doses; .. of·.· ptrenz:epi ne-, · tri·hexyphen:fdyl. and' s.ecoveri'ne' significantly:· inh.i bited~ the:, .. . . effects. of:. ACh~ in·.: the~: pulmonary;· and. systemic vascu:l ature\ In: isola ted:· cat: 1 ung_s:. perfused~- fm: sitw: w.ith; bJ ood.;.free- · medium~. 'ACh'" di-lated... the·· precons.tricted:: pulmonary: artery. bu.t> tt: did·: not ·s,tgnific.antly:· alter- TXB2 ori 6~ Keto~PGF 1 at 1 eveTs:~.. Smal:T: concentrat:i ons:- of:-· secoverfne-': but· not· pJ.renzep.i ne~-- par.t.ially;' i nhi bi'tedr the· ACh;.. induced~· decrease· in: pulinonar,Yvascular ·· resistance· •. The· pros.tacyc;ti n .. · synthetase fnhi bitor· tranylcypromine· si"gn.f$icantly~ i'nhi bited;'; the,· ACh- induced§ decrease:· in:· · pu]monary.:; va-scu..lar· resi.stance·~- . . i fi: ·These· results. ·indi-cate that l) ACh has . opposite actions in. the systemic {dilatory) versus pulmonary· (constrictor) circulation of rabbi-ts, 2r Arachidon-ic· acid.'metabo'lftes·. mediate. the-. pulmonary: . but. :not·· .the" systemic vascular respon.se- to. ACh, in rabbit, 3}. Thromboxane·· A2 mediates,. the:· pulmonary- vasoconstrictor- response·· to, ACh in rabbit·~ 4} The r.abbit pulmonary . v~scular· muscarinic recepto.rs. are· very·,· sensit:ive to·. p_irenzep.i-ne· . and thus be:have m9re · 1 ike Mi recept~rs, _5) · In· the cat·, the· ACh- induced·· decrease in- p~lmonary but not systemic vascular. pressure is partly mediated by prostacycli'n, 6)' In _the cat, the vascula-r· muscarinic receptors both in the· pulmon~ry. and the systemic beds are not selectively sensitive to . .pirenz.ep.ine ·and- thus behave like. non-Mt receptors and,· 7) In the .cat, the· ·muscarinic receptors. in the· pulmonary. and, systemic vasculat~re· represent different or heterogenous populations of M2. receptors since· they exhibit different affinities·. to secoverine.
    • Identification of professional competencies needed for practicing registered nurses in urban and rural hospitals

      Isler, Barbara A.; School of Nursing (1981-05)
      The purpose of this study was to determine if the hospital- location .s i gni fi cantly influenced the regis ter.ed nurses• competency needs .. A. sample of 112 urban and 80 rural .nurses was selected by Nursing Directors in 23 Georgia hospitals. Data were collected by use of a 113 item . competency questionnaire adapted from Clayton'·s (1978) work in thi.s area .. · Findings suggest the urban o~ rural hospital setting significantly . influences the competencies used by the registered nurse. Other findings include the acceptance percentage for ea-ch competency by the urban or. rural nurse indicating thei~ perception of nee~ and demographic characteristics of the two nurse populations.
    • Identification of RAB11-Family Interacting Proteins (RAB11-F1Ps): Integral Components in Plasma Membrane Recycling

      Hales, Chadwick M; Institute of Molecular Medicine and Genetics (2003-05)
      Given the involvement of Rabl la in each of these cellular processes and given the potential impact of Rabl la on human health and disease, we sought to further establish a role for Rabl la in plasma membrane recycling. Since other Rab proteins have numerous characterized interacting proteins and because the repetoire for Rabl la is currently limited to three identified interacting proteins, we hypothesized that other Rabl la binding partners exist as putative downstream effectors for the small GTPase. We therefore proposed the following three aims: Aim 1: Identify R ab lla interacting proteins. Aim 2: Determine the effect of interacting proteins on membrane recycling. Aim 3: Establish an organizational model of a putative Rabl la complex. The progression of studies herein provides insight into the dynamic and complex process of plasma membrane recycling. Yeast two hybrid screening of a parietal cell cDNA library utilizing dominant active Rabl laS20V as the bait identified Rabl 1-Family Interacting Protein 1 (Rabll-FIPl), a novel R ab lla interacting protein. EST database searches with the R abll-FIPl sequence identified three homologous proteins with high carboxyl-terminal identity. Chapter 1 introduces the new family of Rabl la interacting proteins and provides the initial characterization. Interestingly, these studies indicated an interaction between Rabll-Family Interacting Protein 2 (Rabll-FIP2) and myosin Vb tail. Chapter 2 further describes the Rabl l-FIP2/myosin Vb tail binding and provides functional data placing Rabll-FIP2 as an integral component of the plasma membrane recycling system. Finally, recent studies have indicated a recycling system dependence on different kinase activities. Through kinase inhibitor studies and immunofluorescence imaging, evidence presented in Chapter 3 suggests that R ab lla along with multiple Rabll-FIP proteins function as a complex beginning at the process of endocytosis with movement dependent on multiple phosphorylation events. The ultimate goal throughout these studies is to provide a clearer picture of Rabl la function in plasma membrane recycling so that one day a positive impact on human health can be achieved.
    • Identification of Regulatory Elements in a Conserved Upstream Region of the Gene Encoding Interphotoreceptor Retinoid-Binding Protein (IRBP)

      Lu, Haiyan; Department of Ophthalmology (1999-06)
      (First Paragraph) IRBP is a large, single-subunit extracellular glycolipoprotein found in the interphotoreceptor matrix between the photoreceptor cells and retinal pigment epithelium cell layer (Fig. 1.). The protein is synthesized and secreted by the photoreceptor rods and cones, as well as pinealocytes, of all vertebrates. The molecular weight of human IRBP (1230 residues) is 133,400 daltons. This protein consists of four homologous segments of approximately 300 residues each. Each segment contains highly conserved hydrophobic domains among species. Ligands identified as bound to IRBP include retinoid isomers and fatty acids, and IRBP can also bind cholesterol, a-tocopherol and retinoic acid. The ability of IRBP to bind various retinoid isomers, fatty acids and many other hydrophobic ligands suggests multiple functions in the retina .
    • The Identification of the Requisite Knowledge and Relevant Role Activities by Nurse Administrators and the Relationship of These to Their Educational Preparation and Management Level

      Allen, Lori; Department of Nursing (1988-01)
      The purpose.of this descriptive correlational study was to identify the requisite knowledge and relevant role activities needed by nurse administrators to successfully enact their roles (first, mid, and top) and the relationship of these role knowledge areas and role.activities to their educational preparation and management level. To achieve the study purpose, two research questions and three hypotheses were tested. A total of 226 subjects participated in the study: 156 first-line nurse administrators; 44 mid level administrators.; and 26 top level nurse administrators. The study population consisted of a convenience sample of nurse administrators from five hospitals in a Southeastern metropolitan area. Data were collected through the use of the Nurse Administrative Role Aetivities ·and Knowledge (NARAA~) tool. Descriptive statistics were utilized to address the first research question. To test the hypptheses related to the second research question, ANOVA were utilized-. All three hypotheses were supported at the p=.05 level. More specifically, 13 role activities and four requisiteknowlege areas were found to be significantly different for first level .. and .top level administrators. The majority of these items were related to leadership, management, and administration of human resources. Seven role activities and 11 requisite knowledge areas were found to differ on the basis of educational preparation. Nurse administrators at each organizational level identified significantly different role activities·relevant for.their successful role enactment, but did not identify different requisite role knowledge areas. There was no significant difference in how nurses with different academic degrees rated the relevance of role activities to the success of their role enactment on the basis of academic degree. Academic degree did, however, discriminate among respondents in the degree to which they rated the relevance of requisite knowledge areas to the success of their role enactment. Other findings, limitations, implications, and suggestions for further study were discussed.
    • Immune regulation of tumor cell plasticity: A promising molecular target in breast cancer metastasis

      LEE, EUNMI; Department of Biochemistry and Molecular Biology / Cancer Center (2018-11-29)
      It is widely accepted that phenotypic plasticity of malignant cells is required during metastatic cascade. However, the specific mechanism of how the tumor microenvironment regulates tumor cell plasticity in metastasis is under intense investigation. We demonstrate here that monocytic and granulocytic subsets of myeloid-derived suppressor cells (MDSC), hereafter called mMDSCs and gMDSCs, infiltrate in the primary tumor and distant organs with different time kinetics and regulate spatiotemporal tumor plasticity. Using co-culture experiments and mouse transcriptome analyses in syngeneic mouse models, we provide evidence that tumor-infiltrating mMDSCs facilitate dissemination from the primary site by inducing the EMT/CSC phenotype. In contrast, pulmonary gMDSC infiltrates support metastatic growth by reverting the EMT/CSC phenotype and promoting tumor cell proliferation. We also observe that lung-derived gMDSCs isolated from tumor-bearing mice enhance metastatic growth of already disseminated tumor cells. Our ongoing studies reveal that calprotectin (S100A8 and S100A9 heterotetramer) is an important regulator of gMDSCs, which play a critical role in promoting breast cancer metastasis by inducing MET-like CSCs as well as suppressing anti-tumor immunity within the pre-metastatic niche. Furthermore, we develop a novel gMDSC-targeting compound that potentially binds to calprotectin and validate its therapeutic utility in a preclinical breast cancer model. Our goal for this study is to elucidate the molecular co-evolution of tumor and immune cells in cancer development and to identify molecular targets to provide alternative therapeutic options for women with metastatic disease.
    • Immune Response of Hamsters to Adenovirus

      Boudet, Robert; Department of Cell and Molecular Biology (1970-06)
    • Immunometabolic Regulation of Myeloid-Lymphoid Interactions Following Traumatic Brain Injury

      Braun, Molly; Biomedical Sciences (Augusta University, 2019-09)
      Traumatic brain injury (TBI) is a major public health issue, producing significant patient mortality and poor long-term outcomes. Increasing evidence suggests an important, yet poorly defined, role for the immune system in the development of progressive secondary injury. Herein, we tested the hypothesis that peripheral macrophage infiltration initiates long-lasting adaptive immune responses after TBI. Using a murine controlled cortical impact model, we found increased infiltration and pro-inflammatory (M1) polarization of macrophages for up to three weeks post-TBI. Monocytes purified from the injured brain stimulated the proliferation of naïve T lymphocytes, enhanced the polarization of T effector cells (Teff: TH1/TH17), and decreased the production of regulatory T cells (TREG) in a mixed lymphocyte reaction. Similarly, elevated Teff polarization within both blood and brain tissue was attenuated by myeloid cell depletion after TBI. Functionally, C3H/HeJ (TLR4 mutant) mice reversed both M1 macrophage and TH1/TH17 polarization after TBI, as compared to C3H/OuJ (wild-type) mice. Moreover, brain monocytes isolated from C3H/HeJ mice were less potent stimulators of T lymphocyte proliferation and TH1/TH17 polarization, as compared to C3H/OuJ monocytes. To further elucidate the mechanism underlying this myeloid TLR4-mediated Teff activation, we examined the metabolic regulator, 5’-adenosine monophosphate-activated protein kinase (AMPK), within infiltrating macrophages following TBI. We determined that reduced activation of myeloid AMPK induced the generation of pro-inflammatory, myelin reactive T-cells. Similarly, we detected myelin-laden macrophages within the cerebrospinal fluid of severe TBI patients. Administration of the AMPK activator, metformin, attenuated pro-inflammatory Teff cell generation and enhanced counter-inflammatory TREGs in wild-type mice; however, these effects were lost in myeloid-specific AMPKα1 knockout mice. Activation of AMPK restored myeloid expression and activity of Ten-eleven translocase 2 (TET2), a demethylase that regulated the expression of myeloid PD-L1 after TBI. Moreover, TET2-/- mice exhibited exaggerated T-cell activation in response to TBI. In line with these data, activation of myeloid AMPK reduced the loss of white matter and improved neurobehavioral outcomes after TBI. Our studies identify that immunometabolic dysfunction drives epigenetic regulation of the myeloid-lymphoid transition after TBI; suggesting targeted interventions during the early stage of injury may prevent progressive neurodegeneration.
    • Impact of Genetic Predisposition and Environmental Stress on Measures of Preclinical Essential Hypertension

      Poole, Joseph C.; Department of Cellular Biology and Anatomy (2006-06)
      The main objective of this project was to determine the impact of genetic risk and chronic environmental stress on measures of preclinical essential hypertension (EH) (e.g., exaggerated cardiovascular reactivity, increased resting hemodynamics and increased left ventricular mass [LVM]). A secondary objective was to evaluate the moderating and interactive effects of ethnicity, gender, body mass index [BMI] and anger expression on EH risk indices. Two genes with relevance for blood pressure (BP) control (e.g., beta-2 adrenergic receptor [ADRB2] gene and serotonin transporter [5-HTT] gene) were used to define genetic risk. Chronic environmental stress was assessed by socioeconomic status (SES) and subjective social status (SSS). The project consisted of three sequential studies on a large, multiethnic cohort of young adults (N>500). The first two studies were cross-sectional and based on the analysis of cardiovascular reactivity, resting hemodynamics and LVM data collected at a single visit. The third study was longitudinal and involved the tracking of BP and LVM over a 15-year span from childhood to early adulthood. In the first study, ADRB2 haplotype significantly interacted with anger suppression in African Americans such that high anger suppressing carriers had the highest resting SBP (p<.05) and TPR reactivity to a cold pressor task (p<.01). In European Americans, ADRB2 haplotype significantly interacted with BMI to predict resting hemodynamics, such that carriers who were high in BMI showed the highest SBP (p<.05). In the second study, a significant interaction between the 5-HTT promoter region polymorphism (5-HTTLPR) and social status was found for cardiovascular reactivity, such that S allele homozygotes who were low in SES and high in SSS exhibited the greatest BP and TPR reactivity to the video game stressor (p-values<.05). No significant interaction was found between 5- HTTLPR and social status in the longitudinal study, however a significant 5- HTTLPR by BMI interaction was determined for LVM, such that obese LL homozygotes had the greatest LVM over time (p<.001). Results from this project expand what is currently known with regard to EH etiology and carry implications for the prevention of EH through the early consideration of genetic, environmental and demographic risk factors.
    • Impaired Cognition in Spontaneously Hypertensive Rats: Role of Central Cholinergic Receptors

      Gattu, Mahanandeeshwar; Department of Pharmacology and Toxicology (1996-09)
      The cognitive impairment exhibited by the spontaneously hypertensive strain of rat is due to a reduction in the expression fo central cholinergic receptors. Therefore, the specific aims of this study are: 1. To determine whether the hypertensive state present in SHR contributes to impaired performance on spatial memory tasks. 2. To determine whether SHR exhibit altered expression of cholinergic receptor subtypes in brain regions important for memory function 3. To determine whether the decreased expression of central nicotinic-cholinergic receptors observed in SHR have functional significance.
    • Impaired Volume Regulation in Cardiovascular Disease in Two Distinct Populations

      Beavers, Melinda L. C.; Georgia Prevention Institute (2012-05)
      Volume dysregulation leads to congestive heart failure and death. The condition has been well-documented in both obesity-related cardiovascular disease and congenital heart disease. Our hypotheses are as follows: 1) Volume dysregulation, in the form of elevated systolic blood pressure and left ventricular mass index, is related to adiposity and aldosterone in adolescent boys but not girls. 2) Volume dysregulation, in the form of decreased nocturnal decline in blood pressure (non-dipping) is present in patients with tetralogy of Fallot, and is related to decreased left and right ventricular function. To test our first hypothesis, 100 healthy adolescents, recruited from area schools, were studied. Subjects were placed on a sodium-controlled diet for 4 days. Blood and urine samples were collected after one hour of rest. The protocol was repeated twice for each individual. Data were averaged between visits for greater statistical power. Adiposity and echocardiography measures were collected within 1 month of testing. Stepwise regression indicated that race and adiposity both contributed to the effects of aldosterone. For example, body mass index and race contributed to the model for aldosterone (Adjusted R2=0.303, p=0.002). In the aldosterone-hypertension risk relationship, stepwise regression indicated that only aldosterone contributed to the model for systolic blood pressure (Adjusted R2=0.098, p=0.023). To test our second hypothesis, 20 patients with repaired tetralogy of Fallot were recruited from clinic. Subjects completed a submaximal exercise test (modified Bruce) with echocardiography, and then wore an ambulatory blood pressure monitor for 24 hours. Of the 20 subjects with tetralogy of Fallot, 60% were 'non-dippers'. Race was significantly different between the dippers and non-dippers, with 1 of 8 African Americans being a dipper, and the remaining 7 African Americans being non-dippers (t=2.188, p=0.042). Right ventricular stroke volume (t=2.392, p=0.028) and ejection fraction (t=3.484, p=0.003) were significantly different between dippers and non-dippers. In a population of healthy adolescents with a well-distributed range of adiposity, increasing adiposity is associated with increasing aldosterone levels in boys but not girls. This is related to increasing systolic blood pressure and left ventricular mass in boys, but not in girls. These results may indicate an underlying volume dysregulation that contributes to hypertension and cardiovascular disease as a result of prolonged exposure to increased adiposity. In a population of 20 adolescents and young adults with repaired tetralogy of Fallot, nondipping is associated with decreased right ventricular stroke volume and ejection fraction, but is not associated with decreased left ventricular function. Patients with repaired tetralogy of Fallot and non-dipping may be at greater risk for failure due to the combination of pulmonary regurgitation with increased nocturnal pressures.
    • The Implementation of Controlled Physical Training and the Physiological Effects of Exercise in Women with Sickle Cell Disease

      Ramsey, Leigh-Taylor; Department of Physiology (1999-12)
      Sickle cell disease (SCD) comprises a group of genetic disorders of hemoglobin that affect more than 70,000 Americans and is associated with increased morbidity and health care cost (1). W ith improved medical management, individuals with SCD are now surviving well into adulthood. However, adults with SCD are often sedentary and because of medical conservatism are frequently discouraged from exercise fo r fear of precipitating acute vasoocclusive or painful episodes. A review of the literature revealed little baseline data on perceived health status and well-being, physical fitness (cardiovascular and muscular), and body composition in adults with SCD. It is currently unknown whether exercise or physical training (PT) will have beneficial, adverse, or no effect in SCD patients. As a result, exercise recommendations for adults with SCD are vague, and implementation and effectiveness of PT have not been reported. Moreover, there are no reports on the effects of controlled PT on perceived health status, physical fitness, and body composition parameters in patients with SCD. The effect of exercise on inflammatory mediators (i.e., tum or necrosis factor-alpha [TNF-a], interleukin - 6 [IL-6], and C-reactive protein [CRP]) and vasoactive mediators (i.e., nitric oxide metabolites [NOx] and endothelin-1 [ET-1]) has not been reported in patients with sickle cell anemia (SCA). Since changes in these parameters may influence the occurrence of vasoocclusive episodes by increasing adhesion of erythrocytes to the endothelium and by other mechanisms, such as vasoconstriction, it is important to clarify changes that may occur with exercise. Physical training holds the promise of allowing patients with SCD to preserve functional capacity and improve their quality of life, as has been reported for other chronic diseases. Yet, there are no repotted studies assessing the implementation and effectiveness of PT in adults with SCD. The study reported in this thesis provides new information concerning the impact of controlled PT on fitness and well-being in women with SCD. It also provides new information on the effects of three consecutive days of exercise, which might be used in a standard PT program. This project is a first step in the assessment of the role of regular exercise for SCD patients.
    • In search of genetic mutations for familial keratoconus

      Khaled, Mariam Lotfy; Department of Cellular Biology and Anatomy (Augusta University, 2019-05)
      Keratoconus (KC) is the most common corneal degenerative disorder and a leading cause of corneal transplantation in developed countries. KC is a multi-factorial disease with involvement of genetic, environmental, and hormonal factors. Although KC has been widely studied, the main cause of the disease and the molecular mechanism remain unknown. We aimed to study the molecular genetics of KC via utilizing next-generation sequencing technology including RNA-Seq, whole exome sequencing, and whole genome sequencing. We used RNA-Seq to study the KC-affected corneal transcriptome. We identified 436 coding RNAs and 584 lncRNAs with differential expression in the KC-affected corneas with a |fold change| ≥ 2 and a false discovery rate ≤ 0.05. Pathway analysis, using WebGestalt, indicated the enrichment of the genes involved in the extracellular matrix, protein binding, glycosaminoglycan binding, and cell migration. Co-expression analysis revealed 296 pairs of genes with significant KC-specific correlations. The RNA-Seq data analysis highlighted the potential roles of several genes (CTGF, SFRP1, AQP5, lnc-WNT4-2:1, and lnc-ALDH3A2-2:1) and pathways (TGF-β, WNT signaling, and PI3K/AKT pathways) in KC pathogenesis. Next, we used whole genome and exome sequencing to figure out the causal mutation(s) in a four-generation KC family with a linkage locus on Chr5q14.3-q21.1. We found a missense mutation in the phosphatase domain of PPIP5K2 (c.1255T>G, p.Ser419Ala). We found another missense mutation in the same domain of PPIP5K2 (c.2528A>G, p.Asn843Ser) in a second KC family. PPIP5K2 is a bifunctional enzyme involved in the inositol phosphate metabolic pathway. In vitro functional assays indicated the impact of the identified mutations on the enzymatic activity of PPIP5K2. PPIP5K2 expresses at a higher level than its homolog PPIP5K1 in both human and mouse corneas. A transgenic mouse model with the loss of phosphatase activity and elevated kinase activity of Ppip5k2 exhibited corneal structural abnormalities emphasizing the important role of PPIP5K2 in the homeostasis of corneal integrity. This study advances our knowledge of KC genetic etiology and helps in identifying a potential therapeutic target for KC.
    • IN VITRO AND IN VIVO STUDIES DEMONSTRATE A ROLE FOR SH3PX1 IN LAMELLIPODIA FORMATION.

      Hicks, Lawrence Joseph; Department of Cellular Biology and Anatomy (5/22/2018)
      Actin remodeling and endocytosis are essential functions for most cells. Defects in these processes present in a variety of diseases. Sorting nexins are known to contribute to endocytic uptake, cytokinesis, the retromer complex, and autophagy. Sorting nexin 9 (Snx9) interacts with major endocytic factors and proteins involved in regulation of actin cytoskeleton dynamics. Nonetheless, Snx9’s exact in vivo roles in these basic cellular processes and disease mechanisms are not known. By examining the roles of Sh3px1, we can better understand the mechanism by which this protein contributes to endocytosis and actin remodeling in vivo. Two additional paralogs, Snx18 and Snx33, complicate studies in mammalian models due to potential redundant mechanisms. Utilizing the single ortholog in Drosophila, sh3px1, this report describes the function of Sh3px1 in membrane organization and actin dynamics. Drosophila S2 cells that are depleted of Sh3px1 fail to form lamellipodia, a process that is also dependent on the actin nucleation factor, Scar. In addition, over-expression of Sh3px1 in S2 cells results in the formation of tubules and also long membrane protrusions, atypical of a classical BAR domain protein. An intact PX-BAR domain is required for these overexpression phenotypes. sh3px1 null flies are viable; however, mutant females have significantly compromised fertility. Female sh3px1 null egg chambers show many morphological defects. The age-dependent degeneration of the null egg chamber is not likely due to compromised endocytosis. Additionally, collective border cell migration is attenuated in the absence of Sh3px1. These cells are known for their reliance on endocytosis and modulation of actin dynamics for migration. We have found that Sh3px1 is essential in efficient lamellipodia production at the start of border cell migration. Our findings also suggest that Scar directly interacts with Sh3px1 and is upregulated in sh3px1 nulls. Mutation of Scar enhances many reproductive defects in sh3px1 nulls. Thus, our work reveals a main in vivo function of Sh3px1 in actin regulation for the production of structures such as lamellipodia.
    • In Vitro mechanical Analysis of Full-Arch Mandibular Implant-Supported, Complete Fixed Prosthesis Retainer Screws After Cyclic Loading

      Sananez, Andreina J.; Department of Oral Biology (2012-04)
      The use of implant-retained and supported prostheses has become a very successful treatment for completely edentulous patients. One of the most common fixed solutions involving implants consists of 5 to 7 implants supporting a framework upon which either porcelain or prefabricated acrylic resin denture teeth are added. A screw is utilized to attach the framework/prosthesis to the implants. Screw loosening is the second most common clinical complication in the implant-prosthesis system. If clinicians fail to detect worn or loose retaining screws, prosthetic fracture could occur, leading to more complicated, time consuming, and expensive repairs. Unfortunately, there is no established parameter that indicates when to expect these complications, and there is no proven recall-maintenance protocol to prevent them. The aim of this study is to examine and compare differences among de-torque values and prosthetic retention screws, using a simulated 5 implant-supported, mandibular complete fixed prosthesis. Material and Methods: Nine groups, each with its respective control, using five Nobel Biocare implants and a milled titanium framework were fabricated, assembled and tested. Dynamic loading was p on the performed tested groups through a custom made loading device for anterior, posterior, and distal cantilevered segments of the prosthesis, calculated to simulate clinical usage time. Removal of screws after 2 years of simulated oral function was performed. Before and after testing, screws were evaluated with a Scanning Electronic Microscope (SEM), for presence of debris, thread striations and homogeneity. Control groups remained unloaded for the same time the loaded groups were tested. Results: Comparisons of the difference between initial tightening torque and de-torque screw values were performed between loaded/unloaded groups and with respect to implant position. The interaction between loaded and position was significant (p=0.002). The comparison between loaded/unloaded groups was not statistically significant (p=0.518). Loaded and unloaded groups were compared separately at each of the 5 implants position, which showed a significant difference (p=0.0002, α=0.001). The sequencing effect was only seen in the control groups and thus would only be relative to framework insertion. The sequence effect was found to be overcome by from loading and resulted in a totally different position related to screw tightness. Within the limitations of this in vitro study, it was concluded that sequence of torque application could play a role in the preload of screws even with a passive fit, regardless the load applied.