• Primary versus secondary reconstruction of mandibular critical size defects using recombinant human bone morphogenetic protein 2: an experimental study in dogs

      Hussein, Khaled A.; Department of Oral Biology (2012-12)
      Very often, delayed reconstruction becomes the setting of choice in the reconstruction of large segmental defects in the mandible. Our hypothesis is that rhBMP2 delivery would elicit endogenous expression of BMP2 and VEGF in the soft tissue bed of the defect. Such response is expected to be more pronounced in the immediate than the delayed reconstruction, which will correlate with the quantity and quality of bone formation in the two settings. We also hypothesized that vascular endothelial cells (ECs) of the surrounding soft tissue contribute to the endogenous production of BMP2. In this study we used a mandibular canine segmental defect model (35 mm), periosteum was excised and also the delayed reconstruction group was included in this study in addition to the control group. We investigated the effect of different reconstruction settings on the quantity and quality of bony regenerates; on the production of endogenous BMP2 from the soft tissue bed of the defects and finally we tried to explore the source of this rhBMP2- induced endogenous BMP2 production both in vivo and in vitro. This study demonstrated that rhBMP2 delivery is more effective in immediate reconstruction of large mandibular segmental defects. Immediate delivery of rhBMP2 yielded more adequate reconstruction of the defect after 12 weeks, evident by the quantity and quality of the bone regenerate. Only in the immediate reconstruction group, the advantageous bone parameters were associated with significant up-regulation of BMP2 mRNA and protein in the soft tissue bed of the defect. This suggests that endogenous-BMP2 is important in maintaining the short-acting effect of the delivered rhBMP2. Regarding the source of the endogenous-BMP2, protein co-localization with ECs marker suggested that these cells could be the source for the endogenous BMP2 secretion in response to rhBMP2 treatment. This was confirmed by the in-vitro results on both the mRNA and protein levels. The gradual increase in expression of BMP2 mRNA and the significant upregulation of secreted BMP2 protein upon stimulation of human umbilical vein endothelial cells with 100-ng/ml rhBMP2 recognized a new mechanism of positive feed back response of ECs in response to BMP2 treatment.
    • Proactive Readiness Among Parents of Children with Chronic Mental Health Conditions

      McKinnon, Caroline R.; Department of Physiological and Technological Nursing (2013-11)
      Childhood mental health conditions are prevalent, persistent, serious, and complex. Families are expected to be involved in their child’s care, but may vary in their readiness for taking an active role. Proactive readiness is a new concept encompassing role beliefs, knowledge, confidence, and self-efficacy. Understanding the relevance of proactive readiness is a critical first step in supporting family management for mentally ill children. The study purpose was to describe the extent for which mothers of mentally ill children discussed proactive readiness. Data from a social support intervention study for mothers of children with mental health conditions (Scharer et al., 2009) were used in a secondary analysis to examine the extent to which mothers participated in a web-based chat room intervention and the content of their posts. A step-wise quantitative content analysis was conducted using a census sample of transcript data. Trained coders categorized mothers’ posts into one of four proactive readiness categories or as other content. Relationships between content of mothers’ posts, chat room participation, and demographic and health variables were examined. Over 3 years, 24 of 39 mothers posted approximately 5,000 messages. Mothers posted proactive readiness content in an average of 20% of their sentences (n=1190). Knowledge was the dominant content category, followed by role belief and much less frequently confidence of self-efficacy. Mothers in the lowest income group posted significantly more proactive readiness, role belief, and confidence content. Maternal race was significantly associated with chat room attendance and total posts, but not with posts per session attended. Proactive readiness content and chat room participation were unrelated to any other child, maternal, or family demographic or health variable. based on the findings of this study, proactive readiness appears to be a relevant topic among mothers of children with chronic mental health conditions. Further data are needed to provide details about the nature of mothers’ proactive readiness. Future researchers should consider using this study’s coding scheme as well as research designs and statistical analyses that would expand on the limited generalizability of this exploratory study. Nursing practice implications include a particular concern for low-income families receiving child mental health services.
    • The Proficiency-Based Classroom: Building on the Standards

      Watts, Tara; Department of English & Foreign Languages; Sandarg, Jana; Augusta University (2019-02-13)
      Proper communication is the most important element in any classroom, in particular, a foreign language classroom. Foreign language acquisition is essential, with many secondary schools and higher education institutions worldwide requiring foreign language studies in order to receive a degree. Students can learn foreign languages in many ways based on the ACTFL Standards: Communication, Cultures, Connections, Comparisons, and Communities. These standards expand the expectations of teaching methods as the guidelines in which a student is able to use the language outside of the classroom. The most important of these standards is Communication, which is the sending of a message from one individual to another. In this study, I will focus on the Communication standard, in particular, verbal communication in the classroom. Many students in the foreign language classroom struggle with the conversational aspect; therefore, teachers are focusing more on how to aid students in conversational learning. Furthermore, teachers incorporate cultural knowledge as a means to expand language acquisition in the classroom, giving language a context. In this presentation, I will study research on language acquisition and the ACTFL Standards; I will discuss proficiency-based projects I have done in addition to projects I plan on using in the classroom after graduation.
    • PROFILING G PROTEINS USING BIOLUMINESCENCE RESONANCE ENERGY TRANSFER

      Farooq, Maheen; Department of Chemistry and Physics; Department of Pharmacology & Toxicology; Spencer, Angela; Lambert, Nevin; Okashah, Najeah; Augusta University (2019-02-13)
      GPCRs are receptors that act in signal transduction pathways via guanosine nucleotide-binding proteins (G proteins). Extracellular ligands act on GPCRs resulting in activation of one or more G protein subtypes (Gs, Gi/o, Gq/11 and G12/13) affecting the concentration of intracellular second messenger molecules ultimately altering cellular function. Cellular responses to external signals are typically studied indirectly by measuring concentration changes in second messengers. However, this approach can be problematic as many GPCRs can activate multiple G protein subtypes, and many second messenger pathways engage in crosstalk. To address this issue, we used Bioluminescence Resonance Energy Transfer (BRET) to directly measure coupling between 5-hydroxytryptamine (5-HT; serotonin) receptors and different G protein family subtypes. We co-transfected cells with plasmid DNA encoding the 5-HT2B or 5-HT4 receptors fused to the bioluminescent protein nanoluciferase (NLuc) as well as plasmid DNA containing G protein subtypes fused to the fluorescent protein Venus. In BRET assays, we found that mGsq couples to 5-HT2B and mGscouples with 5-HT4 in response to 5-HT activation. These results are consistent with the literature. Interestingly, initial studies suggest that activated 5-HT4 shows secondary coupling to mGsi highlighting the potential novel signaling pathways that can be elucidated using this technique.
    • Profiling G Proteins Using Bioluminescence Resonance Energy Transfer

      Farooq, Maheen; Department of Biological Sciences (Augusta University, 2019-05)
      GPCRs are receptors that act in signal transduction pathways via guanosine nucleotide-binding proteins (G proteins). Extracellular ligands act on GPCRs resulting in activation of one or more G protein subtypes (Gs, Gi/o, Gq/11 and G12/13) affecting the concentration of intracellular second messenger molecules ultimately altering cellular function. Cellular responses to external signals are typically studied indirectly by measuring concentration changes in second messengers. However, this approach can be problematic as many GPCRs can activate multiple G protein subtypes, and many second messenger pathways engage in “crosstalk”. To address this issue, we used Bioluminescence Resonance Energy Transfer (BRET) to directly measure coupling between 5-hydroxytryptamine (5-HT; serotonin) receptors and different G protein family subtypes. We co-transfected cells with plasmid DNA encoding the 5-HT2B or 5-HT4 receptors fused to the bioluminescent protein nanoluciferase (NLuc) as well as plasmid DNA containing G protein subtypes fused to the fluorescent protein Venus. In BRET assays, we found that mGsq couples to 5-HT2B and mGs couples with 5-HT4 in response to 5-HT activation. These results are consistent with the literature. Interestingly, initial studies suggest that activated 5-HT4 shows secondary coupling to mGsi highlighting the potential novel signaling pathways that can be elucidated using this technique.
    • Profiling Insulin Like Factor 3 (INSL3) Signaling in Human Osteoblasts

      Ferlin, Alberto; Perilli, Lisa; Gianesello, Lisa; Taglialavoro, Giuseppe; Foresta, Carlo; Mei, Lin; Department of Neurology; College of Graduate Studies (2011-12-28)
      Background: Young men with mutations in the gene for the INSL3 receptor (Relaxin family peptide 2, RXFP2) are at risk of reduced bone mass and osteoporosis. Consistent with the human phenotype, bone analyses of Rxfp2â /â mice showed decreased bone volume, alterations of the trabecular bone, reduced mineralizing surface, bone formation, and osteoclast surface. The aim of this study was to elucidate the INSL3/RXFP2 signaling pathways and targets in human osteoblasts.
    • Profiling the HCA Receptor Family through BRET Analysis of GPCR-G-Protein and GPCR-Arrestin Interactions

      Saj, Dalia; Department of Biological Sciences; Department of Pharmacology & Toxicology (Augusta University, 2020-05)
      Increasing obesity rates have put the American population at higher risk for developing obesity-related medical conditions such as hypertension, heart disease, and diabetes. The hydroxycarboxylic acid (HCA) receptor family is a family of G protein-coupled receptors (GPCRs) that are expressed in adipose tissue and function as metabolic sensors, making them potential pharmaceutical targets in the treatment of obesity and other metabolic disorders. The HCA receptor family consists of the HCA1, HCA2, and HCA3 receptors, which are activated by hydroxycarboxylic acids such as lactate and 3-hydroxybutyric acid. We utilized bioluminescence resonance energy transfer (BRET) to study agonist-induced coupling of luciferase-tagged HCA receptors to Venus fluorescent protein-tagged G protein heterotrimers or arrestins. Our results indicate that the three HCA receptors couple to the Gαi/o subfamily of G proteins. The data additionally confirms a lack of coupling to the other G protein subfamilies (Gαs, Gαq, and Gα12/13), and lacks evidence of arrestin recruitment to HCA receptors. Overall, our study highlights the use of BRET as a powerful tool for analysis of GPCR signaling and demonstrates its possible use for future studies to determine the potency of potential drugs targeting HCA receptors as a therapy for health-related problems such as obesity.
    • Progesterone Regulation of Proliferation and Regression of Rat Decidua Basalis

      Dai, Donghai; Department of Cellular Biology and Anatomy (1998-07)
      During implantation mesometrial cells o f the uterine stroma become decidualized under the coordinate actions of progesterone (P4 ) and estrogen (E) [1,2]. This process is characterized by transformation o f phenotype and stromal cell proliferation between Days 8-12 of gestation, resulting ultimately in the formation o f the decidua basalis (DB) [3,4], By Day 14. however, the DB begins to regress and a reduced layer o f stromal cells persists to the end of pregnancy [5.6]. The regression of DB is accompanied by development o f two other layers, namely junctional zone (JZ) and labyrinth zone (LZ), which are fetal parts of the placenta and morphologically become predominant at the end stages o f pregnancy. Although the morphological changes have been well documented and numerous functions have been revealed for DB [3-8], the mechanism and factors involved in the regulation of proliferation and regression o f DB have not been elucidated. The transition of DB from proliferation to regression occurs in such a delicate way that the morphological integrity and functional competence of the DB and placenta are maintained even though stromal cells are being lost. The objective o f this study was to identify the intracellular signals initially favoring proliferation and synthetic processes and those promoting remodeling and regression as pregnancy progresses.
    • Progress City - An Honors Thesis Exhibition

      Conway, Baillie; Department of Art (2017-03)
      The purpose of this project is to explore the way sculpture can visually represent history. Progress City is an art exhibition that focuses specifically on the time period during which America transitioned from an industrial to a service-based economy (1950s - 1970s) and the effects this departure had on cities throughout the Southeast. The exhibition comprises a series of twelve buildings with sides featuring screen printed images of actual abandoned and dilapidated structures. Each building reflects an industry or trade that was once considered vital to the growth of southern communities. These buildings are juxtaposed alongside a collection of found objects that have either a direct or indirect correlation to businesses that once occupied the actual structures. For example, a series of empty food packaging boxes from the 1950s are set alongside a structure featuring screen printed images of an abandoned market facade. By juxtaposing the found objects, now considered outdated, alongside a corresponding abandoned building, the viewer is able to see first-hand the effects progress has had on southern cityscapes.
    • Progress City: An Honors Thesis Exhibition

      Conway, Baillie; Department of Art and Design (Augusta University, 2017-05)
      Progress City is a full-scale art exhibition examining the way history can be visually represented through sculpture. Focusing specifically on the time period during which America transitioned from an industrial to a service-based economy, it examines the effects this departure had on cities throughout the Southeast. Comprised of a series of sculptural buildings juxtaposed alongside a collection of found objects, this project mixes different forms of media together to create a conceptual display for the viewer to enjoy. As you will see, the project has undergone many revisions and edits over the course of this process. The final product is nowhere close to the ideas I had initially proposed at the beginning. However, you will come to see that, although this project may have seen many changes, it still revolves around the same theme, and that’s progress. [Introduction]
    • Proliferative Verrucous Leukoplakia (PVL) Expresses High Levels of Toll-Like Receptor 2 (TLR2)

      Koh, Joon; Kurago, Zoya; Georgia Cancer Center (Augusta University, 2019)
      In the current study, we analyzed samples of human oral mucosal PVL and other epithelial disorders to test the possibility that, if TLR2 is involved in early stages of carcinogenesis, then keratinocytes in early-intermediate stages of PVL may express more TLR2 than keratinocytes in non-dysplastic epithelium.
    • Properties of the Force Exerted by Filopodia and Lamellipodia and the Involvement of Cytoskeletal Components

      Cojoc, Dan; Difato, Francesco; Ferrari, Enrico; Shahapure, Rajesh B.; Laishram, Jummi; Righi, Massimo; Di Fabrizio, Enzo M.; Torre, Vincent; Mei, Lin; Department of Neurology; et al. (2007-10-24)
      During neuronal differentiation, lamellipodia and filopodia explore the environment in search for the correct path to the axon's final destination. Although the motion of lamellipodia and filopodia has been characterized to an extent, little is known about the force they exert. In this study, we used optical tweezers to measure the force exerted by filopodia and lamellipodia with a millisecond temporal resolution. We found that a single filopodium exerts a force not exceeding 3 pN, whereas lamellipodia can exert a force up to 20 pN. Using metabolic inhibitors, we showed that no force is produced in the absence of actin polymerization and that development of forces larger than 3 pN requires microtubule polymerization. These results show that actin polymerization is necessary for force production and demonstrate that not only do neurons process information, but they also act on their environment exerting forces varying from tenths pN to tens of pN.
    • Protection of Rat Cardiac Myocytes by Fructose-1,6-Bisphosphate and 2,3-Butanedione

      Wheeler, Thomas J.; Chien, Sufan; McNeil, Paul L.; Department of Cellular Biology and Anatomy; College of Graduate Studies (2012-04-27)
      Earlier studies by our group showed that fructose-1,6-bisphosphate (FBP) enhances the hypothermic preservation of rat cardiac myocytes and the functional recovery of animal hearts after hypothermic storage. However, the mechanisms involved were not clear. We extended the cardiomyocyte studies by testing whether the FBP effects were due to chelation of extracellular calcium, leading to lower intracellular levels. We also tested effects of 2,3-butanedione monoxime (BDM), pyruvate, and adenine nucleotide precursors. Cardiomyocytes were incubated in ischemic suspension at 3°C, and aliquots examined over 48 to 72 hours for retention of rod-shaped morphology, a measure of viability. Cytosolic Ca2+ levels were measured in some experiments. FBP at 5 mM reduced the death rate even when added after one or two days of incubation. It caused cytosolic calcium levels that were 33% lower than controls in freshly-isolated cells and 70% lower after one day of incubation. EGTA protected against cell death similarly to FBP. These results indicated that one of the mechanisms by which FBP exerts protective effects is through chelation of extracellular calcium. BDM was strongly protective and reduced cytosolic calcium by 30% after one day of incubation. As with FBP, BDM was effective when added after one or two days of incubation. BDM may be useful in combination with FBP in preserving heart tissue. Pyruvate, adenine, and ribose provided little or no protection during hypothermia.
    • Protein Kinase D In Keratinocyte Maturation

      Dodd, M. Ernest; Department of Physiology (2004-08)
      The epidermis is important for the body's maintenance of water homeostasis and resistance to environmental stress, and the m ajor cell type of the epidermis is the keratinocyte. Keratinocyte maturation requires proliferation, followed by terminal differentiation, and diseases of the skin often exhibit deregulated epidermal maturation. Protein kinase D (PKD) expression correlates with proliferation in keratinocytes, and PKD activation occurs in response to mitogen stimulation in other cell types. W e have hypothesized that PKD functions as a pro-proliferative and/or anti-differentiative signal in primary mouse keratinocytes and have predicted that agents that stimulate differentiation might also initiate a reduction in PKD expression and/or activation to allow differentiation to proceed. Thus, changes in PKD levels, autophosphorylation and activity were analyzed upon treatment with differentiating agents and with 1 2 -0 - tetradecanoylphorbol-13-acetate, TPA, which stimulates differentiation acutely and proliferation chronically. 1,25-dihydroxyvitamin D3 -, elevated extracellular calcium-, and acute TPA-induced differentiation down-modulated PKD levels and autophosphorylation at serine 916. In addition, elevated extracellular calcium- and acute TPA-induced differentiation down-modulated PKD activity. Chronic TPA treatment stimulated proliferation and caused a recovery o f PKD levels, autophosphorylation and activity. In co-transfection experiments in keratinocytes, co-expression of PKD increased and decreased the promoter activities of keratin 5, a marker of proliferation, and involucrin, a marker of differentiation, respectively, and opposed the effects of elevated extracellular calcium on the expression of these markers. W hile cloning PKD for expression studies, we identified a splice variant of PKD, PKD{3, which is differentially spliced in a region important in activation and subcellular localization. Therefore, we hypothesized that this splice variant may have dissimilar activation properties and/or alternate roles in keratinocyte maturation. However, in vitro activation studies demonstrated equal activation of PK D a (full length) and PKDj3 by TPA and DAG. Co-transfection experiments showed that P K D a and PKDp affected marker expression to the same degree and similarly opposed the effects of elevated extracellular calcium-induced differentiation on marker expression. Our work represents the first demonstration of: 1) down-modulation o f PKD during differentiation, 2) pro-proliferative/anti-differentiative effects of PKD on keratinocyte marker expression and 3) existence of a splice variant of PKD.
    • Protein Kinase D Restrains Angiotensin II-Induced Aldosterone Secretion in Primary Adrenal Glomerulosa Cells

      Shapiro, Brian A.; Department of Physiology (2007-07)
      Misregulation of the renin-angiotensin II (Angll)-aldosterone (Aldo) system is a key feature of cardiovascular disease. A focus of study in this system is the Angll-elicited secretion of Aldo from the adrenocortical zona glomerulosa. An excellent model in which to study this phenomenon is primary cultures of bovine adrenal glomerulosa (AG) cells. These cells secrete detectable quantities of Aldo in response to secretagogues, such as Angll, elevated potassium (K+), adrenocorticotrophic hormone (ACTH) and phorbol 12-myristate 13-acetate (PMA), within 30 minutes. The serine (Ser)/threonine kinase protein kinase D (PKD) is reported to be activated by Angll in several systems, including the adrenocortical carcinoma cell line NCI H295R, and is thought to have a positive role in chronic (24 hours) Angll-evoked Aldo secretion. Because the role of PKD in acute Angll-elicited Aldo secretion has never been examined in a primary culture system, we undertook to study the role of PKD in acute (minutes to one hour) Aldo secretion. Thus, Angll (10 nM) and PMA (100 nM), but not elevated K+ (15 mM) and ACTH (10 nM), induced phosphorylation of PKD on Ser910, a marker of PKD activation, in primary bovine AG cells. This finding was confirmed by an in vitro kinase activity assay. Angll and PMA were also able to induce PKD activation in H295R cells. Furthermore, this activation was concentration dependent, and was rapidly induced (by 5 min). PKD activation was dependent on Angll type 1 (AT-1), but not AT-2 receptor, signaling, and was independent of tyrosine kinase signaling. Finally, we introduced, via adenovirus transduction, wild-type PKDwt and dominant negative PKDS738/742A constructs into primary AG cells and monitored Angll-evoked Aldo secretion. PKDwt -transduced AG cells exhibited decreased Angll-stimulated Aldo secretion, while in the PKDS738A742A - infected AG cells Angll-stimulated Aldo was enhanced. Thus, we hypothesize that PKD has an anti-secretory role in Angll-induced acute Aldo secretion.
    • PROTEIN SYNTHESIS IN PANCREAS OF FASTED PIGEONS

      Black, Owen; Webster, Paul D.; Department of Medicine; Department of Cellular Biology and Anatomy (1973-04-1)
      The regulation of protein synthesis in the pigeon has been studied by comparing the capability of cell-free amino acid incorporating systems of membrane-bound and membrane-free polysomes prepared from fasted and fed birds. New methods were developed for isolating polysomes since techniques used for other tissues did not provide quantitative recovery of polysomal RNA. The sucrose gradient profile of polysomes from pigeon pancreas showed a predominance of trisome species. Although initiation factors are present on polysomes, it was found that polysomes in cell-free systems would not initiate protein synthesis without exogenous initiation factors. This suggested the presence of an inhibitor or regulator of protein synthesis. These studies show that fasting resulted in: (a) decreased amounts of polysomes; (b) disaggregation of polysomes to monosomes; (c) decreased capability of polysomes to synthesize nascent peptides and to initiate additional synthesis, apparently not related to concentration of initiation factors.
    • Protein-Protein Interaction between G protein-coupled receptors and heterotrimeric G proteins

      Qin, Kou; Department of Pharmacology and Toxicology (2011-01)
      G protein-coupled receptors (GPCRs) interact directly with heterotrimeric G proteins to transduce physiological signals. Early studies of this interaction concluded that GPCRs (R) and G proteins (G) collide with each other randomly after receptor activation and that R-G complexes are transient (collision model). More recent studies have suggested that inactive R and G are preassembled as stable R-G complexes in cells (preassembly models). Using fluorescence recovery after photobleaching (FRAP) we examined the stability of complexes formed between cyan fluorescent protein-labeled a2Aadrenoreceptors (C-a2ARs) and G proteins in HEK293 cells. Labeled G proteins diffused in the plasma membrane with equal mobility in the absence and presence of immobile C- a2ARs. In contrast, a stable R-G interaction was detected when G proteins were deprived of nucleotides and C- a2ARs were active. Over-expression of regulator of G protein signaling 4 (RGS4) accelerated the onset of effector activation but did not alter the interaction between C- a2ARs and G proteins. At most a small fraction of C- a2ARs and G proteins exist as R-G complexes at any moment. However, applying similar technique and protocols, we demonstrated that immobilized M3R specifically decreases the mobility of Gaq heterotrimers on the plasma membranes of intact HEK293 cells, suggesting the existence of R-G preassembly. The C-terminus of M3R was determined to be both required and sufficient for preassembly. The M3R C-terminus contains a polybasic region (565KKKRRK570) located distal to the 8th a-helix domain. Substitution of this polybasic region with 6 electroneutral alanines (M3R6A) prevented preassembly. Permeabilization of cells with low ionic strength buffer resulted in enhanced R-G interaction, implicating electrostatic forces as a factor in the preassembly. We examined the functional properties of the mutant M3R6A, which showed decreases in acetylcholine potency compared with M3R. M3R6A produced active Gq at half the rate of M3R. Other Gq-coupled receptors, such as M1 and M5 muscarinic and a1a,a1b, aid adrenergic receptors, contain similar C-terminal polybasic regions. We found that both M5R and alb adrenoceptor (albAR) preassembled with Gq proteins. Our findings suggest that a polybasic regionmediated electrostatic mechanism could be a common mechanism of preassembly between Gq-coupled receptors and Gq proteins.
    • Psychosocial Factors as Predictors for Patient Outcomes in Rehabilitation of Upper Extremity Injury Caused by Trauma:

      Holley, Ashlyn; Saren, Madison; Wygle, Sarah; Deese, Abigail; Payne, Regan; Department of Occupational Therapy (Augusta University, 2020-08-26)
      At the conclusion of this presentation, attendees will be able to: 1) Explain three ways in which psychosocial factors have the ability to alter rehabilitation outcomes in individuals who have sustained a traumatic upper extremity injury, 2) Discuss two gaps in the literature regarding the impact of psychosocial factors on rehabilitation outcomes in individuals recovering from traumatic upper extremity injuries, 3) Identify two methods, strategies, or assessments to be implemented in practice in order to evaluate and address psychosocial factors as a component affecting functional outcomes of clients with traumatic UE injuries.
    • Pulmonary Stress in the Alcoholic and Obese Lung

      Thomas, Amanda Blair; Department Of Undergraduate Health Professions (Augusta University, 2015-05)
      In the United States today, alcoholism and obesity are increasingly becoming issues for a vast age group from young children to geriatric adults. Alcoholism and obesity have been extensively studied as separate entities that have been proven to change metabolism and cause over-lapping health issues, which prompted us to study them in conjunction with one another, especially in the lungs. One major link between these two alarmingly common problems is Krüppel-Like Factor 4 (KLF4), a cell regulatory protein crucial to cellular normality and monocyte differentiation. This particular study highlights the effects alcoholism and obesity have on the lung, specifically epithelial cells (L2) and macrophages (AM). The goal of this project is to measure the KLF4 expression in the L2 and AM cells subjected to high ethanol (EtOH) consumption, high glucose consumption, high EtOH and glucose consumption, as well as a healthy control lung in to explain the mechanism behind decreased immune function in these patients.
    • Purification of Recombinant Human Histone Methyltransferases for Inhibition Studies

      Jahan, Asmat; Shaikh, Zahid; Department of Chemistry & Physics (2017-03)
      Resistance to cellular apoptotic pathways is a major contributing factor in the metastasis of cancer. One such cell-intrinsic pathway which induces programmed cell death is ligand cell surface death receptors, including Fas protein. In primary colorectal cancer, Fas expression is often diminished. The silencing of Fas expression is perhaps a mechanism by which human colorectal cancer cells evade apoptosis. The decrease in Fas expression levels is associated with hypermethylation of histone 3 lysine 9 catalyzed by histone methyltransferases (HMTases) such as SUV39H1 and SUV39H2. Because of the role of methylation in silencing Fas expression, HMTase inhibitors represent potential anti-cancer agents. Verticillin A, a natural compound extracted from wild mushroom, is a broad-based HMTase inhibitor and has been shown to cause toxicity in mice. Our goal in this study is to identify specific SUV39H1 and SUV39H2 inhibitors that are less toxic than Verticillin A. To investigate this, human SUV39H1 was sub-cloned into an expression vector, transformed into an E.coli expression strain and purified using affinity chromatography. Gel electrophoresis and Western blot analysis confirmed the presence of partially purified human SUV39H1. The next phase of the project will involve testing the activity of the purified protein in an in vitro assay.