Graduate Research Day

Abstracts

Graduate Research Day 2005
The Graduate School

Lauren Anton Molecular Medicine

Advisor: Dr. Bridget Brosnihan

Ang-(1-7) Inhibits Angiogenesis in Human Umbilical Vein Endothelial Cells Through the AT 1-7 Receptor.

Lauren Anton , David C. Merrill , Liomar A Neves, David C. Sane, K. Bridget Brosnihan Wake Forest University School of Medicine, Winston-Salem, NC

Ang-(1-7) is increased in normal pregnancy, but its functional role is unknown. Studies suggest that it opposes the action of Ang II on vascular growth. Because angiogenesis is critical to normal embryonic growth and development, these studies assessed the effects of Ang-(1-7) on angiogenesis in human umbilical vein endothelial cells (HUVECs). The effects of the Ang-(1-7) receptor antagonist, D-Ala 7-Ang-(1-7) [Dala], and the Ang II receptor antagonists losartan and PD123319 on angiogenic growth in HUVECs were measured. HUVECs were cultured in EGM-2 media, plated in wells precoated with ECMatrix, and treated with Ang-(1-7) [0.17-17000 nM] for 18 hours. The growth of capillary-like tubules was quantified. Ang-(1-7) inhibited angiogenesis by approximately 24% (p<0.05) at all doses tested. Treatment of HUVECs with 1.7 nM and 1.7 uM Ang-(1-7) followed by 17 nM and 17 uM doses of Dala decreased the Ang-(1-7) mediated inhibition by approximately 81%. These results indicate that Ang-(1-7) may exert a modulatory role in inhibiting angiogenesis through its specific receptor AT 1-7.

Colin G. Barry Chemistry

Advisor: Dr. Ulrich Bierbach

Breaking the Paradigm: Platinum-Acridine Hybrid Agent Targets Adenine-N3 in the Minor-Groove of DNA

We present the first case of adenine-N3 platination by the novel minor groove targeted platinum anticancer agent, [Pt(en)Cl(ACRAMTU-S)] (PT-ACRAMTU), designed produce DNA adducts different from those induced by clinical platinum agents. Previous studies utilizing enzymatic digestions revealed platination of adenine in the sequences 5’-GpA* and 5’-TpA*, however, the exact site of PT-ACRAMTU-adenine binding was unknown. Thus, an acidic depurination assay was developed that allowed the controlled and selective release of platinum-modified adenine from drug-treated nucleic acid samples. The three species isolated from the digestion mixtures were identified as the N7 (A*-I), N3 (A*-II), and N1 (A*-III) linkage isomers of [Pt(en)(ACRAMTU-S)(adenine)] 3+ (A*). Incubations carried out in the single- and double-stranded model sequences d(TA) 5 and d(TA) 15, as well as native DNA indicate that the adduct profiles (A*-1:A*-II:A*-III ratios) are sensitive to the nature of the nucleic acid template. The monofunctional platination of adenine at its N7, N3, and N1 positions in a significant fraction of adducts breaks a longstanding paradigm in platinum-DNA chemistry, the requirement for nucleophilic attack of guanine-N7.

Catherine Bookhout Psychology

Advisor: Dr. Catherine Seta

Social categories as causal dimensions: a modification of the Lewinian Equation?

This study explored the use of social categories as causal dimensions. Do participants use social categories to explain the opinions of group members; does this dimension reflect a type of internal or external attribution, or are social categories a special, orthogonal attribution type?

A 2 (conflict vs. deviance) x 3(Attribution Type) mixed-factorial design included participant ratings of the extent to which a target’s opinion was due to situations, dispositions, and social-categories Participants read descriptions of the target whose opinion was either different from an in-group and consistent with an out-group (deviance), or consistent with an in-group and in opposition to an out-group (conflict).

A mixed-factorial ANOVA revealed a significant main effect of attribution-type, F (2, 150) = 13.47, p < .01, and condition, F (1, 75) = 7.66, p< .01. These effects were qualified by a significant interaction between attribution type and conflict/deviance condition, F (2, 150) = 45.83, p < .01. Contrasts revealed that in the conflict condition, social category dimensions were given more weight than either situational or dispositional dimensions. In the deviance condition both person and situational dimensions were weighted more heavily than were social categories. These results suggest that contextual factors related to a target’s relationship to in-group and out-group members affect the dimensions used to explain his/her behavior. In addition, these data suggest that social categories may represent an attributional dimension that differs from either situational or dispositional variables.

Alan Brown Education

Advisor: Dr. Joseph Milner

Effective Writing in Secondary English Classrooms

Thirty six hours of classroom observations were completed in the classrooms of four English teachers in a representative United States public high school in order to examine possible correlations between instructional techniques, methods of instruction, and the amount of time spent writing. Instructional techniques were divided up into three categories: consequential writing, acquisitional writing, and no writing. Although approximately 28% of classroom time was spent in some form of student writing, students were only engaged in consequential writing, a significant and meaningful writing experience, during 17% of their observed English classes. Acquisitional writing assignments, such as notes and worksheets, made up the other 11%. Overall, 83% of class time was devoted to educational endeavors other than the experience of meaningful writing. This research concludes that assigning consequential writing through student choice and within a structured environment is the best way to encourage effective writing in the classroom.

Michael Budiman Chemistry

Advisors: Dr. Rebecca Alexander

Dr. Ulrich Bierbach

Recognition of Platinum-Acridine Modified TATA Box Sequence by TATA Binding Protein

As the central component of the general transcription machinery, the TATA binding protein (TBP) - TATA box complex has been the subject of intense study. TBP binds the TATA box exclusively through minor-groove contacts that are predominantly hydrophobic in nature and cause a dramatic distortion of the DNA. Alteration within the TATA box sequence usually results in abortion of the preinitiation complex, which may lead to cell death. We have been studying PT-ACRAMTU, a novel non-cisplatin type DNA targeting agent that has been shown to produce damage at 5’-TA. Given the observed modification profile of PT-ACRAMTU, which differs from that of cisplatin, we set out to evaluate how TBP deals with PT-ACRAMTU lesions within the TATA box. We have generated a 24-mer deoxyoligonucleotide containing the palindromic sequence (TA) 4 to use as a probe for TBP binding. Adducts formed were characterized using MALDI-TOF mass spectrometry, circular dichroism spectropolarimetry (CD) and UV-melting curves. The binding affinity between platinated probes and TBP was performed using an electrophoretic mobility shift assay (EMSA) and compared with cisplatin and unplatinated probes.

Brian Carriere Neurobiology & Anatomy

Advisor: Dr. Mark Wallace

Development of Multisensory Cortical Processes

Although a good deal is known about how multisensory processes which subserve orientation behaviors develop in the superior colliculus (SC), far less is known about how multisensory processes subserving higher-order perceptual functions develop in the cerebral cortex. To examine this, we detailed the maturation of neuronal response properties in a well-characterized multisensory area of the cat cortex, the anterior ectosylvian sulcus (AES). At the earliest ages (2-4 weeks postnatal), AES neurons were exclusively modality-specific, with somatosensory and auditory neurons being seen at 2 weeks and visual neurons appearing by 4 weeks. Despite the presence of neurons responsive to each of these modalities at this time, the first multisensory neurons were not encountered until 8 weeks of age, when they comprised < 5% of the sensory-responsive population (as opposed to 25% at maturity). These early multisensory neurons were immature, in that they were unable to integrate their sensory inputs to produce the enhanced multisensory responses that characterize the adult. It was not until 12 weeks of age that the first neurons exhibiting multisensory integration were seen. The incidence of such neurons rose to approximately 15% by 20 weeks of age and to nearly 70% by adulthood (> 6 months). These results indicate that, like SC neurons, the ability to integrate multisensory information is not an inherent capability of AES neurons, but requires considerable postnatal development. The maturation of the cortical multisensory population appears to be delayed relative to the SC, a finding that is consistent with the more gradual maturation of perceptual processes. Whether the acquisition of sensory experience proves to be as important to the maturation of multisensory AES neurons as it is to the maturation of multisensory SC neurons remains to be determined.

Supported by NIH NS36916 and MH63861

Mary E. Cunningham Mathematics

Advisor: Dr. John Baxley

Multiple Solutions To Nonlinear Boundary Value Problems

We seek growth conditions on the nonlinear function f(y) which guarantee the existence of one or more solutions to certain boundary value problems associated with the ordinary differential equation y (n) = f(y) on the interval [0,1]. We illustrate our results for n = 6 and the boundary conditions y(0) = y ¢(0) = y ¢(1) = y ¢ ¢ ¢(0) = y ¢ ¢ ¢(1)= y (4)(0) = 0. Our main tool is the Krasnosel’skiĭ fixed point theorem. We then extend our techniques to the problem y (n) = ±f(y) with various boundary conditions on the more general interval [0,r] for any positive real number r. By imposing conditions on our nonlinear function f(y), we prove the existence of one or more positive solutions to a given boundary value problem on [0,r]. This research provides new techniques for finding positive solutions to a wide range of boundary value problems not previously considered.

Amanda Davis Chemistry

Advisor: Dr. Brad Jones

Determination of urinary cadmium by chelation with ammonium pyrrolidine dithio-carbamate followed by tungsten-coil ICP-AES

A tungsten coil (W-coil) extracted from a 150 W slide projector bulb has been used as a low cost, reproducible, and commercially available atomization device in various spectroscopic techniques. In this work, W-coil is used as an alternative sample introduction device interfaced with an axially-oriented ICP, a high resolution monochromator, and a CCD detector.

The analysis of clinical samples could aid in strengthening or refuting the hypothesis that elevated levels of cadmium correlate to an increased risk of cancer. Unfortunately, interference effects due to the complex nature of the urine matrix is a setback of simple, routine urine analysis. Ammonium pyrrolidine dithio-carbamate (APDC) is a commonly used chelating agent used in conjunction with the organic solvent methyl iso-butyl ketone (MIBK) to successfully extract Cd from urine. This work illustrates the low limits of detection, highly reproducible, and precise data achieved through ETV-ICP-AES following a urinary Cd extraction with APDC.

Robert Davis Mathematics

Advisor: Dr. Hugh N. Howards

Examining Brunnian Links

Mike Freedman and Richard Skora provided proof that the Borromean rings cannot be built out of round circles in 1987. In 2004 Hugh Howards generalized this theorem by proving that the Borromean rings are the unique Brunnian link of 3 or 4 components that bound convex planar disks. This research generalizes Howards’ work. It is proven that the Borromean rings are the unique Brunnian link of 3, 4, or 5 components that bound convex planar disks. Also, an example of a Brunnian link of three 2 -sphere components in R 5 is given.

Dawn Delo Molecular Medicine

Advisor: Dr. Anthony Atala

Engineering of Aging Muscle Tissue

Delo, Dawn M; Eberli, Daniel; Atala, Anthony; Soker, Shay

Wake Forest Institute for Regenerative Medicine

Wake Forest University Health Sciences, Winston-Salem, NC

The goal of tissue engineering is to create biological substitutes useful for repairing tissue dysfunction. Despite recent progress, tissue engineering of muscle still has many obstacles to overcome. The question of whether muscle cells from patients of all ages can be used to engineer functional muscle still remains unclear. In this study, we investigated age-related effects on muscle cells with respect to cell growth, recombinant protein expression, and engineered muscle histology and contractility. This study demonstrated that there is an apparent age-related decline in the ability of muscle precursor cells (MPCs) to create an engineered muscle tissue in vivo. However, we have demonstrated that it is possible to engineer muscle of all ages, but in order to compensate for the decreased performance of the aged MPCs, our results suggest that the initial muscle biopsy should be larger in the elderly. Finally, augmentation of neovascularization can support tissue engineering of muscle.

Daniel Eberli Molecular Medicine

Advisor: Dr. Anthony Atala

An Effective Method To Engineer Corporal Tissues For Erectile Dysfunction

Daniel Eberli , Kuo-Liang Chen, James J Yoo and Anthony Atala

Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC

Erectile dysfunction due to severe corporal fibrosis may require surgical reconstruction. We previously showed that corpora cavernosal structures can be engineered using autologous cells and can be used for corporal replacement in vivo. Although sufficient for erection, penetration and ejaculation, only 50% of normal intracorporal pressures were generated due to the limited muscle cell content which could be loaded within the sinusoidal spaces of the corporal matrix. In this study we investigated a new method of loading cells on matrices in order to achieve anatomically and functionally normal corpora.

Corporal cells were seeded either statically or dynamically on acellular corporal tissue matrices. The cell-matrices were maintained in a bioreactor system. Matrices without cells served as controls. The cell-matrices were analyzed for cell attachment, survival, proliferation and distribution using histological, biochemical and molecular techniques.

The seeded cells attached and proliferated within the sinusoidal walls of the matrices. The DNA and cellular content of the dynamically seeded matrices reached approximately 85% of normal corpora compared to the statically seeded matrices (37% of normal corpora). These findings were confirmed histologically and biochemically.

This study demonstrates that dynamic cell attachment using a bioreactor system may lead to the formation of engineered anatomically and functionally normal corporal tissue, which may be useful for patients requiring penile reconstruction.

Todd Fallesen Physics

With: Anthony Pecorella Advisors: Dr. Jacquelyn Fetrow

Eddie Pryor Dr. David John

Huan Tan

Our program was designed as a tool for researchers investigating unknown proteins that have been tagged for identification using a fluorescent adduct bound to cysteine. To correctly use the program, the unknown protein must be digested using either ASP-n, trypsin, V8, or trypsin and ASP-n at the same time, and then run through mass spectroscopy. This program expects that the input comes from fully protonated mass spectrometry data. While there are currently tools available to determine the identity of unknown proteins from the mass of their digested peptides, they can't account for varying numbers of adducts possibly (but not necessarily) attached to some of the peptides. The program is run on an apache server using a MySQL database so that searches are accessible using the internet/world wide web.

Ryan Farley Computer Science

Advisor: Dr. Erin Fulp

Abstract for Distributed Firewalls for High Speed Networks

Firewalls enforce a security policy by inspecting and filtering traffic arriving or departing from a secure network. This is typically done by comparing an arriving packet to a list of rules and performing the matching rule action, which is accept or deny. Unfortunately packet filtering can impose significant delays on traffic due to the complexity and size of rule sets. Therefore, improving firewall performance is important, given network Quality of Service (QoS) requirements and increasing network traffic loads.

This display introduces a new firewall architecture that can perform packet inspections under increasing traffic loads, higher traffic speeds, and strict QoS requirements. The architecture consists of multiple firewalls configured in parallel that collectively enforce a security policy. Each firewall implements part of the policy and arriving packets are processed by all the firewalls simultaneously. Since multiple machines are used to process every packet, the functional-parallel system has lower delays and a higher throughput, than traditional single machine firewalls and data-parallel (load-balancing) firewalls. These findings will be proven theoretically and demonstrated empirically. Furthermore unlike data-parallel systems, this new function-parallel design allows the stateful inspection of packets, which are critical to prevent certain types of network attacks. Guidelines and methods for distributing rules across the firewalls are introduced to increase performance and provide redundancy.

John Foley Mathematics

Advisor: Dr. Kenneth S. Berenhaut

Discrete Optimization of Linear Recurrences with Coefficients in the Negative Unit Interval

This area of research is concerned with producing optimal bounds on the size of elements of a linear recursion sequence via discrete optimization. Elements of such a sequence are linear functions of previous elements. By discretizing the optimization problem, that is showing the optimal bound is an element of a discrete set, the problem can be resolved by analyzing this more manageable discrete set using accessible, discrete methods. This poster displays the application of this discrete methodology to “Bounds for Linear Recurrences with Coefficients in the Negative Unit Interval and an Appilication to (0,1) Matrices” to be submitted to Discrete Mathematics and discusses related results in discretization and optimization.

Samuel Franklin Neurobiology & Anatomy

Advisor: Dr. Craig Henkel

Maintenance of DNLL Afferents in the Central Nucleus of the Inferior Colliculus of the Rat after Cochlear Ablation Surgery

Samuel R. Franklin, Judy K. Brunso-Bechtold, Craig K. Henkel

The central nucleus of the inferior colliculus (CNIC) is a site of convergence for ascending auditory projections and descending thalamic and cortical projections. During postnatal development, and before the hearing onset (P12), afferent topography within the CNIC is established with remarkable precision. The unique combination of inputs into an afferent band may, in turn, define functional zones (e.g. monaural vs. binaural) within the CNIC. Previous studies in our laboratory indicate that if spontaneous activity from the cochlear is disrupted by unilateral or bilateral cochlear ablation at P2, the development of DNLL afferent bands in the CNIC is disrupted. This suggests that spontaneous activity, of cochlear origin, is required for the establishment of DNLL afferent bands within the developing CNIC. To test if spontaneous activity from the cochlea may also be required for the maintenance of DNLL afferent bands with in the CNIC we preformed cochlear ablations at P9; after the development of DNLL afferent bands within the CNIC. All animals were sacrificed at P12, before hearing onset and pins coated with Carbocyanine Dye DiI (1,1'-dioctodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) were subsequently placed in the commissure of Probst. Preliminary examination has revealed an increase in DiI fluorescence in the CNIC ipsilateral to the cochlear ablation compared to the DiI fluorescence in contralateral CNIC. Interestingly, while DNLL afferent bands appear present within both contralateral and ipsilateral CNIC, the width of afferent bands within the ipsilateral CNIC suggests altered refinement of DNLL afferents ipsilateral to the cochlear ablation at P9.

F. Scott Gayzik Biomedical Engineering

Advisor: Dr. Joel Stitzel

Development of a Finite Element-Based Injury Metric for Pulmonary Contusion

Part I: Results of Preliminary Studies with Rat Lung

F. Scott Gayzik 1,2, Jason Hoth 2 , Jennifer Mercier 2 , H. Donald Gage 2,

Kathyrn A. Morton 3, Stefan M. Duma 1, R. Mark Payne 2 , Joel D. Stitzel 1,2

1Virginia Tech – Wake Forest University Center for Injury Biomechanics

2Wake Forest University School of Medicine

3University of Utah Health Sciences Center

There is a need to develop a finite element (FE) based injury metric for pulmonary contusion. Such a metric will enable FE models to incorporate the role stress and strain play in pulmonary contusion injuries. This study utilizes 15 impacts onto male Wistar rats wherein a weight (46 g) is dropped from a height of 44 cm onto the lungs of intubated, anesthetized rats in situ. Contused lung volume is measured from MicroPET scans and normalized on the basis of liver uptake of 18F-FDG at 24 hours, 7 days, and 28 days post-impact.

An identical impact is simulated with an FE model of the rat lung. The model uses first principal strain as the injury metric for pulmonary contusion. The three volumes of contused tissue measured using PET (1, 7, and 28 days) are used as guidelines to select three corresponding strain thresholds. The first principal strain from the FE model for the three volume levels (6.8, 2.8, and 0.22 cm 3) was 0.5%, 1.35%, and 5.4% strain, respectively.

Shea Gilliam-Davis Physiology and Pharmacology

Advisor: Dr. Debra Diz

Blood Pressure Independent Reduction in Insulin, Leptin and Body Weight in Fisher 344 Rats after AT 1 Receptor Blockade for One-Year

Angiotensin (Ang) II may contribute to development of metabolic syndrome, since long-term AT 1 receptor blockade decreases blood pressure in hypertensive patients and reduces new onset type II diabetes. Fisher 344 rats that develop insulin resistance but not hypertension as they age were treated with the AT 1 receptor blocker L158,809 (20 mg/L) in the drinking water at 13-14 wks of age and measurements or blood samples taken after 1 year of treatment. Systolic blood pressure was not different between control and treated rats. AT 1 receptor-blocked rats exhibited lower serum insulin, leptin and glucose and a 6% reduction in body weight compared with control rats. Treated rats showed the expected increase in plasma Ang II with no difference in food intake. Thus, AT 1 receptor blockade improves metabolic parameters independent of effects on blood pressure.

Eva Goedhart Mathematics

Advisor: Dr. Kenneth S. Berenhaut

Explicit Bounds for Linear Difference Equations

Almost everyone has heard of the Fibonacci sequence, namely 1, 1, 2, 3, 5, 8, 13... generated by a second-order linear difference equation, b(n) = α b(n-1) - β b(n-2), for n > 1, where α = 1 and β = -1. It is more interesting when you consider α and β to be sequences, i.e. α(n) and β(n), close to the values two and one, respectively. In the past, mostly asymptotic bounds have been given for difference equations. Hence the title of our first paper, “Explicit Bounds for Second-Order Difference Equations and a Solution to a Question of Stević”, in which we give explicit, applicable bounds for solutions of a wide class of second-order difference equations with non-constant coefficients. This led to consideration of another second-order difference equation where α(n) and β(n) take values in a restricted interval, [0, A], for some A > 0. Here we obtained not only bounds, but also some interesting results depending on whether A is greater than, less than, or equal to (1/3) 1/3, under the title, “Second-Order Linear Recurrences with Restricted Coefficients and the Constant (1/3) 1/3”.

W. David Hairston Neurobiology & Anatomy

Advisor: Dr. Mark Wallace

Attention-Modulated Gating of Cross-Modal Cortical Deactivation

W. David Hairston 2, Christina Hugenschmidt 1, Mark T. Wallace 2, Robert A. Kraft 3, Joseph A. Maldjian 1, Paul J. Laurienti 1,

1Departments of Radiology, 2Neurobiology and Anatomy, and 3Biomedical Engineering

Wake Forest University School of Medicine, Winston-Salem, NC

Previous human brain imaging studies have revealed that stimulation of one sensory modality can decrease the BOLD signal in the cortex of other non-stimulated sensory modalities (e.g., visual stimulation produces functional deactivations in auditory cortex), a phenomenon referred to as cross-modal deactivation. To distinguish whether these deactivations are stimulus-driven (“bottom-up”), or rather are mediated by higher-order “top-down” processes such as attention, we used a mixed event-related/epoch-based design to direct attention to either the auditory or visual modalities in the presence of a continuous visual stimulus that was identical in both paradigms. To direct attention, in one paradigm subjects performed a visual discrimination task, while in the other they performed an auditory discrimination task. Prior to any other analyses, all event-related (i.e., stimulus-specific) activity was removed from the epoch-based (i.e., attended modality) activity. We found that attention to the visual modality produces robust deactivations in auditory cortex (Fig, Left); in contrast, the same visual stimulus does not produce deactivations in auditory cortex when attention is directed to the auditory modality (Fig, Right). These results demonstrate that when attention is modulated, the simple presence of a stimulus is insufficient for cross-modal deactivations to occur. Rather, directing attention toward one sensory modality deactivates the sensory cortex for the unattended modality.

W. Hairston 1 *; C.E. Hugenschmidt 1; M.T. Wallace 1; R.A. Kraft 1; J.A. Maldjian 1; P.J. Laurienti 1

1. Dept Neurobiology & Anatomy, 2. Radiology, 3. Biomedical Engineering, Wake Forest Univ Sch Med, Winston Salem, NC, USA

Jason Hipp Molecular Medicine

Advisor: Dr. Anthony Atala

Transcriptional Profiling Of Human Amniotic Fluid Derived Stem Cells and Their Multilineage Differentiation

Hipp JD , Siddiqui MM, Hipp JA, Hodges S, Atala A, Soker S

Wake Forest Institute for Regenerative Medicine

Wake Forest University Health Sciences, Winston-Salem, NC

Intro: We have recently isolated a subpopulation of pluripotent stem cells from human amniotic fluid (Amniotic Fluid Stem Cells, AFSC). These cells have a high self renewal capacity (>300 population doublings), with a doubling time of less than 36 hours, are autologus with the fetus, do not require a feeder layer, and can be grown in basic medium supplemented with serum. AFSC can be differentiated in vitro into osteogenic, adipogenic, myogenic, neurogenic, vasculogenic, and hepatic phenotypes. The goal of this study is to understand the genetic processes involved in stem cell differentiation that would identify targets to improve the yield and efficiency of differentiation.

Methods: Microarrays were performed at day 20 and 30 upon myogenic and osteogenic differentiation, day 14 and 30 upon hepatogenic differentiation, and day 30 upon endothelial differentiation using the Affymetrix HU-133A GeneChip. Data files were analyzed with MAS5 for Present detection calls, RMA and Limma for differential gene expression and with EASE for gene ontological analyses.

Results: By combining all day 30 data sets from the myogenic, osteogenic, endothelia and hepatogenic lineages, we identified a genetic signature of 1017 genes as being “universally” up-regulated and 379 genes as “universally” down-regulated. By subtracting the “universally” up-regulated genes from those identified as being up-regulated from each lineage, we were able to identify a “lineage specific” genetic signature of differentiation.

By clustering these “lineage specific” targets based on their Gene Ontology, we were able to identify tissue specific processes such as sterol, lipid, and cholersterol metabolism for hepatogenic differentiation, extracellular matrix production genes (which have a structural function) for osteogenic and endothelia, and muscle developmental processes for the myogenic lineage.

Conclusion: Here we describe how we are using microarrays to understand stem cell differentiation. Our microarray results show how we are able to identify tissue specific processes using a combination of bioinformatics programs. Therefore, genomics may give us insight into the processes that are activated during stem cell differentiation and how a stem cell creates its extracellular environment during development. These results offer new avenues for applying stem cells to tissue engineering.

Kris Huang Biomedical Engineering

Advisor: Dr. Daniel Kim-Shapiro

NO uptake rates of normal and sickle red blood cells

Ivan Azarov, Kris T. Huang, Matthew Cass, Swati Basu, Man Cho, James S. Nichols, Mark T. Gladwin, Neil Hogg, and Daniel B. Kim-Shapiro

Faster NO binding rate by free hemoglobin (Hb) compared to that by red blood cell (RBC) encapsulated Hb is partially responsible for NO scavenging by blood substitutes and cell-free Hb. The reduced scavenging by the RBC is due to an unstirred layer around the RBC and/or a physical diffusion barrier within the RBC membrane. If there is no physical membrane barrier, NO uptake by RBCs at a hematocrit (Hct) of 100% would be expected to scavenge NO at the same rate as free Hb. Sickled cells may be expected to have different NO uptake rates from normal cells due to their shapes and damaged membranes. Using stopped-flow absorption (at low Hct) under anaerobic conditions, we found no cell-type dependence and rates of NO uptake on the order of one-thousand times slower than that for free Hb. We also conducted competition experiments in which limiting amounts of NO are released in the presence of RBCs and free Hb. The relative rates of NO binding were calculated from the amount of NO bound in the RBC and free Hb fractions measured by EPR. Consistent with previous results, we found that at low Hct the relative rate of NO binding to free Hb is 2-3 orders of magnitude faster than to RBC encapsulated Hb. We found no difference between normal and sickle RBCs. Surprisingly, we found that at 50% Hct the relative rate of NO binding is less than 100. Our results do not support a significant role of the RBC membrane or RBC shape in limiting NO uptake, and show that the intrinsic rate of NO scavenging by RBCs is much more dependent on Hct than previously thought. These data have important implications in anemias and polycythemias.

Parul Jain-Shukla Communication

Advisor: Dr. Ananda Mitra

Transforming media: Altering attitudes

The proposed study attempts to gauge the effects of foreign media on Indian culture and the perception of American society by Indians, as conveyed through media images. Authors have compared the globalization attempts in India to neo-colonialism and replacement of imperialism. Some researchers refer to this new culture apparent in Indian metropolitans as “Hybridization”: A mix of Indian and Western way of life. Past academic research has established that the television programming in India is a potpourri of Western and Indian values. This study tries to measure the effects that the fusion in media has brought about in the attitudes and culture of the Indian society by conducting surveys and focus group discussions of people in the urban India. The second question this project tries to explore is how the people in India, who have never been to West, perceive the American society as a result of the media constructions.

Anne Jeffers Physics

Advisor: Dr. Daniel Kim-Shapiro

Nitrite Activation of sGC in Blood

Anne Jeffers, Xiuli Xu, Man Cho, Kris T. Huang, S. Bruce King, Neil Hogg, Rakesh P. Patel, Mark T. Gladwin, and Daniel B. Kim-Shapiro

At high (> 100 micromolar) concentrations, nitrite has been known to activate soluble guanylate cyclase (sGC) and cause vasodilation. Deoxygenated hemoglobin (Hb) reacts with nitrite to form iron nitrosyl Hb and metHb. Recently we have shown that nitrite acts as a vasodilator in vivo at supraphysiological and near physiological (2 micromolar) levels through a mechanism that may involve reduction of nitrite by Hb to NO or some other vasodilating species. In aortic ring assays, red blood cells and cell-free Hb both dramatically potentiated vasodilation by nitrite (0.5 - 2 micromolar). The ability of Hb to react with nitrite to form NO which then activates sGC is challenged by the fact that Hb reacts with NO so fast that NO is not likely to be exported from the red blood cell (RBC). However, deoxygenated RBCs along with nitrite (2 micromolar) increased cGMP levels in rat thoracic aorta. Interestingly, the same reaction system failed to increase cGMP when incubated with recombinant sGC, suggesting cellular localization plays an important role in regulating bioactivity.

We have modeled NO export from RBCs based on measured rates of the reaction of nitrite and NO with Hb, diffusion rates, and membrane permeabilities. We find that only about 0.1 picomolar of NO escapes the RBC at steady state in the presence of one micromolar intracellular nitrite. These results were not significantly different for various two- and three-dimensional models attempted. Assumptions that the reaction of nitrite and NO is compartmentalized in the RBC also did not significantly increase NO export from the RBC. Based on these and previously published results, we hypothesize that the reaction of Hb and nitrite involves an intermediate species that can freely diffuse out of the RBC and into smooth muscle cells (unlike nitrite) which then activates sGC in vivo and in aortic ring bioassays.

JaNae Joyner Molecular Medicine

Advisor: Dr. Bridget Brosnihan

Temporal-spatial Expression of ACE2 and Ang-(1-7) in the Kidney of Normal and RUPP Pregnant Rats

J Joyner, LA Neves, JP Granger, BT Alexander, DC Merrill, CM Ferrario, KB Brosnihan. Hypertension and Vascular Disease Center, Wake Forest University Winston Salem, North Carolina USA.

The renal distribution of angiotensin-(1-7) [Ang-(1-7)] and its processing enzyme ACE2 were examined in virgin, normal pregnant (NP) (gestational day 5, 15, 19) and reduced uterine perfusion pressure (RUPP) pregnant rats, a model of pregnancy induced hypertension (PIH). Immunocytochemical analysis was completed using affinity purified rabbit polyclonal antibodies for Ang-(1-7) or ACE2. Renal Ang-(1-7) and ACE2 immunoreactivity (ir) was localized to proximal and distal tubules of the inner cortex/outer medulla region. Ang-(1-7)-ir at days 15 and 19 of pregnancy and ACE2-ir in NP-19 day rats were significantly increased as compared to virgin and NP-5 day. Ang-(1-7) staining was significantly decreased in RUPP animals as compared to NP-15 and NP-19 day. ACE2 immunoreactivity in the RUPP tended to decrease as compared to NP-19 day. The increase of Ang-(1-7) and ACE2 immunoreactivity during late pregnancy is consistent with Ang-(1-7) playing a significant role in the regulation of blood pressure and/or hydromineral balance, which may be disrupted in PIH.

Sherry Kausch Psychology

Advisor: Dr. Robert Beck

Temporal discounting as a function of achievement motivation

Temporal discounting involves asking participants to choose between a small reward available immediately and a larger reward available at some designated time in the future. 79 subjects chose between a hypothetical $1000 available at seven delays, ranging from one week to five years, and smaller amounts immediately available. After participants completed the discounting task, they filled out the Spence Helmreich achievement motivation questionnaire, which has three subscales: competitiveness, mastery, and work. There was a significant interaction between Delay and Competitiveness (p=.009), with Ss showing less discounting at short delays and more discounting at longer delays. Ss high on the Work scale discounted less than those low on the Work scale (p<.01). There was an interaction between delay and mastery and work (p=.051). The results indicate a relationship between rate of discounting and measures of achievement motivation.

Jie Liu Biomedical Engineering

Advisor: Dr. Joel Stitzel

Electrospinning Fabrication of Collagen-based Scaffolds for Vascular Tissue Engineering

Liu, Jie; Lee, Sang Jin; Yoo, James J; Soker, Shay; Komura, Makoto; Lim, Grace; Atala, Anthony; Stitzel, Joel

Wake Forest Institute for Regenerative Medicine and Biomedical Engineering

Wake Forest University Health Sciences, Winston-Salem , NC

Substantial effort is being invested by the bioengineering community to develop biodegradable polymer scaffolds suitable for cardiovascular applications 1. Electrospinning is a fabrication method for creating fine fibers (nano to micrometer diameter), which have been shown to support cell and tissue growth 2. An ideal scaffold for tissue engineered vessels is required not only to mimic the structure of a native vessel, but also to facilitate vascular tissue growth.

The goal of this study is to create a biomimetic, small-diameter vascular graft using electrospinning technology. To accomplish this goal, vascular graft scaffolds have been fabricated by electrospinning polymer blends of collagen type I, elastin, and poly (D,L-lactide-co-glycolide) (50:50) at a total solution concentration of 12% (w/v). The structure of the scaffold was characterized by scanning electron microscopy (SEM), biochemical and immunohistological analyses. The mechanical properties of the scaffold were also characterized. Bovine smooth muscle cells and endothelial cells were seeded on the scaffold to evaluate the biocompatibility of this electrospun scaffold for use as a synthetic vascular substitute.

Fiber sizes of 0.72±0.35 microns diameter were obtained from the electrospinning process, which mimics the nanoscale dimension of native small diameter vascular matrix. Collagen type I was stained positively on the electrospun scaffolds, demonstrating uniform distribution. The electrospun scaffold with 15% elastin demonstrated a uniform elastin matrix throughout the scaffold wall by Movat staining. The mechanical testing for compliance showed a typical pressure-diameter curve for electrospun scaffolds. The diameter change was approximately 5% for electrospun scaffolds, which is within the physiologic pressure range for human arteries. 82% of smooth muscle cells and 78% the endothelial cells can survive on the scaffold as analyzed by mitochondrial metabolic (MTT) activity assay.

This study shows that electrospun scaffolds exhibit the structure and mechanical behavior similar to native vessels. The results also indicate a favorable interaction between this synthetic nanofibrous scaffold with the two peripheral types of vascular wall cells. Electrospun vascular scaffolds possess ECM components and distribution similar to normal vessels. Collectively, this study demonstrates the promise of electrospinning as an effective fabrication process for cardiovascular grafts.

References:

Weinberg, C.B., and Bell , E. Science, 231, 397, 1986.

Deitzel, J.M., Kleinmeyer, J.D., Hirvonen, J.K., Beck, Tan. Polymer, 42, 8163, 2001.

Wenhua Liu Physics

Advisor: Dr. Martin Guthold

Visualization and Mechanical Manipulations of Individual Fibrin Fibers by combined Microscope AFM/FM.

W. Liu 1, L. Jawerth 2, B. Stephens 1, R. Hantgan 3, S. Lord 4, R. Superfine 2, R. Taylor 5, M. Guthold 1

Department of Physics 1 and Biochemistry 3 Wake Forest University, Winston-Salem, NC 27109, USA
Departments of 5Computer Science, 2Physics and 4Pathology, University of North Carolina, Chapel Hill, NC 27514, USA

Fibrin fibers are the major structural component of blood clots. Thus, determining the strength and mechanical properties of blood clots will provide new insights into the wound healing process and will advance our understanding of heart attacks and strokes. In the study described here, the rupture force and mechanical properties of individual fibrin fibers are being measured. The rupture force, F R, of human fibrin was determined in ambient conditions as a function of the diameter, D, and found to follow a F~D 1.4 dependence. The ultimate strain of fibrin fibers was measured in buffer. It was found that ligated and unligated fibers can be stretched to 450% and 300% of their original length, respectively. It was also found that the deformations are elastic to at least ~170% extension.

Yan Ma Chemistry

Advisor: Dr. Ulrich Bierbach

Synthesis, crystal structures, and reactivity with biological nucleophiles of sterically overloaded platinum and palladium am(m)mine-carboxylato complexes

Yan ma, Cynthia S Day, Marcus W Wright and Ulrich Bierbach, Wake Forest University, Winston-Salem, NC

Sterically hindered nonleaving groups in platinum antitumor compounds have attracted considerable interest because of their ability to reduce the metal’s reactivity toward detoxifying sulfur nucleophiles. One novel bidentate N,O donor ligand, (esterized form: 6-(methylpyridin-2-yl)acetic acid ethylester, PICACEt), two platinum complexes, and one palladium complex with that ligand were synthesized and characterized by NMR spectroscopy, X-ray crystallography, and elemental analyses. With carboxylate serving as a leaving group, these complexes should be able to bind to DNA in a monofunctional manner. These complexes are not expected to induce cross-links (unlike cisplatin and its analogues) but may to bind through a pseudobifunctional mechanism involving platination of guanine-N7 and stacking of the planar pyridine group with adjacent base pairs. Such adducts have been shown to produce a cytotoxic effect (e. g., cis-[Pt(NH3)2(planar base)Cl]Cl, see Hollis et al., J. Med. Chem. 1989, 32, 128.).

Results of a cytotoxicity study using HL-60 leukemia cells of compounds [Pt(NH3)2(PICAC-N,O)]NO3(1), [Pt(en)(PICAC-N,O)]NO3 (2), and [Pd(en)(PICAC-N,O)]NO3 (3) are reported. We also studied the reactivity of compounds 2 and 3 with 5’-GMP by 1H NMR and characterized the 1:1 adduct of complex 2 with 5´-GMP by 2-D dqf-COSY and NOESY spectroscopy.

Andrea Miller Health and Exercise Science

Advisor: Dr. Shannon Mihalko

Physical Activity and Health-Related Quality of Life: A Study of Older Adults Residing in Assisted Living

Andrea N. Miller, Shannon L. Mihalko, Ph.D., Katie L. Wickley, M.S., Anthony P. Marsh, Ph.D., Wake Forest University, Winston-Salem, NC

Regular physical activity has been associated with improved physical function and Health Related Quality of Life (HRQL) among community-dwelling older adults. However, few studies have included older adults residing in assisted living. Therefore, the purpose of this cross-sectional study was to examine the relationship between physical activity and HRQL in older adults (N=130, M age= 81.6 ± 5.3 years) residing in these communities. Specifically, we measured resident physical activity levels using the Physical Activity Scale for the Elderly (PASE) and HRQL using the SF-36. Residents who were more physically active reported significantly higher levels of HRQL (physical component; r = .204, p<.05) than those who were less active. The relationship between physical activity and the mental component of the SF-36, however, was not significant. Indices of physical function were also significantly correlated (p< .01) with HRQL, such that those participants who reported higher levels of HRQL demonstrated better balance (r = .400) and had faster chair rise (r = -.285), timed up and go (r = -.376), and four meter walk (r = -.364) times. Additionally, residents who reported a higher number of comorbidities had significantly (p<.05) lower levels of physical activity (r = -.288) and HRQL (r = -.244). These data emphasize the need to incorporate physical activity into the daily lives of older adults residing in assisted living. Future studies should evaluate changes in psychological and physical function as related to residents’ participation in physical activity over time.

Alixanna Norris Cancer Biology

Advisor: Dr. Karin Drotschmann

Mismatch Repair Proteins as Prognostic Markers for the Transition from Indolent to Aggressive Prostate Cancer

Mismatch repair (MMR) defects are associated with carcinogenesis of both hereditary and sporadic types of cancer. Recently, MMR defects have been suggested to play a role in the development of prostate cancer. This is supported by the fact that most prostate cancer cell lines contain a defect in at least one MMR protein. This research tests the hypothesis that MMR defects assist in the development and progression of prostate cancer. The identification of such defects will be useful as indicators for the severity of disease. Immunohistochemical analysis of prostate tissue reveals the overexpression of the key MMR gene, PMS2. This effect is limited to the epithelial cells of carcinogenic tissue, while stromal cells show normal to underrepresented expression. Since the opposite expression pattern is observed in the surrounding, benign tissue, an interaction between stromal and epithelial cells may be required for the overexpression of this protein. Though the stability of PMS2 is suggested to require MLH1, we do not see a correlation between the expression of these two proteins in the patients investigated thus far. The analysis of MMR gene expression, protein function and molecular mechanisms for progressive prostate cancer will significantly contribute to prognostic and therapeutic measures for prostate cancer patients.

Heather O’Donohue Neuroscience

Advisor: Dr. David Friedman

Ethanol-Induced Changes in GABA A Receptor Alpha Subunit Expression in Cynomolgus Monkey Sensorimotor Areas 1, 2, 3a, 3b, and 4.

O’Donohue, H.A., Davenport, A.T., Daunais, J.B., Grant, K.A., Friedman, D.P.

We performed in vitro autoradiography on cortical sections from monkeys who chronically self-administered ethanol, and characterized the binding of total GABA A receptors in addition to the alpha-1 and -4 subunits in all layers of areas 4, 3a, 3b, 1, and 2. Compared to controls, subjects who self-administered ethanol had significantly lower (p < 0.05) total binding in layer V of area 1 and layers I and V of area 4. The density of alpha-1 subunits in the drinkers was significantly less than controls in layer V of area 1, layer VI of area 2, and layer I of area 4. These data suggest that alcohol self-administration may alter the laminar density of GABA A receptors and some subunits in specific regions. This study was supported by NIH, T32-AA-07565 and P20-AA-11997.

Christian Pacchia Health & Exercise Science

Advisor: Dr. Tony Marsh

Spatiotemporal Characteristics of Treadmill and Overground Walking in Older Adults

C.F. Pacchia, K.L. Koury, A.P. Marsh, L.C. Johnson, W.J. Rejeski, J.A. Katula

Wake Forest University

The purpose of this study was to assess the spatiotemporal characteristics of treadmill walking and overground walking at a preferred pace and an RPE of 13 in older adults. We examined walking velocity, stride rate, and stride length using a Gaitrite instrumented carpet for overground trials and calculated stride length using the relationship, treadmill velocity = stride length x stride rate. Male (n = 9) and female (n = 14) volunteers with an average age of 74 ± 4 yrs served as subjects. The Short Physical Performance Battery (SPPB) was administered and used as an indicator of physical function. SPPB scores for the sample ranged from 4 to 12 with a mean score of 9 ± 2. The preferred or self-selected comfortable walking velocity of the participants was slower on the treadmill (63 ± 26 cm/s) compared to overground (102 ± 18cm/s). Similarly, the walking velocity was slower on the treadmill (90 ± 34 cm/s) than overground (121 ± 25 cm/s) when participants walked at an RPE of 13. Within each mode of walking the walking velocity was greater at an RPE of 13 than at the preferred velocity. However, the preferred velocity overground was greater than the velocity on the treadmill at an RPE of 13. The magnitude of the difference between treadmill and overground walking was greater at the preferred velocity (-41 ± 23 cm/s) than at an intensity equivalent to an RPE of 13 (-30 ± 17 cm/s). The lower velocities were a result of both shorter stride length and slower stride rate on the treadmill than overground at both intensities. These data have relevance when prescribing modes of activity for older adults when the intensity is based on perceived effort.

Pantoja, David Soto Molecular Genetics

Advisors: Dr. Ann Tallant

Dr. Patricia Gallagher

Pantoja DS, GallagherPE, Tallant EA.

" Inhibition of Human Breast Cancer Cell growth by Angiotensin-(1-7)".

Angiotensin-(1-7) [Ang-(1-7)] is an endogenous peptide hormone of the renin-angiotensin system with vasodilator and antiproliferative properties. We propose that Ang-(1-7) serves as a general growth inhibitor and may be an effective cancer treatment. ZR-75-1 cells, a human ductal carcinoma cell line, were mitogen treated, in the presence and absence of Ang-(1-7), to determine whether the heptapeptide inhibits the growth of breast cancer cells in vitro. Ang-(1-7) caused a significant reduction in mitogen-stimulated growth and decreased serum-stimulated DNA synthesis in ZR-75-1 or MCF-7 human breast cancer cells in a dose dependent manner. Ang-(1-7) increased p16 ink a cyclin-dependent kinase inhibitor and reduced CDK4, a cyclin dependent kinase. This correlates with a reduction in hyperphosphorylation of retinoblastoma which causes cell cycle arrest. This study provides the first evidence that Ang-(1-7) inhibits the proliferation of human breast cancer cells, suggesting that it may be an effective chemotherapeutic or chemopreventive agent for breast cancer.

Anthony Pecorella Mathematics

(see Todd Fallesen)

Lu Peng Physics

Advisor: Dr. Martin Guthold

Novel, single-molecule methodology to identify new aptamers

Lu Peng 1, Roger Cubicciotti 2, MartinGuthold 1

1 Department of Physics, Wake Forest University , Winston-Salem , NC 27109 , 2Nanomedica Inc. Newark , NJ 07102

We are developing a novel, single-molecule methodology to identify aptamer molecules in a pool of random oligonucleotides. Unlike SELEX, our approach does not require iterative cycles of selection and partitioning, but aptamers are selected one-by-one. At the core of our methodology is unique instrumentation that combines a nanoManipulator Atomic Force Microscope (nM-AFM) and an inverted optical microscope with TIRF illumination and FRET detection capabilities.

This instrument, in combination with PCR, is used to identify new aptamers as follows. Target molecules are labeled with a donor fluorophore and linked to a cover slip. Random-sequence oligo libraries are labeled with an acceptor fluorophore and a bead (for AFM detection and pick-up). The oligos are then flowed over the target area. High-affinity, target-specific aptamers will bind tightly to the target for relatively prolonged periods resulting in a strong fluorescence signal. After a fluorescence signal has been observed with the optical microscope, the nM-AFM is used to obtain a high-resolution image of the fluorescence signal-generating region. Subsequently, the AFM tip is used as a “gripper” to retrieve the bead plus the attached aptamer. PCR is then used to amplify the extracted aptamer.

We have implemented the instrumentation and completed proof-of-principle experiments with a known thrombin aptamer. We successfully detected (AFM and fluorescence), extracted and amplified thrombin-aptamers from a pool of random oligonucleotides.

Eddie Pryor Computer Science

(see Todd Fallesen)

Adam Reilly Computer Science

Advisor: Dr. Stan J. Thomas

All life depends on complex machinery which operates within cells. The mechanisms which comprise this machinery are chemical reactions which affect and are affected by the cell's environment and its surroundings. Each reaction is typically a link in a chain which culminates in some cellular event. Biologists commonly refer to these chains as "pathways" or "networks". Understanding these networks would likely generate new insights into everything from cancer treatments to pharmaceutical discovery.

Mathematics and computational modelling offer powerful methods for elucidating complex relationships between entities. Additional benefits are reaped from the ability to model the underlying systems. Bayesian Networks are graphical models which are structured to encode relationships between entities. Bayesian Networks also incorporate a probabilistic component which allows flexibility in non-deterministic situations. The main goal of the current research is to explore applications of Bayesian Networks to the problem of recovering cellular regulatory networks from various experimental data.

Kimberly Rogers Psychology

Advisor: Dr. Batja Mesquita

The Perception of Surprise and Sadness in Japanese and American Cultures

In the present study, we examined cultural differences in both the quantity and type of emotions perceived in facial expressions. Participants examined eight series of ten pictures that display a face gradually moving from a neutral state to a strong emotion (either surprise or sadness), and rated these pictures on a series of emotion scales. Despite an equivalent threshold of emotion recognition, differences were found in the secondary emotions members of each culture perceived in the faces. Japanese tended to perceive an overall higher intensity of emotion in the faces than Americans, particularly for ratings of emotions that were similarly valenced to the target emotion (i.e., disgust, anger). The present study shows that beyond the snapshot of similarity provided by the research of theorists such as Ekman and Izard, there are intricate cultural differences in emotion perception.

Anne Sanders Molecular & Cellular Pathobiology

Advisor: Dr. Zheng Cui

Effect of Immune Deficiency on SR/CR Phenotype of Cancer Resistance

This project’s goal was to determine if SR/CR cancer resistance could be abolished by breeding into immune deficient backgrounds, affecting leukocyte maturation or effector mechanisms (perforin, superoxide, nitric oxide) reported to be involved in cancer cell destruction. Upon two rounds of breeding, the offspring were challenged with cancer (S180) and the survival percentage was evaluated for each genotype. Knocking out perforin, Cybb, or Nos2 had no apparent effect on the SR/CR phenotype, the knockout (-/-) survival percentage was similar to the controls (+/-) or (+/+). In contrast, athymic mice were unable to resist the challenges at doses higher than 2x10e5 S180 cells, whereas hairy littermates could resist more than 10e6 S180 cells. This suggested that the thymus may play an important role either in modulating effector activity of innate immune response or in regulating maturation of leukocytes of innate immune system.

Samir Shah Biomedical Engineering

Advisor: Dr. Bruce Rubin

Quantification of biopolymer filament structure

Samir Shah 1, Pete Santago, Ph.D. 1, Bruce K. Rubin 1,2 MEngr, MD, MBA, FRCPC, FCCP

Wake Forest University School of Medicine

Departments of Biomedical Engineering 1 and Pediatrics 2

The quantitative analysis of a polymer network is important for understanding its role in biological function. We developed a Matlab program to recognize and segment filaments in an image, and measure and describe the structure. Our algorithm improved the speed of the Lichtenstein Fiberscore segmentation algorithm by using matrix convolutions, compared filament length by the algorithms of Kulpa, Lichtenstein, and Kimura, and measured the number of branch points and Euler number. A user interface was added to easily manipulate algorithm parameters, select images, and visualize results. We used the program to compare the DNA biopolymer network of cystic fibrosis sputum with mucus from patients without respiratory problems, as well as a fibrin image. The images were taken with a laser scanning confocal microscope after staining the specimens with Yo-Yo-1. Computation using matrix convolutions reduced the executive time (Pentium III) of a 512 x 512 TIF image from 18 minutes to 15 seconds. The Kimura length measurement appeared best at describing filament length. The image of CF sputum showed increased filament length, more branch points, and more negative Euler number compared to the normal sample. These quantitative descriptions of the network can be correlated to material, mechanical, diffusion, or flow properties, physiological processes, or therapy.

Amy Sloat Chemistry

Advisor: Dr. Christa Colyer

Protein Labeling with Red Squarylium Dyes for Analysis by Capillary Electrophoresis and Laser-Induced Fluorescence Detection.

Christa L. Colyer 1, Amy L. Sloat 1, Weiying Yan 1, Hiroyuki Nakazumi 2, Shigeyuki Yagi 2

1. Deparment of Chemistry, Wake Forest University, Winston Salem, NC, 27106, USA

2. Department of Applied Materials Science, Graduate School of Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan

The use of fluorescent probes enables increased sensitivity for detection of biomolecules over UV-visible detection. Squarylium dyes are a class of compounds which non-covalently label proteins and fluoresce in the NIR region. Absorption in the NIR is of particular interest in fluorescent labeling because it offers lower background signal and less scattering than the visible and ultraviolet regions when analyzing biological samples. In addition, lesser expensive, longer lasting, diode lasers can be used for excitation. The stability, along with their ability to form non-covalent bonds with proteins makes Squarylium Red-1c and Red-3 ideal probes for on-column fluorescence labeling thereby resulting in less sample handling and increased the speed of analysis.

Maureen Stanford Education

Advisor: Dr. Raymond Jones

The Effect of Teacher Instruction and Teacher Behaviors on Student Participation

This study investigated the influence teacher instruction and teacher behaviors had on student participation.  In a highly participatory classroom students will internalize the skills of participation, collaboration and interpersonal communication which will transcend into citizenship qualities for future community involvement.  Since the development of citizenship is a goal for teachers, it is important to know what to do to promote this development.  Observational data was collected and categorized in terms of instructional method variation, task completion by student, teacher behaviors witnessed, and student behaviors viewed.  This research found positive attitude towards student effort, use of humor and personal stories to make connections, written and verbal accountability of student responsibility, and instructional method variation produced action and participation with the students which in turn could be used to build skills of participation in one’s community. 

Olivia Stanley Education

Advisor: Dr. Leah McCoy

A Study of Attitude toward Mathematics and Its Relationship to Ethnicity and Gender

This study examined high school mathematics students’ attitudes toward mathematics and how their attitude related to the students’ ethnicity and gender. Students enrolled in different levels of mathematics and different high schools were given a survey to complete that was divided into three sub-categories for attitude measurement. The three categories covered in the survey were Perceived Usefulness of Mathematics, Confidence in Mathematics, and Mathematics Anxiety. Although previous research showed differences in attitudes among males and females as well as different ethnic groups, this research found no significant difference among these groups.

Huan Tan Computer Science

(see Todd Fallesen)

Susan Walker Chemistry

Advisor: Dr. Rebecca Alexander

Protein-RNA interactions are abundant in cells. In many cases, conformational changes in the protein, RNA, or both are required for efficient catalysis. Protein synthesis requires attachment of amino acids to tRNAs through catalysis by aminoacyl-tRNA synthetases (AARSs). Catalysis by methionyl-tRNA synthetase (MetRS) depends on communication between the two domains through a 50 Å distance. We hypothesize that MetRS requires conformational changes in both tRNA and MetRS for efficient catalysis. We will characterize domain-domain communication in the MetRS:tRNA Met through mutagenesis in conjunction with spectroscopic methods and kinetic experiments.

Kelly Weaver Molecular Medicine

Advisor: Dr. Jamal A. Ibdah

Mouse fibroblasts deficient is mitochondrial trifunctional protein are insulin resistance

Kelly Lynn Weaver, Jamal A. Ibah. Internat Medicine, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157

Mitochondrial dysfunction and fatty acids play an important role in the pathogenesis of insulin resistance, but the role of impaired mitochondrial fatty acid oxidation (FAO) remains unknown. Mitochondrial trifunctional protein (MTP) catalyzes the last 3 steps in long chain FAO. MTP deficiency causes metabolic crisis and infantile death if untreated. The goal of our study was to assess insulin resistance in an in vitro cell model for MTP deficiency. Fibroblasts were isolated from MTP-deficient (-/-) and wild-type (+/+) mouse embryos at 19 dpc. We analyzed insulin-stimulated glucose uptake in these cells by measuring [3H]-2-deoxy-D-glucose transport into the cells following 15 minute stimulation with 100nM insulin. We detected significant reduction in total insulin-stimulated glucose uptake in -/- compared to +/+ fibroblasts (652.17±117.3 vs 1022.02±125.5 pmol/min/mg, n=18, P<.0001). This was associated with triglyceride deposition in the MTP-/- fibroblasts. We also compared insulin-stimulated Akt/PKB activation in -/- and +/+ fibroblasts by measuring Akt S473 phosphorylation using immunoblot analysis. MTP-/- fibroblasts showed a 39% reduction in Akt S473 phosphorylation after 10 min incubation with 100nM insulin. Our results suggest that impaired mitochondrial FAO in mouse fibroblasts causes insulin resistance with reduced Akt activation.

Funded by NIH Molecular Medicine Training Grant T32 GM63485.

Kyle Webb Health & Exercise Science

Advisor: Dr. Michael Berry

Level Of Agreement Between Two Commercially Available Pulse Oximeters During Graded Exercise Testing

K.M. Webb, K.J. Hanson, J.M. Sheely, and M.J. Berry, Department of Health and Exercise Science, Wake Forest University, Winston-Salem, NC.

Due to the difficulty of directly measuring oxygen saturation, pulse oximetry is used clinically to monitor oxygen saturation (SpO 2) non-invasively in those with chronic obstructive pulmonary disease (COPD). The purpose of this study was to measure the level of agreement between two commercially available pulse oximeters during rest and exercise. SpO 2 was measured in 33 COPD patients (age=68.7 ± 10.7 yrs) at rest, during a graded exercise test, and during recovery. The Ohmeda pulse oximeter (Biox 3740; Louisville, CO) was placed on the right hand throughout the test, and the Respironics pulse oximeter (Model 950; Kennesaw, GA) was placed on the left hand. Statistical analysis included Pearson correlations, paired-sample t-tests, and Bland-Altman plots. SpO 2 values from Ohmeda and Respironics were significantly correlated (r=0.812, p<0.001). The mean SpO 2 of the Ohmeda (94.5 ± 3.1) was not significantly different from that of the Respironics (94.0 ± 3.2). The Bland-Altman plot showed a mean difference between the oximeters of 0.5%; however, the 95% confidence intervals ranged from 4.2% to -3.4%. The variance in the level of agreement between the Ohmeda and the Respironics pulse-oximeters is clinically significant and suggests potential error and a large level of disagreement between the two pulse oximeters. Given the importance of monitoring SpO 2 in COPD patients, specifically those who are prescribed supplemental oxygen, more research is needed on the reliability of commercially available pulse-oximeters. Supported by NIH grants HL53755 and AG10484.

Shayla West Microbiology & Immunology

Advisor: Dr. W. Edward Swords

Phosphorylcholine Weakens Host Innate Responses to Haemophilus Influenzae

Nontypeable Haemophilus influenzae (NTHi) is a human respiratory commensal that can cause opportunistic infections. NTHi produces a diverse population of lipooligosaccharides (LOS), some of which are substituted with the host glycolipid phosphorylcholine (ChoP). LOS elicits host innate responses via the TLR4 recognition complex, which includes lipopolysaccharide-binding protein (LBP), CD14, and Toll-like receptor 4 (TLR4). The goal of this study is to define how ChoP affects host innate responses elicited by LOS. In this study, we challenged TLR4+ mouse macrophages with either purified ChoP+ or ChoP- NTHi LOS, then measured the concentrations of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α) in the cell supernatants. Our results showed that ChoP- LOS elicit more potent innate responses than ChoP+ LOS, resulting in higher concentrations of NO and TNF-α. No significant responses were observed in TLR4- macrophages. Our interpretation of these results is that ChoP weakens host innate responses by altering a proximal step in LOS recognition. Comparison of the aggregates formed by these LOS’s by light-scattering spectroscopy showed that ChoP+ LOS aggregates are larger and more stable than those formed by ChoP- LOS. The binding affinity of LOS for immobilized LBP was also decreased by ChoP. These findings suggest that ChoP affects the physicochemical properties of LOS in aqueous solutions, resulting a reduction in bioactivity.

Jill Wykosky Molecular Medicine

Advisor: Dr. Waldemar Debinski

EphA2 as a New Molecular Marker and Therapeutic Target in Glioblastoma Multiforme

EphA2, a member of the largest family of receptor tyrosine kinases, is over-expressed in a number of epithelial malignancies. We found previously that ephA2 is also over-expressed in glioblastoma multiforme (GBM), the most invasive and lethal brain tumor of astroglial origin. Hence, we have investigated the expression and role of EphA2 in human GBM. We employed Western blotting and immunostaining to examine the presence of EphA2 in tumor specimens and in established and non-established GBM cell lines. We observed specific and abundant over-expression of the EphA2 protein in both GBM tumors and cell lines. Notably, EphA2 is nearly absent in normal human brain. In addition, ephrinA1, the endogenous ligand for EphA2, caused a dose-dependent inhibition of anchorage-independent growth of GBM cells. Thus, EphA2 is specifically over-expressed in GBM and transduces anti-oncogenic signals. EphA2 represents a new marker and novel target for molecular therapeutics for the treatment of patients with GBM.

Saami Yazdani Biomedical Engineering

Advisor: Dr. Joel Berry

Tissue engineered small-caliber blood vessels

Small caliber synthetic grafts used for Coronary Artery Bypass Graft are compromised by thrombogenicity and accelerated intimal thickening, resulting in early graft occlusion. Synthetic grafts do not posses the mechanical properties and morphology of the native artery. Here we describe the fabrication and physical properties of a tissue engineered small-caliber blood vessels (TEBV). Porcine carotid arteries were decellularized using multiple steps. The TEBV were then coated with endothelial cells (EC) in a bioreactor where they were incubated in physiological flow and pressure conditions for 1 week. The mechanical properties (burst pressure, stress, and strain) of the decellularized vessels were very similar to the native human artery. Chemical staining (Movat) of the decellularized vessels showed that the native matrix architecture, including several collagen layers in between internal and external elastin layers were preserved. Hematoxylin and Eosin staining (H&E) illustrated that Bovine EC, seeded on the luminal side, adhered to the matrix and formed a uniform monolayer. These results reveal that, TEBV coated with EC posses many of the morphologic and functional characteristics of small-caliber vessels. TEBV may potentially be useful clinically as vascular grafts.

Marc Yelle Neurobiology & Anatomy

Advisor: Dr. Robert C. Coghill

Offset Analgesia and the Relationship Between Stimulus Fall Rates and Perceptual Fall Rates

Yelle, M.D *, Rogers, J.M, and Coghill, R.C.

Department of Neurobiology and Anatomy

Wake Forest University School of Medicine, Winston-Salem, NC 27157-1010

Offset analgesia is a transient, yet disproportionately large, decrease in perceived pain intensity elicited by slight decreases in noxious thermal stimulus intensity. In order to better characterize offset analgesia, 13 healthy subjects (9 male, 4 female) were asked to give a real-time rating of perceived pain intensity on a continuous visual analog scale (VAS) (range = no pain to most intense pain imaginable) following decreases in noxious stimulus intensity. Noxious stimuli were delivered to the subjects’ ventral forearms via a 1.6 cm 2 peltier device. Two stimulus paradigms were used in this experiment. The first paradigm used three temperatures presented in a continuous fashion. From a baseline of 35 ° C (approximately skin temperature), stimulus temperatures were first increased to 49 ° C (T1) for 5s, then to 50 ° C (T2) for 5s, and then decreased to either 35 ° or 49 ° C (T3) for 20s. A 30s constant 49 ° C stimulus was used as a control for this paradigm. Following the T2 to T3 transition, subjects rated the T3 in a presentation of 49-50-49 ° C stimulus train much lower than the T3 in a constant presentation of 49 ° C (ANOVA, p=0.0046). Furthermore, subjects could not distinguish between a T2-T3 decrease of 50 ° to 35 ° C and 50 ° to 49 ° C at the same time point (p=0.1638). The second paradigm used only one temperature pulse (48 ° C or 50 ° C) and varied the rate at which the stimulus intensity returned to baseline (0.5, 1.0, 2.0, 3.5, and 5.0 ° /s). VAS fall rates were largely independent of temperature fall rates. VAS fall rates evoked by temperature fall rates of 1, 2, and 3.5 ° /s were not significantly different from those evoked by temperature fall rates of 5 ° /s. Moreover, during temperature decreases, VAS ratings returned to zero at temperatures as high as 49 ° C (0.5 ° /s fall rate from 50 ° C). Data from both of these paradigms further confirm that a transient analgesic mechanism is activated at stimulus offset.

Supported by NINDS NS-39426