The Role of the Protein Hfq in Gene Regulation by Small Regulatory RNAs: Binding of Hfq to the rpoS mRNA 5' Leader Region
Graduate Student Mentor: Taylor Updegrove
Faculty Advisor: Roger Wartell
URS Student: Fazila Aseem (Junior - Agnes Scott College, Biochemistry)
In bacteria such as E.coli, a gene designated rpoS encodes a special protein subunit of RNA polymerase (the RpoS protein or 'sigma-38' subunit). Expression of the RpoS protein is turned on during stress conditions. RpoS changes the set of DNA promoters that RNA polymerase binds to and transcribes from. When RNA polymerase has the RpoS subunit, it turns on more than 30 genes needed for bacterial survival during life-threatening stresses such as stationary phase of growth, high osmolarity, low pH, heat shock, elevated H2O2, and UV light. Recent studies showed that RpoS expression and regulation occurs at the post-transcriptional level and controls virulence for a number of bacteria. Several small regulatory RNAs (sRNA) and a RNA-binding protein called Hfq have been shown to be essential for regulating RpoS expression. Each of the sRNAs is induced by a specific environmental signal. Hfq binds to the sRNAs and is thought to promote their interaction with a target region on RpoS mRNA that controls translation initiation. How this Hfq-sRNA-mRNA interaction occurs is not very well understood. Mutational studies suggest that Hfq alone, or in a complex with an sRNA, binds to one or more regions of the rpoS mRNA leader region, thereby possibly altering the mRNA secondary structure and facilitating ribosome access. Information on Hfq interaction with the sRNAs and the rpoS mRNA leader region is needed to understand the role of Hfq in this molecular mechanism. Our goal is to study the binding of Hfq to the sRNA called RprA and to the rpoS mRNA 5' leader region. In vitro transcription methods will be used to produce the RNA regions hypothesized to interact with Hfq. Fluorescence spectroscopy and gel electrophoresis will be used to characterize Hfq binding to these RNAs. This information will provide new insights into its mode of action.
Bioreactor's Role for Improved Growth In Vivo of Engineered Cartilage
Graduate Student Mentor: Tanya M. Farooque
Faculty Advisor: Barbara Boyan
URS Student: Sean Connell (Senior - Georgia Tech, BME)
Millions of Americans are diagnosed with osteoarthritis, a debilitating and painful disease of the wearing away of articular cartilage. Current treatment options do not offer any long-term solutions, treating only the painful symptoms or small lesions. The emergence of the engineering of cartilage tissue for implantation offers a promising and long-term solution. The objective of this proposal is to characterize the influence of the bioreactor environment on tissue-engineered cartilage development in vivo. Native articular cartilage tissue has adapted to and is heavily influenced by its surrounding mechanical forces (shear, perfusion, and compression). These observations demonstrate that in vivo characteristics of cartilage can be important bioprocessing factors in the in vitro development of cartilage. Bioreactors can serve as the central processing unit, applying specific mechanical forces in a suitable culture medium to stimulate chondrogenesis in seeded scaffolds, creating tissue-engineered cartilage suitable for implantation, potentially healing defects. A novel bioreactor has been developed in this lab employing orthogonal fluid regimes promoting cartilage growth. This perfusion concentric cylinder (PCC) bioreactor provides shear stress on the surface of the scaffolds seeded with chondrocytes and forced convection of fluid media through the scaffolds. In turn, the tissue-engineered cartilage grown in this bioreactor is hypothesized to be further developed in vivo. The goal for this project is to show that applying both low shear stress and perfusion in a PCC bioreactor will enhance tissue-engineered cartilage development compared to static culture conditions. The proposed studies will also show those that cartilage constructs grown under bioreactor conditions that enhance cartilage development, will develop further into cartilage-like tissue in vivo. Analyses will include gene expression and histological examination for collagen type II and aggrecan, markers for functional articular cartilage.
In Vitro Fluid Dynamics of the Superior Cavopulmonary Anastomosis Using Particle Image Velocimetry
Graduate Student Mentor: Diane A. de Zelicourt,
Faculty Advisor: Ajit P. Yoganathan
URS Student: Quantez Freeman (Junior - Morehouse College, Biology)
Fluid dynamic efficiency is vital in surgically created superior cavopulmonary anastomoses of patients with single ventricle congenital heart defects to maximize quality of life. Previous studies of flow analysis of the total cavopulmonary connection as in Figure 1 have shown that certain geometries and flow structures can cause increased stress on the single ventricle, making normal circulation more difficult. Single ventricle pathologies are encountered in 2 per 1000 live births and leads to mixing between systemic and pulmonary circulations, creating a cyanotic condition that requires surgical intervention to prevent loss of life. The Fontan repairs have been developed to bypass the right heart, using the single ventricle as a systemic pump. The total cavopulmonary connection (TCPC) is the present procedure of choice for single ventricle patients. The TCPC is an anastomosis of the superior and inferior venae cavae (SVC and IVC) to the left and right pulmonary arteries (LPA and RPA). In a 1988 report by de Leval, two components for the TCPC repair are proposed: the bi-directional Glenn stage as a superior cavopulmonary anastomosis of the SVC to the RPA, and the Fontan stage to channel the IVC to the main pulmonary artery. The bi-directional Glenn connection features an end-to-side anastomosis of the SVC to the RPA. As a variant of the bi-directional Glenn connection, the hemi-Fontan connection reported by Douville et al. in 1991 incorporates a patch to create a side-to-side anastomosis to seal the SVC and the RPA. Advocates of the bi-directional Glenn contend that hemodynamics are smoother at the Glenn stage because the hemi-Fontan connection is inclined to create a bulbous region that may promote flow stasis. Still, literature by Bove et al. in 2003 alleges that the flow differences are negligible. Thus, optimal geometry for a superior cavopulmonary anastomosis has yet to be determined.
Self-assembled Insulative Coatings for Micro-electrode Arrays
Graduate Student Mentor: Maxine McClain
Faculty Advisor: Michelle LaPlaca
URS Student: Vaun Greer (Junior - Morehouse College, BME)
Electrical signals generated by neurons are a primary area of study because while they are not fully understood, they are the functional hallmark of neural systems. Microscale electrodes are commonly used to record neural activity and arrays of electrodes are useful for recording activity at multiple locations simultaneously. Creating and benchmarking electrode arrays is the focus of several projects in the LaPlaca group. Additionally, we are developing microfabrication processes, focusing on flexible electrodes and electrode arrays. The proposed project below would greatly enhance the throughput of device production and would be publishable because once optimized, processing could be done in parallel, while it is now done serially. The goal of the project is to use SAMs and polymer grafting to insulate and open electrically active sites on microneedle arrays. Selectively creating electrically active sites based on microneedle topography will expedite the electrode fabrication process for 3-D MEAs. Only one electrode will be possible per tower, but that is not anticipated to be a significant limitation for the desired application of the device.
Identify the Sequence Differences that are Responsible for "Species Barrier"
Graduate Student Mentor: Buxin Chen
Faculty Mentor: Yury Chernoff
URS Student: Stefka Gyoneva (Junior - Georgia Tech, Biology)
Prions are self-perpetuating infectious protein aggregates, propagated by converting non-prion proteins of the same amino acid sequences into a prion. Mammalian prion protein PrP causes neurodegenerative diseases, such as sheep scrapie, "mad cow" disease, and human Creutzfeldt-Jacob disease (CJD).
Efficient prion conversion requires the precise correspondence of the amino acid sequences of the interacting protein molecules. A lack of precise correspondence leads to a "species barrier", preventing the transmission of the prion state between the phylogenetically divergent isolates of prion proteins. If the "species barrier" is overcome, it results in a cross-species prion infection, such as in "mad cow" disease. Yeast [PSI+], a self-perpetuating amyloid-like prion isoform of the translation termination factor Sup35, provides a useful model for studying mechanisms of the "species barrier". Sup35 has three domains. The N-proximal region (Sup35N), which is called prion forming domain (PFD), is evolutionarily variable and important for [PSI+] induction and propagation. The PFD could be sub-divided into a QN-rich stretch (QNR) and the oligopeptide repeats region (ORs). The function of the M domain (Sup35M) is unknown. Evolutionarily conserved C-proximal region (Sup35C) is required and sufficient for translation termination and cell viability. (Fig.1) The strain we construct had an ade1-14 nonsense mutation. The [PSI+] strain, in which the Sup35 is in a prion form, could not terminate protein synthesis completely once the ribosome encounters the stop codon, and we see growth on -Ade medium and a white/pink color on YPD. However, the [psi-] strain which has the Sup35 normal protein, could terminate protein synthesis completely which causes no growth on -Ade and red color on YPD.
Bio-systems Modeling in Lipidomics for Cancer Research
Graduate Student Mentor: Chang Quo
Faculty Advisor: May D. Wang
URS Student: Chanchala Kaddi (Junior - Georgia Tech, BME)
We propose a two-step process to deal with the problem of bio-systems modeling in lipidomics for cancer research - (1) a comprehensive evaluation of existing systems modeling approaches and techniques relevant for biological systems i.e. knowledge acquisition step and (2) development of a robust mathematical model for the sphingolipid de novo synthesis pathway i.e. application and innovation step. Biological system models are useful to assist researchers in conducting genomics, proteomics and systems biology research. These models are proposed and developed by many research groups around the world to conduct specific biological and medical studies and are the critical components to bridge molecular biology with predictive medicine in the future. However, many of these models produce different results for the same input and operating conditions. This inconsistency raises confusion in the research community and becomes a serious roadblock for the models and tools to be used in medicine. The long-term clinical goal for developing biological system models is to reduce the uncertainty in any clinical decision for cancer disease diagnosis and prognosis. By using such models to simulate the projected patient response to a few clinical treatment options, hopefully, physicians will be better informed to prescribe the optimal one. In the near-term, in vitro experiments that are time-consuming and expensive to perform may be simulated in silico to aid experimental design.
Tensile Culture to Promote Formation of Tissue-engineered Tendon/Ligament
Graduate Student Mentor: Derek Doroski
Faculty Advisor: Johnna Temenoff
URS Student: Brian Nguyen (Junior - Georgia Tech, BME)
Tissue engineered therapies employing autologous cell transplantation offer distinct advantages to traditional methods of tendon and ligament reconstruction by reducing donor site morbidity while invoking minimal immune response. Both tendon/ligament fibroblasts and marrow stromal progenitor cells (MSCs) have been explored for these applications. Although it is known that tensile loading of constructs containing these cells promotes differentiation and extracellular matrix production, the mechanical properties of the cell delivery vehicle can have a large impact on response of the resident cells. In our lab we employ a novel synthetic hydrogel carrier, oligo(poly(ethylene glycol) fumarate) (OPF) along with encapsulated fibroblasts and MSCs in an effort to develop a tissue-engineered tendon/ligament construct. Although, the OPF biomaterial is resistant to cell and protein adhesion, through incorporation of adhesion peptides such as the integrin binding motif RGD, the interactions of cells with the biomaterial can be modulated and tensile forces imparted on the biomaterial may be more directly transduced to the cultured cells. The objective of this project is to aid in the development of a tissue-engineered tendon construct. This research will seek to determine the effects of tensile forces on the degradation of OPF hydrogels and the response of cells (MSCs and fibroblasts) grown on this material.
In Vitro Fluid Dynamics of the Superior Cavopulmonary Anastomosis using Particle Image Velocimetry
Graduate Student Mentor: Murali Padala
Faculty Mentor: Ajit Yoganathan
URS Student: Moses Nyaribo (Junior - Georgia Tech, ME)
Mitral valve disease is a leading cause of mortality in heart disease patients, with approximately 2 million patients treated for valvular incompetence every year. Mitral valve repair compared to replacement has become the procedure of choice in the recent years. Today, mitral valve annuloplasty and leaflet plication are the only accepted or practiced procedures in the operating room. However, the efficiency of these surgical devices or procedures in managing mitral regurgitation or backflow of blood through the valve is alarmingly low, especially in the patients with ischemic heart disease and/or dilated cardiomyopathy. Various clinical investigators including our lab have extensively studied the mechanics and the geometrical determinants of valve regurgitation after these repair procedures. However, most of these studies have focused on the geometry of the valve as a determinant of valve incompetence considering the mitral valve leaflets to be passive scallops analogous to a barn door. In this study, we hypothesize that mitral valve leaflets and chordae tendineae with their interstitial cells, and myriad of proteins that make up the extra-cellular matrix continuously remodel in ischemic disease and cardiomyopathy patients. Recent key studies on the release of various vasoactive substances into the blood plasma in the setting of ischemic heart failure has interested us in looking at the effects of these vasoactive substances on mitral valve tissue tone, material properties and mechanics. In this study the focus will be on the vasoactive substances Endothelin-1 and 5-Hydroxytriptomine (5-HT).
Effects of Sequence Mutations on the Hybridization Activity of DNA
Graduate Student Mentor: Chris Tison
Faculty Advisor: Valeria Milam
URS Student: Sonya Parpart (Junior - Georgia Tech, BME)
DNA is a useful materials assembly tool for the programmed assembly of colloidal particles. This technique was pioneered by Mirkin, et al, using surface coupled DNA strands to assemble gold nanoparticles. Recently, studies by Milam and Kim have investigated DNA assemblies using micron-sized polystyrene colloids. In each of these previous studies, perfectly matched sequences were used for hybridization and assembly purposes. The proposed research will expand upon these studies to observe the effects that sequence mutations have on the hybridization activity of DNA both in solution and coupled to one micron polystyrene microspheres; as well as the effect mutations have on the DNA-mediated assembly of polystyrene colloids. The overall goal is to use DNA to form a complete, but ultimately reversible colloidal assembly. We hypothesize that sequences with a single nucleotide mismatch would have the desired weak attraction for its partner strand. Preliminary results obtained during a SURF fellowship this summer indicate that a mutation in the center of a probe sequence results in a lower surface density of hybridized strands than mutations near either end of a sequence, indicating that the location of a mutation on DNA affects hybridization activity.
Assessment of a Tissue Engineered Construct for Treatment of Spinal Cord Injury
Graduate Student Mentor: Crystal Simon
Faculty Advisor: Michelle LaPlaca
URS Student: Yasamin Rahmani (Junior - Georgia State University, Chemistry)
Cell types derived from human embryonic stem cells (hESCs) have the potential to restore function following spinal cord injury (SCI), but these treatments are currently limited by poor donor cell survival and control of differentiation. In this study, neuroepithelial cells (hNEPs, the precursors to neurons, astrocytes, and oligodendrocytes) were derived from hESCs in vitro. hNEPs were then transplanted into the immunocompromised injured rat spinal cord with or without a bioactive scaffold composed of the extracellular matrix proteins collagen and laminin. A collagen-based scaffold was chosen for its advantageous thermoreversible property, which allows it to be injected at room temperature then gel at physiologic temperature and pH once inside the injury cavity. This phase transition provides a scaffold for cellular adhesion to laminin, a crucial protein for development and cell migration. It is hypothesized that the tissue engineered construct can improve hNEP survival rates and influence differentiation.
Drug Delivery Systems Based on a Novel Acid-sensitive, Biodegradable Material: Polyketals
Graduate Student Mentor: Stephen Yang
Faculty advisor: Niren Murthy
URS Student: Ben Solomon (Junior - Emory, Chemistry)
There is currently great interest in biodegradable materials with acid sensitive linkages in their backbone, because of their potential to deliver therapeutics to the acidic environment of macrophages, tumors and sites of inflammation. Synthetic polymers with ketal linkages in their backbone are a new class of biodegradable materials recently developed in our lab that have attracted great interest in the field of drug delivery because of their acid sensitivity and neutral degradation products. These polyketals that have tunable hydrolysis kinetics, hydrolyze under acidic conditions, and also can encapsulate protein therapeutics in 1-3 micron sized particles and deliver them to macrophages. These properties of polyketals offer numerous advantages over existing biodegradable materials for many drug delivery applications. The objectives of this URS project are (1) to formulate nano-sized and micro-sized particles with a hydrophobic or hydrophilic dye loaded, (2) to characterize and optimize the formulation and loading of these particles, (3) to study the release of loaded dye from these particles at various pH values, and (4) to study the degradation of the polymers and erosion of the particles during the process of dye release.
Biomimetic Micropatterned Biomaterials
Graduate Student Mentor: Timothy A. Petrie
Faculty Advisor: Andrés J. García
URS Student: Brandon Stanley (Junior - Morehouse College, BME)
Cell adhesion to extracellular matrices regulates the organization, maintenance and repair of numerous tissues, and abnormalities in adhesive interactions are often associated with pathological states. Furthermore, adhesive interactions regulate cellular and host responses to implanted biomedical devices, tissue-engineered constructs, and biotechnological systems. The adhesive process comprises integrin receptor binding to their extracellular ligand, integrin clustering, and assembly of discrete supramolecular adhesive structures containing cytoskeletal and signaling molecules. These focal adhesion complexes function as structural links and signal transduction elements between the cell and its extracellular environment. We previously used micropatterned substrates to analyze the contributions of cell-substrate adhesive area to adhesion strengthening. The objective of this project is to expand our micropatterning approaches to engineer biomaterial surfaces for which cell adhesive area and density of adhesive ligands can be independently modulated. Microcontact printing of functionalized alkanethiolates will be used to generate these well defined substrates. These advanced biomaterial surfaces will provide robust platforms to analyze the contributions of adhesive area and integrin binding to adhesion strengthening as well as promising materials to regulate cell responses to biomaterials.
Biomolecular Distribution within Embryonic Stem Cell Derived Spheroids
Graduate Student Mentor: Rich Carpenedo
Faculty Advisor: Todd McDevitt
URS Student: Ranni Tewfik (Junior - Georgia Tech, Biology)
Embryonic stem cells (ESCs) have shown great potential as a cell source for regenerative medicine applications, yet various limitations have prevented their use clinically, including the inability to induce controllable, homogenous differentiation to a targeted cell type. Differentiation of ESCs is commonly induced by cell aggregation into non-adherent spheroids referred to as embryoid bodies (EBs), which recapitulate aspects of the developing embryo including endoderm, mesoderm, and ectoderm formation. Various signaling molecules such as growth factors and small molecules, including retinoic acid and DMSO, have been reportedly used to direct differentiation of cells within EBs toward specific cell lineages. However, because EBs can be relatively large (tens of thousands of cells, up to 500 µm diameter), the degree to which signaling molecules readily diffuse throughout EBs and interact with all of the cells is not known. Cells on the exterior of EBs may be induced to differentiate toward a cell type specified by interactions with exogenous signaling molecules while cells on the interior spontaneously differentiate based on cell-cell interactions and endogenous signals, resulting in a heterogeneous cell population. Therefore the objectives of this project are to: 1) determine the relationship between the size of signaling molecules and their ability to diffuse within EBs; 2) assess the influence of EB size and formation method on permeability to signaling molecules, and 3) investigate the presence of cell-cell interactions, such as tight and gap junctions, which may contribute to diffusional barriers in EBs. The project will identify which signaling molecules are appropriate for soluble delivery to EBs, as well as those that require novel delivery approaches to allow interaction with all cells within an EB.
Bioactivity Assay Development and Analysis for a Novel Stem Cell-derived Acellular Biomaterial
Graduate Student Mentor: Alyssa Ngangan
Faculty Advisor: Todd McDevitt
URS Student: James Waring (Junior - Georgia Tech, ME)
The extracellular matrix (ECM) is a complex assembly of proteins and polysaccharides that serve dual structural and functional purposes to maintain normal tissue homeostasis and in the event of injury, direct tissue morphogenesis. Embryonic stem cells (ESCs) are pluripotent cells capable of differentiating into all somatic cell types and thus may be useful for regenerative cell therapies. ESCs are sensitive to cues presented within the ECM of their microenvironment that can promote either stem cell self-renewal or differentiation into multiple cell lineages. In addition, ESCs are also capable of autonomously producing ECM molecules that may direct embryonic cell fate decisions. Similar to biological development, the regeneration of tissues is a complex biological process requiring numerous molecular factors be presented in an appropriate spatiotemporal manner in order to successfully restore tissue function and prevent scar tissue formation. A number of cellular responses, including cell migration, are also required for functional tissue regeneration to occur by recruiting endogenous cells to a site of injury in need of cellular restoration. Thus, the objective of this project is to examine the bioactivity of a soluble form of a novel acellular biomaterial-derived from ESCs by assessing its effects on cell migration. This project will involve the development and characterization of in vitro cell migration assays and quantitative analytical tools to assess the stimulatory effects of the solubilized biomaterial on cell migration.