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School of Biological Sciences

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https://hdl.handle.net/1853/70750

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College of Sciences

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Center for the Study of Systems Biology

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https://finding-aids.library.gatech.edu/agents/corporate_entities/1131

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Publication Search Results

Now showing 1 - 10 of 14

Optimization of over-expression and purification of human leukotriene C4 synthase mutant R104A for structure-function studies by two-dimensional crystallization and electron crystallography

(Georgia Institute of Technology, 2012-11-15) Kim, Laura Yaunhee

Membrane proteins are involved in a number of disease pathologies and thus comprise a large number of drug targets. Determination of the high-resolution three-dimensional structure is essential for rational drug design, but several hurdles need to be overcome, primarily the over-expression and purification of said membrane proteins. Human leukotriene C4 synthase (hLTC4S), an 18 kDa integral membrane protein localized in the outer nuclear membrane of eosinophils and basophils, catalyzes the conjugation of LTA4 and reduced glutathione to produce LTC4. LTC4 and its metabolites LTD4 and LTE4 are the cysteinyl leukotrienes implicated in bronchoconstriction and inflammation pathways. The focus of my project involves optimizing the over-expression and purification of hLTC4S, which was heterologously expressed in Schizosaccharomyces pombe, purified by immobilized affinity chromatography, and finally "polished" with a buffer exchange step to remove excess co-purified lipids. The optimized protocol yielded ~1 mg of ~90% homogenous, pure protein per liter of cell culture. The finalized purified protein can then be used for further investigation of two-dimensional crystals by electron crystallography with the overall goal of structure determination.
Seaweed allelopathy against coral: surface distribution of seaweed secondary metabolites by imaging mass sepctrometry

(Georgia Institute of Technology, 2012-08-16) Andras, Tiffany D.

Coral reefs are in global decline, with seaweeds increasing as corals decrease. Though seaweeds have been shown to inhibit coral growth, recruitment, and survivorship, the mechanism of these interactions is poorly known. Here we use field experiments to show that contact with four common seaweeds induces bleaching on natural colonies of Porites rus. Controls in contact with inert, plastic mimics of seaweeds did not bleach, suggesting treatment effects resulted from allelopathy rather than shading, abrasion, or physical contact. Bioassay-guided fractionation of the hydrophobic extract from the red alga Phacelocarpus neurymenioides revealed a previously characterized antibacterial metabolite, Neurymenolide A, as the main allelopathic agent. For allelopathy of lipid soluble metabolites to be effective, the metabolites would need to be deployed on algal surfaces where they could transfer to corals on contact. We used desorption electrospray ionization mass spectrometry (DESI-MS) to visualize and quantify Neurymenolide A on the surface of P. neurymenioides and found the metabolite on all surfaces analyzed. The highest concentrations of Neurymenolide A were on basal portions of blades where the plant is most likely to contact other benthic competitors.
Experimental phylogenetics: a benchmark for ancestral sequence reconstruction

(Georgia Institute of Technology, 2012-07-05) Randall, Ryan Nicole

The field of molecular evolution has benefited greatly from the use of ancestral sequence reconstruction as a methodology to better understand the molecular mechanisms associated with functional divergence. The method of ancestral sequence reconstruction has never been experimentally validated despite the method being exploited to generate high profile publications and gaining wider use in many laboratories. The failure to validate such a method is a consequence of 1) our inability to travel back in time to document evolutionary transitions and 2) the slow pace of natural evolutionary processes that prevent biologists from ‘witnessing’ evolution in action (pace viruses). In this thesis research, we have generated an experimentally known phylogeny of fluorescent proteins in order to benchmark ancestral sequence reconstruction methods. The tips/leaves of the fluorescent protein experimental phylogeny are used to determine the performances of various ASR methods. This is the first example of combining experimental phylogenetics and ancestral sequence reconstruction.
Adverse effects of bone morphogenic protein-2 during osseointegration

(Georgia Institute of Technology, 2012-05-21) Hyzy, Sharon Leigh

Modifications of biomaterial surface properties are employed to increase osteoblast differentiation and bone formation. Microtextured metallic surfaces promote osteoblast differentiation and high surface energy- achieved by controlling surface hydrocarbon contamination- increases osteoblast differentiation and peri-implant bone formation. Recombinant human bone morphogenic protein 2 (BMP2) is approved to induce bone formation in a number of applications. It is used clinically in combination with biomaterials to improve peri-implant bone formation and osseointegration. The amount of BMP2 that is required is large and inflammatory (swelling/seroma) and bone-related (ectopic bone/bone resorption) complications have been reported after BMP2 treatment. The aim of this study was to examine potential deleterious effects of BMP2 on the inflammatory environment and apoptosis of osteoblasts. Surface roughness and energy decreased pro-inflammatory interleukins and increased anti-inflammatory interleukins. In contrast, BMP2 abolished the surface effect, increasing pro-inflammatory interleukin (IL) 6, IL8, and IL17 in a surface roughness-dependent fashion and decreasing anti-inflammatory IL10 on rough surfaces. 5Z-7-Oxozeaenol and Dorsomorphin, but not H-8, blocked the effect of BMP2 on IL1A expression. There was an increase in expression of IL6 when treated with BMP2 for the control and H-8 groups, but both 5Z-7-Oxozeaenol and Dorsomorphin blocked the effect. Both 5Z-7-Oxozeaenol and H-8 blocked the effect of BMP2 on IL10 expression. BMP2 treatment had little effect on apoptosis in human mesenchymal stem cells (MSCs). Exogenous BMP2 had no effect on TUNEL. Caspase-3 activity was increased only at 200ng/ml BMP2. BAX/BCL2 decreased in MSCs treated with 50 and 100ng/ml BMP2. In contrast, BMP2 increased caspase-3 activity and TUNEL at all doses in normal human osteoblasts (NHOst). BAX/BCL2 increased in NHOst treated with BMP2 in a dose-dependent manner. Cells treated with 200 ng/ml BMP2 had an 8-fold increase in BAX/BCL2 expression in comparison with untreated cells. Similarly, BMP2 increased DNA fragmentation in NHOst cells. The BMP2-induced increase in DNA fragmentation was eliminated by 5-Z7-Oxozeaenol and Dorsomorphin. The results suggest that while surface features modulate an initial controlled inflammatory response, the addition of BMP2 induces a pro-inflammatory response. The effect of BMP2 on apoptosis depends on cell maturation state, inducing apoptosis in committed osteoblasts. BMP2 together with microtextured orthopaedic and dental implants may increase inflammation and possibly delay bone formation. Dose, location, and delivery strategies are important considerations in BMP2 as a therapeutic and must be optimized to minimize complications.
Principles of fin and flipper locomotion on granular media

(Georgia Institute of Technology, 2012-05-04) Mazouchova, Nicole

Locomotion of animals, whether by running, flying, swimming or crawling, is crucial to their survival. The natural environments they encounter are complex containing fluid, solid or yielding substrates. These environments are often uneven and inclined, which can lead to slipping during footsteps presenting great locomotor challenges. Many animals have specialized appendages for locomotion allowing them to adapt to their environmental conditions. Aquatically adapted animals have fins and flippers to swim through the water, however, some species use their paddle-like appendages to walk on yielding terrestrial substrates like the beach. Beach sand, a granular medium, behaves like a solid or a fluid when stress is applied. Principles of legged locomotion on yielding substrates remain poorly understood, largely due to the lack of fundamental understanding of the complex interactions of body/limbs with these substrates on the level of the Navier-Stokes Equations for fluids. Understanding of the limb-ground interactions of aquatic animals that utilize terrestrial environments can be applied to the ecology and conservation of these species, as well as enhance construction of man-made devices. In this dissertation, we studied the locomotion of hatchling loggerhead sea turtles on granular media integrating biological, robotic, and physics studies to discover principles that govern fin and flipper locomotion on flowing/yielding media. Hatchlings in the field modified their limb use depending on substrate compaction. On soft sand they bent their wrist to utilize the solid features of sand, whereas on hard ground they used a rigid flipper and claw to clasp asperities during forward motion. A sea turtle inspired physical model in the laboratory was used to test detailed kinematics of fin and flipper locomotion on granular media. Coupling of adequate step distance, body lift and thrust generation allowed the robot to move successfully forward avoiding previously disturbed ground. A flat paddle intruder was used to imitate the animal's flipper in physics drag experiments to measure the forces during intrusion and thrust generation.
Evolutionary synthetic biology: structure/function relationships within the protein translation system

(Georgia Institute of Technology, 2011-09-06) Cacan, Ercan

Production of mutant biological molecules for understanding biological principles or as therapeutic agents has gained considerable interest recently. Synthetic genes are today being widely used for production of such molecules due to the substantial decrease in the costs associated with gene synthesis technology. Along one such line, we have engineered tRNA genes in order to dissect the effects of G:U base-pairs on the accuracy of the protein translation machinery. Our results provide greater detail into the thermodynamic interactions between tRNA molecules and an Elongation Factor protein (termed EF-Tu in bacteria and eEF1A in eukaryotes) and how these interactions influence the delivery of aminoacylated tRNAs to the ribosome. We anticipate that our studies not only shed light on the basic mechanisms of molecular machines but may also help us to develop therapeutic or novel proteins that contain unnatural amino acids. Further, the manipulation of the translation machinery holds promise for the development of new methods to understand the origins of life. Along another line, we have used the power of synthetic biology to experimentally validate an evolutionary model. We exploited the functional diversity contained within the EF-Tu/eEF1A gene family to experimentally validate the model of evolution termed ‘heterotachy’. Heterotachy refers to a switch in a site’s mutational rate class. For instance, a site in a protein sequence may be invariant across all bacterial homologs while that same site may be highly variable across eukaryotic homologs. Such patterns imply that the selective constraints acting on this site differs between bacteria and eukaryotes. Despite intense efforts and large interest in understanding these patterns, no studies have experimentally validated these concepts until now. In the present study, we analyzed EF-Tu/eEF1A gene family members between bacteria and eukaryotes to identify heterotachous patterns (also called Type-I functional divergence). We applied statistical tests to identify sites possibly responsible for biomolecular functional divergence between EF-Tu and eEF1A. We then synthesized protein variants in the laboratory to validate our computational predictions. The results demonstrate for the first time that the identification of heterotachous sites can be specifically implicated in functional divergence among homologous proteins. In total, this work supports an evolutionary synthetic biology paradigm that in one direction uses synthetic molecules to better understand the mechanisms and constraints governing biomolecular behavior while in another direction uses principles of molecular sequence evolution to generate novel biomolecules that have utility for industry and/or biomedicine.
A test of optimal defense theory vs. the growth-differentiation balance hypothesis as predictors of seaweed palatability and defenses

(Georgia Institute of Technology, 2011-08-31) Heckman, Melanie L.

Because organisms have limited resources to allocate to multiple life history traits, the Optimal Defense Theory (ODT) and the Growth-Differentiation Balance Hypothesis (GDBH) were developed by terrestrial plant ecologists to predict intraindividual defense allocation based on the cost of defense and these life history trade-offs. However, these theories have garnered equivocal experimental support over the years and are rarely experimentally extended from predictions of plant physiology to the palatability of the tissues an herbivore experiences. We therefore examined tissue palatability, nutritional value, and defense mechanisms in multiple Dictyotalean seaweeds in two Caribbean locations, using two herbivores. Relative palatability of tissues varied greatly with algal species, grazer species, and location. Because older bases were not consistently defended, GDBH did not predict relative palatability. We could not reject ODT without intensive measures of tissue fitness value and herbivore risk, and this theory was therefore not useful in making broad predictions of tissue palatability. In testing the physiological predictions of these theories, we found the young, growing apices of these seaweeds to be generally more nutritionally valuable than the old, anchoring bases and found organic-rich apices to be more chemically deterrent, thus supporting ODT. However, the combined chemical, nutritional, and structural traits of these algae all influenced herbivore choice. As a result, these patterns of apical value and chemical defense reflected palatability of live tissues for only one of five algal species, which rendered ODT and GDBH poor predictors of relative palatability for most algae.
Genetic and physical interaction of Sgt2 protein with prion-chaperone machinery

(Georgia Institute of Technology, 2011-08-10) Pan, Tao

The word "Prion" refers to self-perpetuating protein aggregates that cause neurodegenerative diseases in mammals. It is a protein isoform that has undergone a conformational change which converts the normal form of the protein into the infectious form with the same amino acid sequence. Yeast [PSI+] prion is the prion isoform of Sup35 protein, a translation termination factor eRF3. It has been suggested that prion [PSI+] is controlled by the ensemble of chaperones with Hsp104 playing the major role. The previous work performed in the Chernoffs lab showed that the defective GET pathway caused by get led to the defect in [PSI+] curing by excess Hsp104. The GET pathway is a system responsible for transporting newly synthesized TA-protein to the ER membrane, and the components which have been proven to be involved in this pathway include: Get1, Get2, Get3, Get4, Get5 and Sgt2. In this study we describe the mechanism underlying the effect of the defective GET pathway on [PSI+]. We demonstrate that Sgt2, one of the components of GET pathway, interacts with Sup35 in both [PSI+] and [psi-] strains through its prion domain. Overproduction of Sgt2 and Hsp70-Ssa is triggered by the defective GET pathway and leads to the protection of [PSI+] aggregates from curing by excess Hsp104. We show that the direct interaction between Sgt2 and Hsp70-Ssa is not required for this protective effect.
Freshwater red algae use activated chemical defenses against herbivores

(Georgia Institute of Technology, 2011-07-12) Goodman, Keri M.

Chemically mediated interactions have important ecological and evolutionary effects on populations and communities. Despite recognition that herbivory can significantly affect the biomass and composition of freshwater macrophyte communities, there are few investigations of chemical defenses among freshwater vascular plants and mosses and none of freshwater red algae. This study compares the palatability of five species of freshwater red algae (Batrachospermum helminthosum, Boldia erythrosiphon, Kumanoa sp., Paralemanea annulata, and Tuomeya americana) that occur in the southeastern United States relative to two co-occurring macrophytes (the chemically defended aquatic moss Fontinalis novae-angliae and the broadly palatable green alga Cladophora glomerata). We assessed the potential role of structural, nutritional, and chemical traits in reducing macrophyte susceptibility to generalist crayfish grazers. Both native and non-native crayfish significantly preferred the green alga C. glomerata over four of the five species of red algae. B. erythrosiphon was palatable, while the cartilaginous structure of P. annulata reduced its susceptibility to grazing, and chemical defenses of B. helminthosum, Kumanoa sp., and T. americana rendered these species as unpalatable as the moss F. novae-angliae. Extracts from these latter species reduced feeding by ~30-60% relative to solvent controls if tissues were crushed (simulating herbivore damage) prior to extraction in organic solvents. However, if algae were first soaked in organic solvents that inhibit enzymatic activity and then crushed, crude extracts stimulated or had no effect on herbivory. B. helminthosum, Kumanoa sp., and T. americana all exhibited "activated" chemical defenses in which anti-herbivore compounds are produced rapidly upon herbivore attack via enzymatic processes. In an additional accept/reject behavioral assay, B. helminthosum extracts reduced the number of crayfish willing to feed by >90%. Given that three of the five red algal taxa examined in this study yielded deterrent crude extracts, selection for defensive chemistry in freshwater rhodophytes appears to be substantial. Activated chemical defenses are thought to be an adaptation to reduce the resource allocation and ecological costs of defense. As such, activated chemical defenses may be favored in freshwater red algae, whose short-lived gametophytes must grow and reproduce rapidly. Roughly 20% of the known chemical defenses produced by marine algae are activated; further examination is needed to determine whether the frequency of activated chemistry is higher in freshwater red algae compared to their marine counterparts. Continued investigation of chemical defenses in freshwater red algae will contribute to among-system comparisons, providing new insights in the generality of plant-herbivore interactions and their evolution.
RNA secondary sturcture prediction using a combined method of thermodynamics and kinetics

(Georgia Institute of Technology, 2011-07-07) Pan, Minmin

Nowadays, RNA is extensively acknowledged an important role in the functions of information transfer, structural components, gene regulation and etc. The secondary structure of RNA becomes a key to understand structure-function relationship. Computational prediction of RNA secondary structure does not only provide possible structures, but also elucidates the mechanism of RNA folding. Conventional prediction programs are either derived from evolutionary perspective, or aimed to achieve minimum free energy. In vivo, RNA folds during transcription, which indicates that native RNA structure is a result from both thermodynamics and kinetics. In this thesis, I first reviewed the current leading kinetic folding programs and demonstrate that these programs are not able to predict secondary structure accurately. Upon that, I proposed a new sequential folding program called GTkinetics. Given an RNA sequence, GTkinetics predicts a secondary structure and a series of RNA folding trajectories. It treats the RNA as a growing chain, and adds stable local structures sequentially. It is featured with a Z-score to evaluate stability of local structures, which is able to locate native local structures with high confidence. Since all stable local structures are captured in GTkinetics, it results in some false positives, which prevents the native structure to form as the chain grows. This suggests a refolding model to melt the false positive hairpins, probable intermediate structures, and to fold the RNA into a new structure with reliable long-range helices. By analyzing suboptimal ensemble along the folding pathway, I suggested a refolding mechanism, with which refolding can be evaluated whether or not to take place. Another way to favor local structures over long-distance structures, we introduced a distance penalty function into the free energy calculation. I used a sigmoidal function to compute the energy penalty according to the distance in the primary sequence between two nucleotides of a base pair. For both the training dataset and the test dataset, the distance function improves the prediction to some extent. In order to characterize the differences between local and long-range helices, I carried out analysis of standardized local nucleotide composition and base pair composition according to the two groups. The results show that adenine accumulates on the 5' side of local structure, but not on that of long-range helices. GU base pairs occur significantly more frequent in the local helices than that in the long-range helices. These indicate that the mechanisms to form local and long range helices are different, which is encoded in the sequence itself. Based on all the results, I will draw conclusions and suggest future directions to enhance the current sequential folding program.