Organizational Unit:
School of Biological Sciences

Permanent Link
https://hdl.handle.net/1853/70750
Research Organization Registry ID
Description
Previous Names
Parent Organization
Parent Organization
Organizational Unit
Includes Organization(s)
ArchiveSpace Name Record
https://finding-aids.library.gatech.edu/agents/corporate_entities/1131

Filters

2000 - 2009 68
68
68
68
Reset filters

Settings
End date: 2009 × Start date: 2000 × search.filters.applied.f.resourcesubtype: Dissertation ×

Publication Search Results

Now showing 1 - 10 of 68
Thumbnail Image
Item

Mechanisms and implications of sodium loss in sweat during exercise in the heat for patients with cystic fibrosis and healthy individuals

2009-11-17 , Brown, Mary Beth

Our aim was to understand mechanisms responsible for excessive electrolyte loss in the sweat gland and the potential impact on fluid balance during exercise in heat stress conditions. Human physiological testing under exercise/heat stress and immunofluorescence staining of sweat glands from skin biopsies were compared between healthy individuals (with normal and high sweat sodium chloride concentration, [NaCl]) and with cystic fibrosis patients (CF), who exhibit excessively salty sweat due to a defect of Cl- channel cystic fibrosis transmembrane conductance regulator (CFTR). Three novel findings are presented. First, excessively salty sweat may be associated with reduced expression of CFTR in the sweat gland reabsorptive duct of healthy individuals in addition to in those with CF; however, although a link to a CF gene mutation in healthy individuals with high sweat [NaCl] was not demonstrated, the possibility of an undetected CFTR mutation or polymorphism remains to be investigated as an underlying mechanism. Two, CF and healthy individuals with excessively salty sweat respond to moderate dehydration (3% body weight loss during exercise) with an attenuated rise in serum osmolality, greater relative loss in plasma volume, but similar perceived thirst compared to healthy individuals with "normal" sweat [NaCl]. However, individuals with CF respond to rehydration with hypotonic beverage by drinking less ad libitum in response to reduced serum [NaCl], suggesting that thirst-guided fluid replacement may be more appropriate for this population rather than restoring 100% of sweat loss following dehydration as is often recommended in healthy individuals.

View
Thumbnail Image
Item

Lysophosphatidic acid, vitamin D, and p53: a novel signaling axis in cell death and differentiation

2009-09-09 , Hurst-Kennedy, Jennifer Lynne

Lysophosphatidic acid (LPA) is the simplest of the glycerol lipids and regulates a number of cellular processes such as morphological changes, migration, proliferation, and inhibition of apoptosis. LPA exerts these effects through activation of the G-protein coupled receptors (GPCRs) LPA1-6 and the intracellular fatty acid receptor peroxisome proliferator-activated receptor-gamma (PPARγ). The overall goal of this thesis was to determine the mechanisms by which LPA enhances cell survival by inhibiting apoptosis. The project was divided into three studies: 1) to determine the mechanism of LPA-mediated inhibition of p53 in A549 lung carcinoma cells, 2) to investigate the regulation of growth plate chondrocytes by LPA, and 3) to determine the mechanisms of LPA-mediated effects in the growth plate. In the first study, evidence is provided that LPA reduces the cellular abundance of the tumor suppressor p53 in A549 lung carcinoma cells. The LPA effect depends upon increased proteasomal degradation of p53 and it results in a corresponding decrease in p53-mediated transcription. The result of LPA-mediated inhibition of p53 in A549 cells is enhanced resistance to chemotherapeutic-induced apoptosis. In the second study, the role of LPA in resting zone chondrocytes (RC cells) was investigated. RC cells are regulated by 24,25-dihydroxyvitamin D3 [24,25(OH)[subscript2]D [subscript 3]] via a phospholipase D-dependent pathway, suggesting downstream phospholipid metabolites are involved. In this study, we showed that 24R,25(OH)[subscript 2]D[subscript 3] stimulates rat costochondral RC cells to release LPA. Additionally, we demonstrated that RC cells respond to LPA with increased proliferation, maturation, and inhibition of apoptosis. In the final study, the mechanism of LPA and 24R,25(OH)[subscript 2]D[subscript 3]-mediated inhibition of chondrocyte apoptosis was further investigated. Our data show that 24R,25(OH)[subscript 2]D[subscript 3] inhibits apoptosis through Ca⁺⁺, PLD, and PLC signaling and through LPA/Gαi/PI[subscript 3]K/mdm2-mediated degradation of p53, resulting in decreased caspase-3 activity. Collectively, our data establish LPA, vitamin D, and p53 as an anti-apoptotic signaling axis.

View
Thumbnail Image
Item

Functional identification and initial characterization of a fish co-receptor involved in aversive signaling

2009-05-18 , Cohen, Staci Padove

Chemoreception plays an important role in predator-prey interactions and feeding dynamics. While the chemoreception of attractant or pleasant tasting compounds has been well studied, aversive chemoreceptive signaling has been difficult to investigate behaviorally in an ecological context because these interactions are species- and context- specific and deterrent compounds vary among prey. Using the coral reef system, this thesis explores on a molecular level the deterrent mechanism underlying detection by fish predators of an aversive compound, in order to gain a greater understanding of predator-prey interactions in this community. Like other organisms that are sessile or slow-moving, marine sponges have special mechanisms for defense from predation, commonly containing aversive-tasting compounds that defend these organisms from predation. To this end, we sought to identify and characterize a fish chemoreceptor that detects one or more of these compounds. We isolated a single cDNA clone encoding RAMP-like triterpene glycoside receptor (RL-TGR), a novel co-receptor involved in the signaling of triterpene glycosides. This co-receptor appears to be structurally and functionally related to receptor activity-modifying proteins (RAMPs), a family of co-receptors that physically associate with and modify the activity of G protein-coupled receptors (GPCRs). Expression in Xenopus oocytes showed that it responds to triterpene glycosides in a receptor-mediated manner and requires co-expression of a GPCR to enable signaling in oocytes; both of these receptors may be components of a larger signaling complex. A 40 bp portion of the gene is conserved across multiple fish species, but is not found in any other organism with a sequenced genome, suggesting that the expression of this receptor is limited to fish species. RL-TGR is the first identified gene encoding a co-receptor that responds to a chemical defense. This finding may lead the way for the identification of many other receptors that mediate chemical defense signaling in both marine and terrestrial environments, as this protein has the potential to represent the first of an entire family of co-receptors that respond to aversive compounds.

View
Thumbnail Image
Item

Genome instability induced by triplex forming mirror repeats in S.cerevisiae

2009-04-07 , Kim, Hyun-Min

The main goal of this research is to understand molecular mechanisms of GAA/TTC-associated genetic instability in a model eukaryotic organism, S. cerevisiae. We demonstrate that expanded GAA/TTC repeats represent a threat to eukaryotic genome integrity by triggering double-strand breaks and gross chromosomal rearrangements. The fragility potential strongly depends on the length of the tracts and orientation of the repeats relative to the replication origin and to block replication fork progression. MutSbeta complex and endonuclease activity of MutLalpha play an important role in facilitation of fragility. In addition to GAA/TTC triplex forming repeats, non-GAA polypurine polypyrimidine mirror repeats that are prone to the formation of similar structures were found to be hotspots for rearrangements in humans and other model organisms. These include H-DNA forming sequences located in the major breakpoint cluster region at BCL2, intron 21 of PKD1, and promoter region of C-MYC. Lastly, we have investigated the effect of the triplex-binding small molecules, azacyanines, on GAA-mediated fragility using the chromosomal arm loss assay. We have found that in vivo, azacyanines stimulate (GAA/TTC)-mediated arm loss in a dose dependent manner in actively dividing cells. Azacyanines treatment enhances the GAA-induced replication arrest. We discovered that also, azacyanines at concentrations that induce fragility also inhibit cell growth. Over 60% of yeast cells are arrested at G2/M stage of the cell cycle. This implies an activation of DNA-damage checkpoint response.

View
Thumbnail Image
Item

Development of an assay for fatty acyl-CoAs using liquid chromatography-electrospray ionization-tandem mass spectrometry and its application to the stable isotope labeling and quantitation of sphingolipid metabolism

2009-11-16 , Haynes, Christopher Allen

Fatty acyl-Coenzyme As are metabolites of lipid anabolism and catabolism. A method was developed for their quantitation in extracts of cultured mammalian cells using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Palmitoyl-CoA (C16:0-CoA) is utilized for de novo sphingolipid biosynthesis catalyzed by serine palmitoyltransferase (SPT), which condenses palmitoyl-CoA and serine to form 3-ketosphinganine. After reduction to form sphinganine (Sa), dihydroceramide synthase (CerS) can N-acylate the Sa using a second fatty acyl-CoA molecule, forming dihydroceramide (DHCer). The CerS enzyme family utilizes different acyl chain lengths of fatty acyl-CoAs in an isoform-specific manner, resulting in DHCer with N-acyl chains ranging from C16 to C26 [and even longer] in mammalian tissues. DHCer is trans-4,5-desaturated to yield ceramide, which is further metabolized by the addition of moieties at the 1-O-position, forming sphingomyelin (SM) and ceramide monohexose (CMH). The rates of fatty acyl-CoA and sphingolipid biosynthesis were determined using stable isotope-labeling and LC-ESI-MS/MS analysis of the analyte isotopologues and isotopomers. Isotopic labeling of palmitoyl-CoA with [U-13C]-palmitate in HEK293 and RAW264.7 cells was robust and rapid (~ 60% labeling of the metabolite pool in 3 hr). Isotopic labeling of sphingolipids indicated utilization of [M + 16]-palmitoyl-CoA by SPT and CerS isoforms in both cell types. Metabolic flux modeling was applied to the data for [U-13C]-palmitate activation to [M + 16]-palmitoyl-CoA and its subsequent utilization in de novo sphingolipid biosynthesis, and this analysis indicated rapid turn-over rates for palmitoyl-CoA and ceramide in both cell types. Palmitate treatment of cultured cells alters their metabolic status and gene expression, therefore labeling of palmitoyl-CoA by treatment with [1-13C]-acetate was employed. A distribution of mass-shifted palmitoyl-CoA species (isotopologues) is observed based on the number of incorporations of [1-13C]-acetate during de novo biosynthesis, requiring computational analysis to derive two parameters: the isotopic enrichment of the precursor pool, and the fraction of palmitoyl-CoA that was biosynthesized during the experiment. Previous reports by others describe mass isotopomer distribution analysis (MIDA) and isotopomer spectral analysis (ISA) for this purpose, and both calculation approaches indicated concurrent results. In summary, the quantitation of fatty acyl-CoAs and their isotopic enrichment during stable isotope-labeling studies of lipid metabolism can provide data that significantly change the interpretation of analyte quantitation in these experiments, as demonstrated here for investigations of de novo sphingolipid biosynthesis.

View
Thumbnail Image
Item

Queen-specific selective pressures and caste dimorphism in the social wasp Vespula maculifrons

2009-08-19 , Kovacs, Jennifer L.

Within social insect colonies, sterile workers are responsible for brood care, while queens are the primary egg-layers. These reproductive caste differences are often accompanied by pronounced morphological distinctions. Queen and worker phenotypic differences are particularly remarkable considering caste is environmentally, rather than genetically, determined. Environmental caste determination can produce intralocus genetic conflict between castes, particularly when homologous traits are highly dimorphic. Therefore, when studying the evolution of social insect caste dimorphism, one must consider the genetic architecture underlying phenotypic expression as well as the selective pressures that have shaped caste morphology. This dissertation presents the results of four studies that investigated factors affecting caste morphology in the social wasp Vespula maculifrons. The first two studies focused on identifying queen morphological traits that were positively associated with queen fitness and would therefore be subject to selection. Queen length, specifically gaster length, was positively associated with overwintering survival and was consistently associated with mating success. Both of these findings suggest that queen gaster length is under selection during two life-history events, mating and overwintering, in which workers do not participate. These findings provide empirical support for the adaptive evolution of a caste dimorphic trait. The third and fourth studies used classical quantitative genetic and morphological analyses to examine the genetic architecture underlying caste dimorphism in V. maculifrons. I determined which traits were under caste-specific selection by analyzing trait allometries and the levels of genetic control, variation, and dimorphism of traits between castes. Little genetic variation for morphological trait size was detected for most worker and queen traits, suggesting a strong influence of environment on phenotypic variation. Additionally, analyses of trait allometries indicated that several queen traits (mass, thorax width and length) were under queen-specific selection. The relationship between thorax length, gaster length, and overall body size is further evidence of selection on length in queens. Overall, these studies provide evidence for the importance of queen-specific selection in the evolution of caste dimorphism. When placed in the broader context of caste evolution, they point to the importance of life-history in shaping the genetic architecture underlying caste dimorphism.

View
Thumbnail Image
Item

Characterization of ceramide synthases (Cers) in mammalian cells

2009-05-13 , Park, Hyejung

This thesis describes the characterization of ceramide (Cer) biosynthesis by mammalian cells. The possibility that Cer undergo developmental changes was explored using mouse embryonic stem cells versus embryoid bodies by analysis of the Cer subspecies by liquid chromatography, electrospray ionization-tandem mass spectrometry (LC ESI-MS/MS) and of the transcript levels for enzymes involved in Cer biosynthesis by qRT-PCR. Cer of embroid bodies had higher proportions of very-long-chain fatty acids, which correlated with the relative expression of mRNA for the respective Cer synthases (CerS) and fatty acyl-CoA elongases, as well as changes in the fatty acyl-CoA's of the cells. Therefore, it is clear that Cer subspecies change during embryogenesis, possibly for functionally important reasons. One CerS isoform, CerS2, was studied further because it has the broadest tissue distribution and a remarkable fatty acyl-CoA specificity, utilizing longer acyl-chain CoAs (C20-C26) in vitro. The fatty acid chain selectivity was refined by analysis of the Cer from livers from CerS2 null mice, which displayed very little Cer with fatty acyl chains with 24 + 2 carbons. Another interesting structural variation was discovered in studies of cells treated with fumonisin B1 (FB1), which inhibits CerS. Under these conditions, cells in culture and animals accumulate substantial amounts of a novel sphingoid base that was identified as 1-deoxysphinganine. This compound arises from utilization of L-alanine instead of L-serine by serine palmitoyltransferase (SPT) based on the inability of LYB cells, which lack SPT, to make 1-deoxysphinganine. In the absence of FB1, 1-deoxysphinganine is primarily acylated to 1-deoxydihydroceramides. These are an underappreciated category of bioactive sphingoid bases and "ceramides" that might play important roles in cell regulation and disease. In summary, cells contain a wide variety of Cer subspecies that are determined by changes in expression of CerS, enzymes that produce co-substrates (such as fatty acyl-CoAs), and the types of amino acids utilized by SPT, the initial enzyme of de novo sphingolipid biosynthesis. One can envision how these changes might impact membranes structure as well as signaling by this family of highly bioactive compounds.

View
Thumbnail Image
Item

Transactivation of Beta 2 Adrenergic Receptor by Bradykinin type 2 Receptor via heterodimerization

2009-11-10 , Vincent, Karla Kristine

Although a long standing convention maintained that G Protein Coupled Receptors (GPCRs) exist in the plasma membrane solely as monomers, substantial work over the last two decades has demonstrated that these ubiquitous receptors can and in many cases, preferentially, exist as homodimers, heterodimers, or higher order oligomers. Often, two GPCRs of the same class heterodimerize; it is less common for two GPCRs of different signaling pathways to interact. The work presented here studied the physical and functional interaction of two GPCRs from discrete classes, the Beta 2 Adrenergic Receptor (β2AR), a Gαs-coupled receptor, and Bradykinin type 2 Receptor (Bk2R), a Gαq coupled receptor. These data show that Bk2R and β2AR are physically coupled when heterologously expressed in Xenopus oocytes, and in pheochromocytoma (PC12) cells and in freshly isolated murine ventricular myocytes, two systems that endogenously express these receptors. This physical coupling led to functional consequences in heterologous and endogenous expression systems, as Bk2R was able to transactivate β2AR signaling via its direct interaction with the receptor. Furthermore, coexpression of Bk2R shifted the dose response curve of β2AR for its selective agonist rightward in Xenopus oocyte electrophysiology experiments, suggesting the presence of Bk2R negatively affected β2AR native pharmacology. Up to thirty minutes of either bradykinin (BK) or isoproterenol exposure did not change the relative amount of Bk2R/β2AR heterodimer in PC12 cells, a rat adrenal medulla tumor cell line that endogenously expresses these receptors. Despite the obvious signaling consequences, the Bk2R/β2AR heterodimer accounted for only 10% of the total β2AR protein detected and 20% of the total Bk2R protein detected. When other Bk2R-specific ligands were also tested to examine the extent of β2AR transactivation, our data showed that both Lys-des-Arg-Bradykinin, a Bk2R partial agonist and NPC 567, a Bk2R antagonist, transactivated β2AR to the same extent as BK. Taken together, our data provide a novel mode of receptor regulation and signaling via Bk2R/β2AR heterodimerization. Because a large percentage of therapeutics target GPCRs, a greater understanding of how a GPCR heterodimer functions could be beneficial for targeting new drugs and refining existing drugs. Understanding the Bk2R/β2AR heterodimer provides a new perspective on the myriad of fucntional consequences that occur when a GPCR is activated.

View
Thumbnail Image
Item

Genetic elements and molecular mechanisms driving the evolution of the pathogenic marine bacterium Vibrio parahaemolyticus

2009-07-06 , Hazen, Tracy Heather

Vibrio parahaemolyticus is an opportunistic human pathogen that occurs naturally in a non-pathogenic form in coastal estuarine and marine environments worldwide. Following the acquisition of virulence-associated genes, V. parahaemolyticus has emerged as a significant pathogen causing seafood-borne illnesses. The mechanisms and conditions that promote the emergence of disease causing V. parahaemolyticus strains are not well understood. In addition, V. parahaemolyticus clinical strains isolated from disease-associated samples and environmental strains from sediment, water, and marine organisms have been identified with considerable diversity; however, the evolutionary relationships of disease-causing strains and environmental strains are not known. In the following research, the evolutionary relationships of V. parahaemolyticus clinical and environmental strains are examined. In addition, the contribution of genetic elements and molecular mechanisms such as deficiency of DNA repair to the evolution of V. parahaemolyticus clinical and environmental strains is shown. Molecular analysis of the evolutionary relationships of V. parahaemolyticus clinical and environmental strains demonstrated separate lineages of pathogenic and non-pathogenic strains with the exception of several environmental strains that may represent a reservoir of disease-causing strains in the environment. Sequence characterization of plasmids isolated from diverse environmental Vibrios indicated a role of plasmids in strain evolution by horizontal transfer of housekeeping genes. In addition, analysis of plasmids from V. parahaemolyticus clinical and environmental strains indicated the existence of a plasmid family distributed among V. parahaemolyticus, V. campbellii, and V. harveyi environmental strains. Sequence characterization of a plasmid of this family from a V. parahaemolyticus environmental strain indicated the contribution of these plasmids to the emergence of the clonal pandemic strains. Investigation of the role of molecular mechanisms to the evolution of V. parahaemolyticus strains showed that inactivation of the DNA repair pathway methyl-directed mismatch repair (MMR) increased the accumulation of spontaneous mutations leading to increased nucleotide diversity in select genes. The research findings in the following chapters demonstrate a considerable contribution of genetic elements and molecular mechanisms to the evolution of genetic and phenotypic diversity.

View
Thumbnail Image
Item

The effects of plume property variation on odor plume navigation in turbulent boundary layer flows

2009-05-13 , Page, Jennifer Lynn

A significant body of research has focused on tracking behaviors of predators responding to prey odor plumes, yet little is known about the specific mechanisms by which predators make decisions during tracking that lead them to a source. This dissertation advances the current knowledge of plume tracking behavior by examining blue crab tracking behavior over a large range of bed-roughnesses (thereby manipulating ambient levels of turbulence), and interpreting these results with respect to chemical signal structure derived from separate examinations of plume characteristics as determined by planar laser induced fluorescence (PLIF). Foraging success and the speed of blue crabs attempting to locate the odorant source both decline consistently with increasing bed roughness. In contrast, steering (path linearity) appears unaffected by bed roughness induced turbulence. The spatial arrangement of blue crab chemosensors combined with the three-dimensional structure of odorant plumes accounts for the differential effects of turbulence on the speed and success of crab tracking behavior. Separate examinations of tracking behavior and plume properties cannot directly examine hypotheses concerning the utility of specific chemical signal properties. In order to make a direct link between cue and behavior, three-dimensional laser induced fluorescence (3DLIF) was used to analyze three-dimensional plume structure and concentration of odor filaments that reach blue crab sensory structures. The corresponding tracking behavior was simultaneously recorded and then analyzed with a motion analysis system. These data provide the most comprehensive examination of odor signal input-behavioral output functions for animals in turbulent plumes. Crabs do not react differentially in response to the absolute concentration of antennule spikes above threshold at their antennules but do show a state-dependent acceleration response to antennule spikes. Signals arriving at the leg sensors of blue crabs help mediate upstream motion and signal change across a single set of leg sensors is sufficient to induce turning during upstream motion. Blue crabs decrease the height of their antennules in correspondence with changing plume properties as they approach the source and the timing of signals arriving at the antennules appears to affect upstream motion.

View