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End date: 2009 × Start date: 2000 × search.filters.applied.f.isOrgUnitOfPublicationTitle: College of Sciences × search.filters.applied.f.isOrgUnitOfPublicationTitle: School of Biological Sciences × search.filters.applied.f.resourcesubtype: Thesis ×

Publication Search Results

Now showing 1 - 10 of 23
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Bone marrow regeneration follwing tibial marrow ablation in rats is age dependent

2008-11-19 , Fisher, Maya

Objective: Injuries to the marrow cavity result in rapid bone formation followed by regeneration of the marrow. It is not known whether this process is affected by age, although the quality of marrow is markedly different in young and old animals. To test if marrow restoration differs with age, we used the rat tibial bone marrow ablation model, which has been used to examine calcification, osteointegration of metal implants, and remodeling of bone graft substitutes. Methods: Marrow was ablated in the left tibia of seven rats (rNu/rNu) per time point. At 0,7,14,21,28,35 and 42 days post-surgery, treated tibias and contralateral tibias were harvested and fixed in buffered formalin. Both tibias were scanned using microCT and trabecular and cortical BVF/TV calculated. Mid-sagittal sections of decalcified bones were stained with H&E and BVF/TV calculated. Results: MicroCT analysis of 1-month animals showed increased bone formation on day-7 and on day-21 the marrow was restored. Increased bone was seen in 3-month animals on day-7 and day-14, but it was significantly less than in 1-month rats. By day-21, trabecular bone was reduced by 50%. 10-month animals had less trabecular bone at day-7 and 14, but bone remained in the medullary canal through day-1. Histomorphometry indicated that bone formation peaked at day-7 in 1-month rats with remodeling underway by day-14. Bone formation in 3-month rats also peaked at day-7, but restoration occurred by day-21. However, in 10-month rats, peak bone occurred on day-14, with remodeling on day-28. Conclusions: Aged animals produced less primary bone than younger animals and remodeling was initiated later. Differences in micro-CT and histomorphometric analyses may reflect a reduction in calcification of the osteoid in the 10-month old animals. (Supported by Boston Scientific, Inc.)

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Effects of the components of the Get pathway on prion propagation

2007-11-15 , Bariar, Bhawana

Yeast prions e.g. [PSI+], [PIN+] and [URE3] are similar to mammalian amyloids that cause neurodegenerative diseases. [PSI+] is the aggregated self-perpetuating (prion) isoform of Sup35, a translation termination factor. The molecular chaperone Hsp104 plays a crucial role in the maintenance and propagation of [PSI+]. Deletion of the GET2 gene has been shown to cause a [PSI+] curing defect by excess Hsp104 and [PSI+] instability on synthetic medium (S. Muller, J. Patterson and Y. Chernoff, unpublished data; and J. Patterson Honors Thesis). Get2 is a membrane protein working in a complex with Get1 and Get3 proteins. This complex, called GET (Golgi-to-ER Traffic), is known to retrieve resident ER proteins from Golgi. In this particular study we provide further evidence for the connection between the GET pathway and yeast prions. The get2 deletion also leads to a detectable loss of [PIN+] prion on synthetic medium. The role of the other two members of the Get complex in prion propagation is also explored. The levels and the activity of Hsp104 in the get2 mutants is analyzed. The size of [PSI+] aggregates in the get2Δ strain is compared to that found in wild type. Finally, other possible mechanisms for the effect of get2 on prion maintenance and propagation are addressed.

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Chemical cues affecting susceptibility of gorgonian corals to fungal infection

2005-11-28 , Hicks, Melissa Kathryn

Coral diseases have become more prevalent and destructive over the past 20 years, possibly due to an increase in stressful environmental factors that may weaken corals defenses against disease. Aspergillosis is a disease caused by the fungus Aspergillus sydowii, which apparently infects only two species of gorgonian corals in the Caribbean Ocean (Gorgonia ventalina and G. flabellum). We hypothesized that the differential resistance to infection is caused by differences in chemical defenses among gorgonians. Freeze-dried gorgonian powders and extracts deterred fungal growth, but potencies varied among gorgonian species and among fungi. Extracts and powders generated from G. ventalina all strongly inhibited fungal growth. Since G. ventalina was predicted to have weak antifungal chemical defenses compared to gorgonians not known to suffer from aspergillosis, we concluded that gorgonian susceptibility to fungal infection is determined by factors other than, or in addition to, chemical defenses. In order to investigate specific gorgonian antifungal strategies, we attempted to use bioassay-guided fractionation to isolate antifungal compounds from four gorgonians: Gorgonia ventalina, Briareum asbestinum, Eunicea succinea, and Pseudopterogorgia americana. We succeeded in isolating two antifungal compounds, diastereomers of 9,11-seco-24-hydroxydinosterol, from the gorgonian Pseudopterogorgia americana. This compound was previously identified by other groups, but this study is the first to establish its antifungal activity. At natural concentration, one diastereomer of 9,11-seco-24-hydroxydinosterol inhibited the growth of three different fungi, suggesting that at least this diastereomer may possess broad-spectrum antifungal activity. The results from our survey of gorgonian chemical defenses indicate that susceptibility to aspergillosis cannot be explained by chemical growth inhibition alone. Further areas of investigation include induction of gorgonian chemical defenses, examination of growth-inhibiting mechanisms of antifungal metabolites, and identification of non-chemical factors affecting gorgonians vulnerability to fungal infection.

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Interactions of the chaperones and components of UB system in the formation and propagation of the yeast prion [PSI+]

2005-06-28 , Tennant, Esther Paula

Three of the best-characterized prions of Saccharomyces cerevisiae are [PSI+], [URE3], and [PIN+]. This study focuses on the prions [PSI+] and [PIN+]. [PSI+] is the prion isoforms of the protein Sup35 that functions as a eRF3 translational termination factor. The presence of [PSI+] is detected by the partial loss of function of Sup35. The prion [PIN+] is the isoform of the protein Rnq1, and this proteins function is unknown. The presence of the prion [PIN+] is necessary for the de novo formation of the prion [PSI+] (Derkatch et al., 1997). The chaperone, Hsp104, belongs to an evolutionary conserved Hsp100 family of proteins that participate in a various number of cellular processes (Schirmer et al., 1996). Hsp104, in particular, is responsible for the cells adaptation to heat shock, it controls spore viability and the long-term viability of starving vegetative cells. (Sanchez and Linquist, 1990; Sanchez et al.,1992) It is an ATPase that has been shown to promote solubilization of aggregated protein (Parsel et al., 1991). A unique relationship exists between Hsp104 levels within the cell and the maintenance of the prion [PSI+]. The over production of Hsp104 eliminates [PSI+] (Chernoff 1995). This seems logical considering Hsp104 is a disaggregase, and it is reasonable to assume that the over production provides sufficient resources to break the aggregates into portions that are accessible to either other chaperones which would facilitate the proper folding or perhaps the system responsible for the elimination of unusable proteins, such as the ubiquitin-proteasome system. This study examines the role of the ubiquitin-proteasome system in curing of [PSI+] by Hsp104. The role of alternate pathways, in which the prion isoform is refolded into it correct, functional conformation by the action of the chaperones Ssb1 and Ssb2 is examined. These results suggest that the combination of both the degradation pathway and the refolding of proteins are involved in curing of [PSI+] by Hsp104 over production.

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Molecular Characterization of Microbial Communities Fouling Concrete Infrastructures

2008-07-10 , Giannantonio, David John

The objective of this study was to identify and characterize naturally-occurring communities of Bacteria and Fungi fouling the surfaces of concrete structures in Georgia, USA, through the use of culture-independent and culture-dependent approaches. Genomic DNA was extracted and ribosomal RNA genes were PCR amplified from 4 biofouled sites located in or around the cities of Atlanta, Gainesville, LaGrange, and Savannah. Bacterial and fungal community composition was determined by phylogenetic analysis. Molecular analysis revealed five bacterial phyla, and representatives of the phylum Cyanobacteria and the classes Betaproteobacteria and Gammaproteobacteria dominated the bacterial clone libraries. Fungal clone libraries showed the dominant phylotypes to be most closely related to Alternaria, Cladosporium, Epicoccum and Udeniomyces. Phylogenetically distinct microbial populations were present at each of the biofouled sites. In addition, cultured isolates were obtained from sites and tested for their ability to foul concrete of varied compositions under laboratory-controlled conditions. Biofouling tests revealed that fungal isolates obtained from the field were able to colonize concrete surfaces when supplied with moisture (95-100% relative humidity) and a nutrient source, and that fouling was affected by concrete water/cement ratio, surface roughness, and the presence of photocatalytically-activated cement added to inhibit microbial growth.

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Regulation of Galactosylceramide Biosynthesis

2006-07-12 , Kollmeyer, Jessica Elaine

An important branchpoint of mammalian sphingolipid metabolism occurs at the step where ceramides are glycosylated to glucosylceramide (GlcCer) versus galactosylceramide (GalCer), which are precursors of all mammalian glycosphingolipids. Relatively few studies have focused on this branchpoint because these monohexosylceramides are somewhat difficult to resolve chromatographically and because molecular biology tools have only recently become available to follow expression of these genes. The goal of this thesis is to better understand the mechanisms of cell regulation determining galactosylceramide synthesis.

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The Role of Sphingolipids in Cortisol Synthesis in the Adrenal Cortex

2005-11-27 , Ozbay, Tuba Selcuk

In the human adrenal cortex, adrenocorticotropin (ACTH) activates steroid hormone biosynthesis by acutely increasing cholesterol delivery to the mitochondria and chronically up-regulating the transcription of steroidogenic genes (including CYP17). Sphingolipids are a diverse family of phospholipids and glycolipids that mediate a wide variety of cellular processes, including apoptosis, proliferation, and survival. Sterol regulatory element binding proteins (SREBPs) are a family of transcription factors that regulate genes that are involved in cholesterol biosynthesis and fatty acid metabolism. In this study, we investigated the role of sphingolipids in ACTH-dependent steroidogenesis. H295R human adrenocortical cells were treated with ACTH or dibutyryl cAMP (Bt2cAMP) for various time periods and the content of several sphingolipid species was quantified by mass spectrometry. Both ACTH and Bt2cAMP decreased cellular amounts of sphingomyelin, ceramides, sphingosine (So) and sphingosine-1-phosphate (S1P). However, both ACTH and Bt2cAMP increased the activity of sphingosine kinase and the amounts of S1P released into the media. Both So and S1P increased CYP17 mRNA expression and increased cortisol biosynthesis. This increase in CYP17 transcription occurs by promoting SREBP binding to an SRE at -450/-436 basepairs upstream of the transcription initiation site. Furthermore, chromatin immunoprecipitation (ChIP) assays revealed that Bt2cAMP and S1P treatment results in an increase in acetylation of histone H3 and SREBP1 binding to CYP17 promoter. Additionally, transient transfection studies using wild type or mutated hCYP17 promoters and RNA interference (RNAi) assays confirmed the role of SREBP1 in mediating the stimulatory effect of S1P on CYP17 transcription. In summary, our studies demonstrate a link between sphingolipid metabolism and ACTH-dependent steroidogenesis which requires the activation of SREBP1 in human adrenal cortex.

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Contribution of Nitrogen Fixation to Planktonic Food Webs North of Australia

2007-11-16 , Drexel, Jan Peter

Nitrogen fixation is no longer considered to be a minor factor of the nitrogen cycle in oceanic ecosystems. Recent geochemical and biological efforts have led to a significant increase in the estimated input of nitrogen to marine ecosystems by biological fixation, while molecular studies have increased our knowledge of the number and diversity of nitrogen fixers known to be active in the ocean. Although Trichodesmium spp. have long been viewed as the primary marine nitrogen fixers, recent efforts have shown that various members of the picoplankton community are also actively involved in nitrogen fixation. The relative abundance of different nitrogen fixers is an important ecosystem parameter since nitrogen fixers may differ significantly in their physiology, life history and ecology. Here we combine rate measurements and stable isotope natural abundance measurements to constrain the impact of N2 fixation in the waters north of Australia. Samples were collected in the Coral, Arafura, and East Timor Seas, thus spanning three distinct hydrographic regions. Our data show that Trichodesmium has a significant influence on the stable nitrogen isotope ratios of particulate and zooplankton biomass and suggest that Trichodesmium is a significant source of nitrogen for the pelagic ecosystem. Based on stable carbon isotope ratios, it is also likely that the pathways are indirect and nitrogen fixed by Trichodesmium enters the higher trophic levels via decomposition as dissolved organic and inorganic nitrogen. Picocyanobacteria showed high diazotrophic activity at some stations, but unlike Trichodesmium, their N2 fixation rate was not reflected in the stable N isotope ratios of particulate and zooplankton biomass. Our results suggest an important N contribution to biomass by diazotrophs in the Coral Sea, Arafura Sea and East Timor Sea.

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Fenretinide increases dihydroceramide and dihydrosphingolipids due to inhibition of dihydroceramide desaturase.

2006-07-11 , Zheng, Wenjing

N-(4-Hydroxyphenyl) retinamide (4-HPR) is a derivative of all-trans-retinoic acid that induces apoptosis in cancer cell lines and is being tested in clinical trials as a relatively non-toxic anti-cancer agent. 4-HPR induces de novo sphingolipid biosynthesis and production of ceramide has been suggested to contribute to the growth arrest and apoptosis. To characterize the types of ceramides that might be involved, we used liquid chromatography, electrospray ionization tandem mass spectrometry (LC ESI-MS/MS) to analyze the sphingolipids, and found that 4-HPR increased total sphingolipid amounts, but unexpectedly, ceramides (i.e., N-acylsphingosines) changed very little, and in some cases decreased. Instead, dihydroceramides (i.e., N-acylsphinganines) increased as much as 10-fold, both as the free species and as the backbones of dihydrosphingomyelins and dihydrohexosylceramides. To determine if 4-HPR inhibits dihydroceramide desaturase, we synthesized NBD-dihydroceramide and treated Hek293 cells with 4-HPR and analyzed the metabolites by HPLC. These analyses showed that NBD-dihydroceramide was taken up by the cells and converted to NBD-ceramides and more complex NBD-sphingolipids in control cells, however, within one hour of treatment with 10 ~{ and L~}M 4-HPR, the production of NBD-ceramide was blocked. In vitro assays of the desaturase using NBD-dihydroceramide also showed rapid and complete inhibition by 4-HPR. Interestingly, when Hek cells were treated with 4-HPR for one hour then the medium was changed, the recovery of dihydroceramide desaturase activity was very slow (i.e., t1/2 > 66 h); therefore, either 4-HPR is difficult to remove from cells or the inhibition is essentially irreversible. These findings establish that 4-HPR not only induces de novo sphingolipid biosynthesis but also inhibits dihydroceramide desaturase, resulting in production of abnormally high proportions of sphingolipids with dihydroceramide as the backbone. This raises the possibility that some of the effects of 4-HPR on cell behavior may be due to the presence of these abnormal species.

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Reciprocal binding of sphingosine and phosphatidic acid to steroidogenic factor 1 regulates the transcription of CYP17

2005-11-22 , Urs, Aarti N.

Steroidogenic factor (SF1) is an orphan nuclear receptor that is essential for steroid hormone-biosynthesis and endocrine development. Recent studies have demonstrated that phospholipids are ligands for SF1. In the present study our aim was to identify endogenous ligands for SF1 and characterize their functional significance in mediating cAMP-dependent transcription of human CYP17. Using mass spectrometry we show that in H295R adrenocortical cells SF1 is bound to sphingosine (SPH) under basal conditions and that cAMP stimulation decreases the amount of SPH bound to the receptor. We also show that silencing both acid and neutral ceramidases using siRNA induces CYP17 mRNA expression, suggesting that SPH acts as an inhibitory ligand. In vitro analysis of ligand binding using scintillation proximity assays show that several sphingolipids and phospholipids, including phosphatidic acid (PA), can compete with [3H]SPH for binding to SF1, suggesting that SF1 may have more than one ligand and binding specificity may change with the changes in intracellular fluxes of phospholipids. Further, phosphatidic acid (PA) induces SF1-dependent transcription of CYP17 reporter constructs. Inhibition of diacyglycerol kinase (DAGK) activity using R59949 and silencing DAGK- expression attenuates SF1-dependent CYP17 transcriptional. We propose that PA is an activating ligand for SF1 and that cAMP-stimulated activation of SF1 takes place by displacement of SPH.

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