This project was developed to identify novel methods for high-throughput culturing and screening of C. elegans to investigate age-related metabolic changes and to survey the proteomic and metabolomic factors associated with age-related changes. To accomplish these goals we developed a novel way to grow C. elegans in liquid culture in 96-well microplates for several weeks without suffering significant fluid loss due to evaporation and without needing to shake or unseal the plates for aeration. We also developed methods for assaying the total volume of live C. elegans in microplate cultures using a fluorescence microplate reader and for performing RNAi experiments with dead instead of live bacteria, which allows for the measurement of nematode metabolic parameters without bacterial interference. Using these methods, along with established methods for the global identification of metabolites and proteins by mass spectroscopy, we observed an integrated pattern of changes that occurred at the molecular level in aged C. elegans. Specifically, we found protein changes suggesting muscle dysfunction and sarcopenia, an increase in free fatty acids, a decrease in the S-adenosylmethionine cycle, altered or impaired protein synthesis, changes in free amino acid levels consistent with an increase in cell size, indications of epigenetic changes and alteration of DNA repair, and a shift toward a more oxidizing cellular environment, as well as a decrease in NAD+ relative to NADH. Through the use of an automated RNAi screen targeting potential EF-hand Ca2+ binding proteins, we identified genes that are associated with high culture medium Ca2+ toxicity. In addition, from a screen of X chromosome RNAi clones, we identified clones that partially prevented the age-dependent decline in nematode ATP levels, oxygen consumption, and reductive capacity. The set of genes targeted by these RNAi clones is enriched in both anti-longevity genes and negative regulators of cellular processes and are potential targets for anti-aging interventions.

Structured RNAs are pervasive in biology, spanning a functional repertoire that includes messengers, regulators of gene expression and catalysts of translation and splicing. From the relatively simple tRNAs and riboswitches to the highly structured ribosomal RNAs, the ability of RNAs to function is dependent on well-defined secondary and tertiary structures. However, studies of RNA folding in vitro have revealed an extreme propensity to form alternative structures, which can be long-lived and interfere with function. In the cell, a diverse array of RNA binding proteins and RNA chaperones guide RNAs towards the correct structure and disrupt misfolded intermediates. Among these proteins, DEAD-box protein family stands out as one of the largest groups, with its members ubiquitously involved in RNA metabolism across all domains of life. DEAD-box proteins can function as both specific and general RNA chaperones by disrupting RNA structures in an ATP-dependent manner. Here I describe my work studying the general RNA chaperone mechanism of the Neurospora crassa protein CYT-19, a model DEAD-box protein and a biological RNA chaperone that is required for efficient folding of self-splicing group I intron RNAs in vivo. After an introduction to DEAD-box proteins and their mechanisms as RNA remodelers (Chapter 1), I will first describe studies of group I intron unfolding by CYT-19, focusing on the effects of RNA tertiary structure stability on CYT-19 activity and targeting to RNA substrates (Chapter 2). I will then describe the characterization of ATP-dependent mechanisms during CYT-19-mediated refolding of the misfolded group I intron (Chapter 3). In Chapter 4, I will present small-angle X-ray scattering (SAXS) studies of structural features of DEAD-box proteins that allow them to efficiently interact with large structured RNA substrates. Finally, I will turn to studies of DEAD-box protein involvement during early steps of RNA compaction and folding, using SAXS and activity-based approaches (Chapter 5). I will conclude with a general discussion of superfamily 2 RNA helicases, which include DEAD-box and related proteins, and their functions and mechanisms as remodelers of structured RNAs and RNPs. / text

<p><font size="3"> <p>The blood-brain barrier (BBB) is an interface between the brain parenchyma and the circulating system. This barrier plays a vital role in protecting the CNS by restricting free paracellular diffusion of molecules from the systemic circulation. Methamphetamine (MA) is a highly addictive psychostimulant and has demonstrated neurotoxic properties as well as the ability to compromise the BBB. MA exposure is strongly linked with increased oxidative stress which can result in a decrease in the integrity of the BBB.<font size="3">The aim of this study was to investigate </font><i><font size="3" face="Times New Roman,Times New Roman"><font size="3" face="Times New Roman,Times New Roman">in vitro </font></font><font size="3">effects of pure and street MA &quot / tik&quot / on DNA proliferation and cell cycles in mouse brain endothelial (bEnd5) cells. <font size="3">Trypan blue was used to determine effects of MA (0.0001M-1mM) on cell viability and % cell growth. The Cell Titer Glo&reg / luminescent assay and nonradioactive analogue, 5-bromo-2'-deoxyuridine (BrdU) was used to detect ATP and DNA levels, respectively. Cell cycles (propidium iodide incorporation) were analysed using flow cytometry. Statistical analysis was performed using Wilcoxin Rank Sum Test in which P&lt / 0.05 was denoted as significant. <font size="3">Results of this study showed that:&nbsp / <font size="3">1. Viability of bEnd5 cells exposed to all selected concentrations of MA were unaffected when compared to controls (P&gt / 0.05). <font size="3">2. % Cell growth was suppressed by MA exposure at 96hrs in comparison to that of controls (P&le / 0.03). 3. Cells exposed to MA had significant higher ATP concentrations than control cells at 96hrs (P &le / .0.03) 4. DNA synthesis was markedly suppressed in cells exposed to pure MA and street MA sample 4 (P&le / 0.03), while was similar and higher in cells exposed to street MA sample 1 (P=0.39), and street MA sample 2 and 3 (P&le / 0.04), respectively at 96hrs. 5. bEnd5 cell were arrested between 72 and 96hrs at the G1-S phase. <font size="3">In conclusion, this study demonstrated pure and illicit samples of MA obtained from forensic police did not affect the viability of bEnd5 cells, however resulted in the significant suppression of their cell numbers. This growth inhibition may be due to MA-induced cell cycle arrest at the G1-S phase. The study also showed that compounds found in the samples of street MA produced results significantly different to that of pure MA. </font></font> <p>&nbsp / </p> </font>&nbsp / </font></font></font> <p>&nbsp / </p> </i></p> </font></p> <p>&nbsp / </p>

<p><font size="3"> <p>The blood-brain barrier (BBB) is an interface between the brain parenchyma and the circulating system. This barrier plays a vital role in protecting the CNS by restricting free paracellular diffusion of molecules from the systemic circulation. Methamphetamine (MA) is a highly addictive psychostimulant and has demonstrated neurotoxic properties as well as the ability to compromise the BBB. MA exposure is strongly linked with increased oxidative stress which can result in a decrease in the integrity of the BBB.<font size="3">The aim of this study was to investigate </font><i><font size="3" face="Times New Roman,Times New Roman"><font size="3" face="Times New Roman,Times New Roman">in vitro </font></font><font size="3">effects of pure and street MA &quot / tik&quot / on DNA proliferation and cell cycles in mouse brain endothelial (bEnd5) cells. <font size="3">Trypan blue was used to determine effects of MA (0.0001M-1mM) on cell viability and % cell growth. The Cell Titer Glo&reg / luminescent assay and nonradioactive analogue, 5-bromo-2'-deoxyuridine (BrdU) was used to detect ATP and DNA levels, respectively. Cell cycles (propidium iodide incorporation) were analysed using flow cytometry. Statistical analysis was performed using Wilcoxin Rank Sum Test in which P&lt / 0.05 was denoted as significant. <font size="3">Results of this study showed that:&nbsp / <font size="3">1. Viability of bEnd5 cells exposed to all selected concentrations of MA were unaffected when compared to controls (P&gt / 0.05). <font size="3">2. % Cell growth was suppressed by MA exposure at 96hrs in comparison to that of controls (P&le / 0.03). 3. Cells exposed to MA had significant higher ATP concentrations than control cells at 96hrs (P &le / .0.03) 4. DNA synthesis was markedly suppressed in cells exposed to pure MA and street MA sample 4 (P&le / 0.03), while was similar and higher in cells exposed to street MA sample 1 (P=0.39), and street MA sample 2 and 3 (P&le / 0.04), respectively at 96hrs. 5. bEnd5 cell were arrested between 72 and 96hrs at the G1-S phase. <font size="3">In conclusion, this study demonstrated pure and illicit samples of MA obtained from forensic police did not affect the viability of bEnd5 cells, however resulted in the significant suppression of their cell numbers. This growth inhibition may be due to MA-induced cell cycle arrest at the G1-S phase. The study also showed that compounds found in the samples of street MA produced results significantly different to that of pure MA. </font></font> <p>&nbsp / </p> </font>&nbsp / </font></font></font> <p>&nbsp / </p> </i></p> </font></p> <p>&nbsp / </p>

<p><span style="font-size: 11.5pt">The blood-brain barrier (BBB) is an interface between the brain parenchyma and the circulating system. This barrier plays a vital role in protecting the CNS by restricting free paracellular diffusion of molecules from the systemic circulation. Methamphetamine (MA) is a highly addictive psychostimulant and has demonstrated neurotoxic properties as well as the ability to compromise the BBB. MA exposure is strongly linked with increased oxidative stress which can result in a decrease in the integrity of the BBB. </span></p> <div><span style="font-size: 11.5pt">The aim of this study was to investigate <i>in vitro </i>effects of pure and street MA &ldquo / tik&rdquo / on DNA proliferation and cell cycles in mouse brain endothelial (bEnd5) cells. </span></div> <div><span style="font-size: 11.5pt">Trypan blue was used to determine effects of MA (0.0001M-1mM) on cell viability and % cell growth. The Cell Titer Glo&reg / luminescent assay and nonradioactive analogue, 5-bromo-2'-deoxyuridine (BrdU) was used to detect ATP and DNA levels, respectively. Cell cycles (propidium iodide incorporation) were analysed using flow cytometry. Statistical analysis was performed using Wilcoxin Rank Sum Test in which P&lt / 0.05 was denoted as significant. </span></div> <div><span style="font-size: 11.5pt">Results of this study showed that: </span></div> <div><span style="font-size: 11.5pt">1. Viability of bEnd5 cells exposed to all selected concentrations of MA were unaffected when compared to controls (P&gt / 0.05)&nbsp / </span><span style="font-size: 11.5pt">&nbsp / </span></div> <div><span style="color: windowtext / font-size: 11.5pt">2. % Cell growth was suppressed by MA exposure at 96hrs in comparison to that of controls (P&le / 0.03). </span></div> <div style="margin: 0cm 0cm 25pt"><span style="color: windowtext / font-size: 11.5pt">3. Cells exposed to MA had significant higher ATP concentrations than control cells at 96hrs (P &le / .0.03) </span><span style="color: windowtext / font-size: 11.5pt">4. DNA synthesis was markedly suppressed in cells exposed to pure MA and street MA sample 4 (P&le / 0.03), while was similar and higher in cells exposed to street MA sample 1 (P=0.39), and street MA sample 2 and 3 (P&le / 0.04), respectively at 96hrs. </span><span style="color: windowtext / font-size: 11.5pt">5. bEnd5 cell were arrested between 72 and 96hrs at the G1-S phase.&nbsp / </span></div> <div style="margin: 0cm 0cm 25pt"><span style="line-height: 115% / font-size: 11.5pt">In conclusion, this study demonstrated pure and illicit samples of MA obtained from forensic police did not affect the viability of bEnd5 cells, however resulted in the significant suppression of their cell numbers. This growth inhibition may be due to MA-induced cell cycle arrest at the G1-S phase. The study also showed that compounds found in the samples of street MA produced results significantly different to that of pure MA.</span></div>

No description available.

<p><span style="font-size: 11.5pt">The blood-brain barrier (BBB) is an interface between the brain parenchyma and the circulating system. This barrier plays a vital role in protecting the CNS by restricting free paracellular diffusion of molecules from the systemic circulation. Methamphetamine (MA) is a highly addictive psychostimulant and has demonstrated neurotoxic properties as well as the ability to compromise the BBB. MA exposure is strongly linked with increased oxidative stress which can result in a decrease in the integrity of the BBB. </span></p> <div><span style="font-size: 11.5pt">The aim of this study was to investigate <i>in vitro </i>effects of pure and street MA &ldquo / tik&rdquo / on DNA proliferation and cell cycles in mouse brain endothelial (bEnd5) cells. </span></div> <div><span style="font-size: 11.5pt">Trypan blue was used to determine effects of MA (0.0001M-1mM) on cell viability and % cell growth. The Cell Titer Glo&reg / luminescent assay and nonradioactive analogue, 5-bromo-2'-deoxyuridine (BrdU) was used to detect ATP and DNA levels, respectively. Cell cycles (propidium iodide incorporation) were analysed using flow cytometry. Statistical analysis was performed using Wilcoxin Rank Sum Test in which P&lt / 0.05 was denoted as significant. </span></div> <div><span style="font-size: 11.5pt">Results of this study showed that: </span></div> <div><span style="font-size: 11.5pt">1. Viability of bEnd5 cells exposed to all selected concentrations of MA were unaffected when compared to controls (P&gt / 0.05)&nbsp / </span><span style="font-size: 11.5pt">&nbsp / </span></div> <div><span style="color: windowtext / font-size: 11.5pt">2. % Cell growth was suppressed by MA exposure at 96hrs in comparison to that of controls (P&le / 0.03). </span></div> <div style="margin: 0cm 0cm 25pt"><span style="color: windowtext / font-size: 11.5pt">3. Cells exposed to MA had significant higher ATP concentrations than control cells at 96hrs (P &le / .0.03) </span><span style="color: windowtext / font-size: 11.5pt">4. DNA synthesis was markedly suppressed in cells exposed to pure MA and street MA sample 4 (P&le / 0.03), while was similar and higher in cells exposed to street MA sample 1 (P=0.39), and street MA sample 2 and 3 (P&le / 0.04), respectively at 96hrs. </span><span style="color: windowtext / font-size: 11.5pt">5. bEnd5 cell were arrested between 72 and 96hrs at the G1-S phase.&nbsp / </span></div> <div style="margin: 0cm 0cm 25pt"><span style="line-height: 115% / font-size: 11.5pt">In conclusion, this study demonstrated pure and illicit samples of MA obtained from forensic police did not affect the viability of bEnd5 cells, however resulted in the significant suppression of their cell numbers. This growth inhibition may be due to MA-induced cell cycle arrest at the G1-S phase. The study also showed that compounds found in the samples of street MA produced results significantly different to that of pure MA.</span></div>

The KATP channel is a key regulator of the pancreatic β-cell, effectively linking metabolic status to electrical activity. Syntaxin-1A has been previously reported by our lab to both bind and inhibit the KATP channel via the nucleotide-binding folds (NBFs). The purpose of this thesis project was to elucidate the precise regions within the NBFs responsible for the Syn-1A- KATP interaction. In vitro binding assays revealed that Syn-1A associates with the Walker domains of both NBF1 and NBF2. Furthermore, site directed mutagenesis of the conserved lysine in Walker A of both NBFs abolishes Syn-1A affinity for this region. Electrophysiological recordings indicate that channel inhibition was mediated primarily through interaction with NBF1-Walker B and both Walkers of NBF2. Based on these results, we propose a model by which Syn-1A acts as an inhibitory clamp on the KATP channel, effectively buffering minor fluctuations in ATP/ADP concentration to prevent unnecessary channel activity.

The KATP channel is a key regulator of the pancreatic β-cell, effectively linking metabolic status to electrical activity. Syntaxin-1A has been previously reported by our lab to both bind and inhibit the KATP channel via the nucleotide-binding folds (NBFs). The purpose of this thesis project was to elucidate the precise regions within the NBFs responsible for the Syn-1A- KATP interaction. In vitro binding assays revealed that Syn-1A associates with the Walker domains of both NBF1 and NBF2. Furthermore, site directed mutagenesis of the conserved lysine in Walker A of both NBFs abolishes Syn-1A affinity for this region. Electrophysiological recordings indicate that channel inhibition was mediated primarily through interaction with NBF1-Walker B and both Walkers of NBF2. Based on these results, we propose a model by which Syn-1A acts as an inhibitory clamp on the KATP channel, effectively buffering minor fluctuations in ATP/ADP concentration to prevent unnecessary channel activity.

ABCA1 is a key plasma membrane protein required for the efflux of cellular cholesterol to extracellular acceptors, particularly to apoA-I. This process is essential to maintain cholesterol homeostasis in the body. The detailed molecular mechanisms, however, are still insufficiently understood. Also, the molecular identity of ABCA1, i.e. channel, pump or flippase, remains unknown. In this study we analyzed the extracellular ATP levels in the medium of ABCA1-expressing BHK cells and RAW macrophages and compared them to the medium of relevant non-expressing cells. We found that the extracellular ATP concentrations are significantly elevated when cells express ABCA1. Importantly, a dysfunctional ABCA1 mutant (A937V), when expressed similarly as WT-ABCA1, is unable to raise extracellular ATP concentration. This suggests a causal relationship between functional ABCA1 and elevated extracellular ATP. To explore the physiological role of elevated extracellular ATP, we analyzed ABCA1-mediated cholesterol efflux under the conditions where extracellular ATP levels were modulated. We found that increasing extracellular ATP within the physiological range, i.e. < μM, promotes cholesterol efflux to apoA-I. On the other hand, removing extracellular ATP, either by adding apyrase to the medium or by expressing a plasma membrane bound ecto-nucleotidase CD39, abolishes cholesterol efflux to apoA-I. Based on these results we conclude that, through direct or indirect mechanisms, ABCA1 functions to raise ATP levels in the medium. This elevated extracellular ATP is required for ABCA1-mediated cholesterol efflux to apoA-I.