Thesis (Ph.D)--Aerospace Engineering, Georgia Institute of Technology, 2009. / Committee Chair: Costello, Mark. Part of the SMARTech Electronic Thesis and Dissertation Collection.

A recurring theme in biology is the aggregation of mRNA-protein complexes (mRNPs) into higher order assemblies. Often these complexes play important roles in the regulation of gene expression, but the function of the conserved cytoplasmic mRNP assemblies - P bodies and stress granules, is not known. It is believed that the misregulation of granule assembly is related to disorders like Amyotrophic Lateral Sclerosis and Frontotemporal Lobe Degeneration. Determining the complete composition of these granules may hold the key to understanding the function and mechanism of assembly of these granules. This work describes multiple approaches taken to identify new protein and mRNA components of P bodies and stress granules. New members of the P body and stress granule proteome reveal a role for these granules in diverse cellular processes including signal transduction, transcription and metabolism. Additionally, a new stress granule resident complex - the CCT complex, was also identified as a novel regulator of granule disassembly. This work also describes the first purification scheme for stress granules and presents a new system for in vitro study of stress granules. Together, the findings shed new light on the composition, function, structure and regulation of P bodies and stress granules in yeast.

Generally Hepatitis C Virus tropism is limited to hepatocytes. This limited tropism is a result of the receptors HCV requires for cellular entry and other host cellular factors including, uniquely, a liver specific miRNA, miR-122. The relationship between HCV and miR-122 is interesting, as commonly, miRNA are associated with suppression of function, but in the case of HCV, miR-122 actively promotes HCV proliferation. In-depth studies have demonstrated that miR-122 along with the RNA induced silencing complex (RISC) protein Argonaute 2 (Ago2) binds directly to two seed sequences separated by 8-9 nucleotides on HCV 5’UTR. Binding to the 5’UTR results in an increase in viral replication and translation. The method by which miR-122 promotes HCV translation and replication is not fully understood but evidence suggests that part of the function of miR-122 is to stabilize the HCV genome and protect it from exonuclease degradation by Xrn1, but other mechanisms remain to be identified. The reliance of HCV on miR-122 is best exemplified by the fact that removal of miR-122 by a miR-122 antagonist drastically impedes HCV viral titers in Chimpanzees and humans with no indication of escape mutants. The observation that HCV augmentation of the HCV life cycle by miR-122 requires Ago2 suggests that other components downstream in the miRNA suppression pathway may also be part of the mechanism of action. Our studies focused specifically on the processing body (p-body) associated DEAD-box helicase DDX6. DDX6 is essential for p-body assembly, required for robust miRNA suppression activity and elevated in HCV associated hepatocellular carcinomas. As such we hypothesized that DDX6 and p-bodies were directly or in-directly associated with the mechanism of action of miR-122. Knocking down DDX6 with siRNA indicated that DDX6 augments both HCV replication and translation. To examine whether DDX6 augmentation of HCV replication was related to the effects of miR-122 on the HCV life cycle, HCV replication and translation were assessed in the presence or absence of miR-122 when DDX6 was knocked down. Our data indicated that HCV replication and translation were augmented equally by miR-122 whether DDX6 was present or not. Our data also demonstrated that HCV replication and translation that was occurring independent of miR-122 was also still affected by DDX6 knockdown. Taken together our observations strongly suggest that the role DDX6 has on HCV is independent of HCV and miR-122’s relationship. In order to better understand miR-122’s relationship with HCV, we hypothesized that targeting the miR-122 binding region with siRNA would inhibit HCV replication initially, but that over the course of several rounds of treatment with the same siRNA, HCV would mutate to escape the siRNA, producing escape mutants that replicate without a dependency on miR-122. These escape mutants could be evaluated on how they replicate without using miR-122, shedding light on miR-122 and HCV’s relationship. Conversely if no escape mutants arose the siRNA could be further studied as a potential therapeutic for HCV. siRNA designed to target the miR-122 binding region inhibited HCV replication, confirming that the designed siRNAs could access the miR-122 binding region and function as an siRNA. Interestingly, when the siRNAs were used against a replication competent HCV RNA having a single nucleotide mutation in the first miR-122 binding site, instead of abolishing siRNA knockdown, two of the siRNA showed enhanced inhibition activity. The target sequences of these siRNAs spanned both miR-122 binding sites and we speculate that their inhibitory activity was due to competition for miR-122 binding to site 2. This observation indicates that siRNA targeting the miR-122 binding region have dual activity, by siRNA induced cleavage, and as a competitive inhibitor of miR-122 binding. Selection for viral escape mutants of the miR-122-binding site targeting siRNAs revealed viral RNAs having mutations within the miR-122 binding sites, in the surrounding region, and to other areas within the HCV IRES. The mutant viruses will be used to assess the influence of miR-122 binding site mutations on HCV replicative fitness, and to determine if the virus can evolve to replicate independent from augmentation by miR-122.

Interactions between the aerodynamics and dynamics of bluff bodies are important in many engineering applications, including suspension bridges, tall buildings, oil platforms, wind turbine towers, air drops, and construction with cranes. In the rotorcraft field, bluff bodies are commonly suspended underneath the vehicle by tethers. This approach is often the only practical way to deliver a payload in a reasonable amount of time in disaster relief efforts, search-and-rescue operations, and military operations. However, currently a fundamental understanding of the aerodynamics of these bluff bodies is lacking, and accurate dynamic simulation models for predicting the safe flying speed are not available. In order to address these shortcomings, two main advancements are presented in this thesis. The aerodynamics of several three-dimensional canonical bluff bodies are examined over a range of Reynolds numbers representative of wind-tunnel-scale to full-scale models. Numerical experiments are utilized, with a focus on uncertainty analysis and validation of the computations. Mean and unsteady forces and moments for these bluff bodies have been evaluated, and empirical models of the shear layer characteristics have been extracted to quantify the behaviors and provide predictive capability. In addition, a physics-based reduced-order simulation model has been developed for bluff bodies. The physics-based approach is necessary to ensure that the predicted behavior of new configurations is accurate, and it is made possible by the breakthroughs in three-dimensional bluff body aerodynamics presented in this thesis. The integrated aerodynamic forces and moments and dynamic behavior predicted by model are extensively validated with data from wind tunnels, flight tests, and high-fidelity computations. Furthermore, successful stability predictions for tethered loads are demonstrated. The model is applicable to the simulation of any generic bluff body configuration, is readily extensible, and has low computational cost.

In recent years, biocatalytical steps in chemical synthesis are becoming increasingly important for economical and environmental-friendly production. In order to evaluate the use of enzymes in a process at Cambrex Karlskoga AB, an expression system was developed for a dehydrogenase. A synthetic gene was cloned into Escherichia coli DH5a cells, using the pTZ19R expression vector, as previously described in the literature. Protein expression was carried out at 25°C, 30°C and 37°C and results were measured using SDS-PAGE and activity assays. To improve expression, the gene was modified in three ways using PCR, yielding eight clones: It was inserted into the pSE420 expression vector, shortened to avoid inclusion body formation and a missing nucleotide was inserted into the sequence. A protocol for inclusion body screening was also developed. Finally, an assay for determining the kinetic constants of dehydrogenase was designed. It is concluded that further experiments must be done to obtain expression of the dehydrogenase and recommendations for additional work are given. / Biokatalytiska processteg har de senaste åren blivit ett allt viktigare inslag i kemisk syntes för att åstadkomma ekonomisk och miljövänlig produktion. För att utvärdera användandet av enzymer i en process hos Cambrex Karlskoga AB utvecklades ett expressionssystem för ett dehydrogenas. En syntetisk gen klonades in i Escherichia coli DH5a och uttrycktes med hjälp av expressionsvektorn pTZ19R, som tidigare finns beskrivet i litteraturen. Proteinuttrycket utfördes vid 25°C, 30°C och 37°C och resultatet mättes med hjälp av SDS-PAGE och aktivitetsmätningar. Genen för dehydrogenaset modifierades på tre sätt, vilket gav upphov till åtta varianter. Genen fördes över till expressionsvektorn pSE420, kortades för att undvika bildning av inklusionskroppar och en nukleotid som fattades från gensekvensen återinfördes. Ett protokoll utarbetades även för undersökning av inklusionskroppar. Till sist sammanställdes en metod för att undersöka de kinetiska konstanterna hos dehydrogenaset. Slutsatsen av arbetet är att fortsatta studier måste utföras för att erhålla uttryck av dehydrogenaset och rekommendationer ges för framtida undersökningar.

Modification by glycosylation gives proteins a range of diverse functions reflecting their structural variability. N-glycans regulate many biological outcomes in mammalian cells under both normal and pathological conditions. They play a major role in various pathologies such as cancer and lysosomal storage diseases. Interplay between N-glycans and other regulators, such as membrane lipid domains, in the control of signaling pathways remains poorly understood. My thesis therefore focuses on how N-glycans and membrane lipid domains oppose and/or work together at different cellular levels to regulate various processes such as receptor signaling and diffusion, endocytosis and lysosomal organelle biogenesis. Mgat5 encodes for ß1,6-N-acetylglucosaminyltransferase V that produces N-glycans, the preferred ligand for galectins. In tumor cells, galectins bind glycosylated receptors at the cell surface forming a lattice, that restricts receptor endocytosis and enhances its residency at the plasma membrane. In the first part of my thesis, I report that Galectin/receptor crosslinking opposes receptor sequestration by oligomerized caveolin-1 (Cav1) domains overriding its negative regulation of epidermal growth factor receptor (EGFR) signaling, cell surface diffusion and tumor growth. These results identify Cav1 as a conditional tumor suppressor. I also demonstrate that Cav1 is a negative regulator of lipid raft-mediated endocytosis. Cav1 indirectly regulates the internalization of cholera toxin b subunit to the Golgi apparatus independently of caveolae formation. That identifies a new role for caveolin-1 outside caveolae in the regulation of raft-dependent endocytosis Finally, Mgat5 overexpression in pneumocytes is associated with the expression of a lysosomal organelle, the multilamellar body (MLB), via autophagy. MLB expression is also a characteristic of various lysosomal storage diseases. I demonstrate that cholesterol accumulation can override the need for Mgat5 overexpression in MLB formation indicating that they may form via multiple mechanisms. However, I also demonstrate that a contribution of the autophagic pathway is a common determinant of biogenesis of MLB of various lipid compositions. In conclusion, Mgat5-dependent protein glycosylation and Cav1/raft domains therefore both function as regulators of plasma membrane interactions, endocytosis and lysosomal organelle biogenesis. Understanding of this interplay is crucial for the understanding of the mechanisms involve in various pathologies such as cancer and lysosomal storage diseases.

Loss of the promyelocytic leukemia (PML) protein is associated with genomic instability/cancer. There are several isoforms of the PML protein that localize in PML nuclear bodies (PML NBs). How each individual isoform contributes to the functions of PML NBs is unknown. The objective of this study was to identify and characterize PML isoform-I (PML-I) specific protein-protein interactions. Using yeast two-hybrid screens, several interacting partners of PML-I were identified that play roles in translational regulation, including eukaryotic initiation factor 3 subunit K (eIF3K). Our studies demonstrated that eIF3K interacts with PML-I in vitro and in vivo. Through its interaction with eIF3K, overexpression of PML-I resulted in the concomitant increase in eIF3K protein levels in mammalian cells. This suggests that PML-I may be involved in regulating eIF3K protein translation or stability, which in turn could affect translation of specific mRNAs or global translation in cancer cells with reduced expression of PML-I.

No description available.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Certaines maladies neurodégénératives sont associées au mauvais repliement de la protéine prion (PrP[indice supérieur c]) et sont connues sous le nom de maladies à prion ou les encéphalopathies spongiformes transmissibles (ESTs). Récemment, il a été démontré que PrP[indice supérieur c] augmente l'efficacité des micro-ARN (miARN) via son interaction directe avec la protéine Argonaute 2 (Ago2) au niveau de la membrane des corps multivésiculaires (multivesicular bodies (MVBs)). Ago2 est une des protéines centrales du complexe RISC (RNA-induced silencing complex) qui est responsable de l'efficacité des ARNi. Certaines formes pathologiques de PrP[indice supérieur c] induisent l'activation de la réponse au stress eiF2a dépendante, une étape déterminante pour le développement de la pathologie. Cependant, l'activation de la réponse au stress eiF2a dépendante, favorise normalement la survie cellulaire via la formation de deux types de granules d'ARN, les granules de stress (GSs) et les P-Bodies. Aucune étude n'a investigué le rôle de ces deux types de granules d'ARN et de la régulation de l'efficacité des miARN par PrP[indice supérieur c] dans la neurotoxicité associée aux ESTs. Mon premier objectif est d'approfondir nos connaissances sur le nouveau rôle de PrP[indice supérieur c] dans la régulation du système miARN et de l'implication des miARN et du système endosomal dans les ESTs génétiques. Mon second objectif est d'investiguer le rôle des GSs et des P-Bodies, dans la neurotoxicité reliée aux ESTs et établir un lien entre la dérégulation de la réponse au stress et du système ARNi. PrP[indice supérieur c] augmente l'efficacité des miARN via l'interaction de sa région octapeptidique répété (OR, octapeptide repeat region) avec la protéine Ago2. L'effet d'un mutant cytoplasmique artificiel nommé CyPrP (connu comme agent neurotoxique potentiel) et de cinq mutants familiaux de PrP[indice supérieur c] sur le système endosomal et miARN a été caractérisé. Les mutants de PrP[indice supérieur c] affectent la maturation des MVBs et, conséquemment, induisent une délocalisation de GW182, une diminution l'efficacité des miARN et de la production des exosomes. Nous proposons que cette dérégulation de la voie endosomale et miARN par les mutants familiaux de PrP[indice supérieur c] contribue à la neurodégénérescence observée dans les ESTs. Les mutants de PrP[indice supérieur c] induisent également l'expression de la protéine PACT qui est responsable de l'activation de la kinase de stress PKR et de la phosphorylation d'eiF2a. Cependant, malgré la phosphorylation d'eiF2a, les mutants familiaux de PrP[indice supérieur c] et PrP[indice supérieur Sc] inhibent la formation des GSs et des P-Bodies augmentant la susceptibilité des cellules aux différents stress. L'inhibition des P-bodies par les mutants confirment également que les mutants de PrP[indice supérieur c] induisent une diminution de l'efficacité des miARN. Je propose que le maintien de l'activation de la voie PACT-PKR-eiF2a et l'inhibition des GSs et des P-Bodies contribuent à la dérégulation du système d'ARNi, à la susceptibilité des neurones et à la neurodégénérescence observée dans les ESTs. Une meilleure compréhension des mécanismes de neurotoxicité plus particulièrement de l'inhibition du système ARNi et de la réponse au stress par les formes mal repliées de PrP[indice supérieur c] peut mener au développement de médicaments afin de contrer l'évolution de la pathologie. [symboles non conformes]