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.

No description available.

<p>Genetic modification of dendritic cells with plasmid DNA is plagued with low transfection efficiencies because DNA taken up by non-dividing dendritic cells rarely reaches the nucleus. But this difficulty can be overcome by the use of messenger RNA (mRNA), which exerts its biological function in the cytoplasm and obviates the need to enter the nucleus. Since pioneering work of Boczkwoski et al, the ex-vivo application of mRNA-transfected dendritic cells as a vaccine has been evaluated in numerous phase I trials worldwide and is still currently being actively optimized in clinical trials. </p><p> However, a major disadvantage of using mRNA-transfected DCs as a vaccine is that it requires patients to undergo at least one 4-hour leukapheresis procedure, followed by separation of the peripheral blood mononuclear cells (PBMCs), from which monocytes are isolated and cultured for a week in a defined medium with cytokines. The resulting DCs are matured after being loaded with mRNA and frozen for storage. Aliquots are subsequently thawed prior to administration to patients. This process of harvesting, culturing and loading DCs is more time- and resource-intensive than Provenge, the first FDA approved cell based tumor vaccine in 2011.Recent evidence has confirmed a lack of broad translation of Provenge due to complexity and cost of treatment. This predicates a similar fate for mRNA-transfected dendritic cell vaccine going forward. </p><p> This thesis presents alternative delivery strategies for mRNA mediated tumor vaccination. Through the application of synthetic and natural biomaterials, this thesis demonstrates two viable approaches that reduce or eliminate the need for extensive manipulation and cell culture.</p><p> The first approach is the direct in vivo delivery of mRNA encapsulated in nanoparticles for tumor vaccination. A selected number of synthetic gene carriers that have been shown to be effective for other applications are formulated with mRNA into nanoparticles and evaluated for their ability to transfect primary DCs. The best performing formulation is observed to transfect primary murine and human dendritic cells with an efficiency of 60% and 50% (based on %GFP+ cells) respectively. The in vivo transfection efficiency and expression kinetics of this formulation is subsequently evaluated and compared with naked mRNA via various routes of delivery. Following this, a proof-of-concept study is presented for a non-invasive method of mRNA tumor vaccination using intranasally administered mRNA encapsulated in nanoparticles. Results show that intranasally administered mRNA induces tumor immunity only if it is encapsulated in nanoparticles. And anti-tumor immunity is observed in mice intranasally immunized under both prophylactic as well as therapeutic models. </p><p> The second approach evaluates whole blood cells as alternative cell based mRNA carriers. A method is developed to encapsulate intact and functional mRNA in murine whole blood cells. Whole blood cells loaded with mRNA not only include erythrocytes but also T cells (CD3+), monocytes (CD11b), antigen presenting cells (MHC class II) as well as plasmacytoid DCs (CD45R-B220). Mice immunized with mRNA-loaded whole blood cells (intravenously) develop both humoral and cellular antigen-specific immune responses, and demonstrate delayed tumor onset and progression in a melanoma therapeutic immunization model (using tyrosinase related protein -2, TRP-2, as an antigen). Importantly, the therapeutic efficacy of mRNA-loaded whole blood cell vaccine formulation is found to be comparable to mRNA-transfected dendritic cell vaccine.</p><p> In conclusion, this thesis presents new methods to the delivery of mRNA tumor vaccines that reduce or eliminates the need for extensive cell manipulation and culture. Results presented in this thesis reveal viable research directions towards the development and optimization of mRNA delivery technologies that will address the problem of broad translation of mRNA tumor vaccines in the clinics.</p> / Dissertation

Plant polyadenylation factors have proven difficult to purify and characterize, owing to the presence of excessive nuclease activity in plant nuclear extracts, thereby precluding the identification of polyadenylation signal-dependent processing and polyadenylation in crude extracts. As an alternative approach to identifying such factors, a screen was conducted for activities that inhibit the non-specific activity of plant poly(A) polymerases (PAP). One such factor (termed here as Putative Polyadenylation Factor B, or PPF-B) was identified in a screen of DEAE-Sepharose column fractions using a partially purified preparation of a plant nuclear poly(A) polymerase. This factor was purified to near homogeneity. Surprisingly, in addition to being an effective inhibitor of the nuclear PAP, PPF-B inhibited the activity of a chloroplast PAP. In contrast, this factor stimulated the activity of the yeast PAP. Direct assays of ATPase, proteinase, and nuclease activities indicated that inhibition of PAP activity was not due to depletion of substrates or degradation of products of the PAP reaction. The major polypeptide component of PPF-B proved to be a novel linker histone (RSP), which copurified with inhibitory activity by affinity chromatography on DNA-cellulose. The association of inhibitory activity with a linker histone and the spectrum of inhibitory activity, raise interesting possibilities regarding the role of PPF-B in nuclear RNA metabolism. These include a link between DNA damage and polyadenylation, as well as a role for limiting the polyadenylation of stable RNAs in the nucleus and nucleolus. The Arabidopsis genome possesses genes encoding probable homologs of most of the polyadenylation subunits that have been identified in mammals and yeast. Two of these reside on chromosome III and V and have the potential to encode a protein that is related to the yeast and mammalian Fip1 subunit (AtFip1-III and AtFip1-V). These genes are universally expressed in Arabidopsis tissues. AtFip1-V stimulates the non-specific activity of at least one Arabidopsis nuclear PAP, binds RNA, and interacts with other polyadenylation homologs AtCstF77 and AtCPSF30. These studies suggest that AtFip1- V is an authentic polyadenylation factor that coordinates other subunits and plays a role in regulating the activityof PAP in plants.

Selenium (Se) is an important trace mineral in the diet of cattle. Our objective was to determine whether the form of supplemental Se fed to the dam would affect the expression of genes regulating steroidogenesis in the newborn testis. Twenty-four Angus-cross cows were assigned randomly (n=8) to individual ad libitum access of a mineral mix containing 35 ppm of Se supplied as sodium selenite (inorganic, ISe; Prince Se), Sel-Plex (organic, OSe; Sel-Plex, Alltech) or a 50/50 mix of ISe/OSe (MIX) 4 months prior to breeding and throughout gestation. All male calves were castrated within 2 days of birth and total testis RNA was subjected to microarray analysis using the Affymetrix Bovine 1.0 ST array. Ingenuity Pathway Analysis of differentially expressed genes, separated by one-way ANOVA (p<0.05) and a post-hoc LSD test, identified eight mRNAs associated with steroidogenesis. Specifically, mRNAs involved in the conversion of testosterone to estradiol were increased in the testis of OSe-supplemented when compared to ISe-supplemented animals, and mRNAs encoding genes regulating the conversion of androstenedione to testosterone or androstenedione to estradiol were decreased in the testis of MIX-supplemented when compared to ISe-supplemented cows.Expression in the neonate may equate to differences in fertility in the adult animal.

Chez les Eucaryotes, l'étape d'initiation est de loin la plus complexe et la plus lente du processus de traduction. Elle nécessite l'intervention de 12 facteurs protéiques, d'une coiffe m7GpppN située à l'extrémité 5' des ARNm et d'une queue poly(A) en 3'. Les ARNm des histones " réplication-dépendantes " sont particuliers car dépourvus d'extrémité 3' polyadénylée et dotés d'une extrémité 5' non traduite extrêmement courte, de 9 nt seulement chez l'ARNm H4 de la souris. Pour traduire ces ARNm, un processus d'initiation non conventionnel a été décrit au laboratoire. L'objectif de ma thèse a été d'établir les bases structurales de ce mécanisme en combinant différentes approches expérimentales. Deux protocoles originaux de repliement ont été mis au point afin d'isoler l'ARNm H4 dans deux conformations distinctes et stables. Une caractérisation fonctionnelle et structurale de ces deux formes de l'ARNm a ensuite été réalisée. La stabilité et la structure de ces deux formes ont été étudiées par DLS, par SAXS et par équilibre de sédimentation. Puis, nous avons étudié la capacité de ces deux formes d'ARNm H4 à fixer le facteur d'initiation eIF4E et les ribosomes assemblés sur le codon d'initiation ainsi que leur aptitude à être traduits in vitro. Un modèle de repliement de la structure secondaire de l'ARNm H4 a été construit après sondage enzymatique et chimique des deux formes de l'ARNm. Ce modèle a servi de base pour le travail d'ingénierie de l'ARNm H4 qui a conduit à son découpage en sous-domaines. Des essais de cristallisation ont porté sur 18 de ces fragments ainsi que sur les deux formes de l'ARNm H4 complet.

Transcription elongation by RNA polymerase II is regulated by an array of protein complexes. Among various elongation factors, Spt5 is conserved in the three kingdoms of life. I investigated functional interactions of its C-terminal repeats (CTR) domain with several elongation protein complexes in Saccharomyces cerevisiae. By using genetics and molecular biology methods, I established two major pathways in this thesis. The first describes how BUR kinase-mediated phosphorylation of CTR domain leads to co-transcriptional recruitment of the PAF complex to regulate histone modifications on active genes. The second describes how CTR phosphorylation facilitates recruitment of capping enzymes to enhance gene splicing. Finally, several Spt5-associated protein complexes were studied, and potential molecular mechanisms underlying these observations are proposed and discussed.

Transcription elongation by RNA polymerase II is regulated by an array of protein complexes. Among various elongation factors, Spt5 is conserved in the three kingdoms of life. I investigated functional interactions of its C-terminal repeats (CTR) domain with several elongation protein complexes in Saccharomyces cerevisiae. By using genetics and molecular biology methods, I established two major pathways in this thesis. The first describes how BUR kinase-mediated phosphorylation of CTR domain leads to co-transcriptional recruitment of the PAF complex to regulate histone modifications on active genes. The second describes how CTR phosphorylation facilitates recruitment of capping enzymes to enhance gene splicing. Finally, several Spt5-associated protein complexes were studied, and potential molecular mechanisms underlying these observations are proposed and discussed.

No description available.

Tris(1-chloro-2-propyl) phosphate (TCPP) and tris(1,3-dichloro-2-propyl) phosphate (TDCPP) are added to polyurethane foams in a variety of industrial and consumer products to prevent flame ignition. The gradual release of these flame retardants (FRs) from such products leads to contamination of various abiotic and biotic media, including wild birds. Recent studies demonstrated endocrine-disrupting effects of TCPP and TDCPP, including alteration of circulating thyroid hormone (TH) levels. The TH-pathway is essential for normal growth and development in birds. There are limited data on the toxicological effects of TCPP and TDCPP in avian species and, prior to this work, no study has examined their effects in avian embryos. This M.Sc. thesis investigates the developmental, molecular and biochemical effects of TCPP and TDCPP in chicken (Gallus gallus domesticus) embryos via egg injection studies. TCPP delayed pipping at doses ≥9.24 μg/g, both TCPP and TDCPP reduced embryo growth at the highest dose (51.6 μg TCPP/g and 45 μg TDCPP/g), and TDCPP decreased free plasma thyroxine and gallbladder size at 7.64 μg/g and 45 μg/g, respectively. Real-time reverse transcription polymerase chain reaction was used to measure changes in mRNA levels of hepatic genes that were responsive to these FRs in a previous in vitro study. TCPP dysregulated the expression of TH-responsive genes and xenobiotic metabolizing enzymes (cytochrome P450s; CYPs), whereas TDCPP only affected CYPs. Less than 1% of the administered TCPP or TDCPP was detected in egg contents following 19 days of incubation, indicating extensive metabolism of the parent compounds. DNA microarrays were used to perform a global transcriptional analysis on liver samples from embryos that exhibited adverse effects following TDCPP injection. 47 differentially expressed genes were identified at the 45 μg/g dose. Functional analysis revealed that immune function and lipid and steroid metabolism were major targets of TDCPP toxicity and indicated a state of cholestatic liver/biliary fibrosis. Since the TH-pathway is a key regulator of metabolic homeostasis, its disruption early in development is a potential cause of the observed adverse effects. This thesis demonstrates, for the first time, developmental and endocrine-disrupting effects of TCPP and TDCPP in an avian species and attempts to link phenotypic changes to molecular-level disruptions in hopes to improve the understanding of their modes of action.