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Understanding Small Rna Formation In Drosophila Melanogaster: A Dissertation, Elif Sarinay Cenik Jul 2012

Understanding Small Rna Formation In Drosophila Melanogaster: A Dissertation, Elif Sarinay Cenik

GSBS Dissertations and Theses

Drosophila Dicer-2 generates small interfering RNAs (siRNAs) from long double-stranded RNA (dsRNA), whereas Dicer-1 produces microRNAs from premicroRNA. My thesis focuses on the functional characteristics of two Drosophila Dicers that makes them specific for their biological substrates. We found that RNA binding protein partners of Dicers and two small molecules, ATP and phosphate are key in regulating Drosophila Dicers’ specificity. Without any additional factor, recombinant Dicer-2 cleaves pre-miRNA, but its product is shorter than the authentic miRNA. However, the protein R2D2 and inorganic phosphate block pre-miRNA processing by Dicer-2. In contrast, Dicer-1 is inherently capable of processing the substrates of ...


Therapeutic Silencing Of Mutant Huntingtin By Targeting Single Nucleotide Polymorphisms: A Dissertation, Edith L. Pfister Jul 2012

Therapeutic Silencing Of Mutant Huntingtin By Targeting Single Nucleotide Polymorphisms: A Dissertation, Edith L. Pfister

GSBS Dissertations and Theses

Huntington’s disease (HD) is an autosomal dominant, progressive neurodegenerative disorder. Invariably fatal, HD is caused by expansion of the CAG repeat region in exon 1 of the Huntingtin gene which creates a toxic protein with an extended polyglutamine tract 1. Silencing mutant Huntingtin messenger RNA (mRNA) is a promising therapeutic approach 2-6. The ideal silencing strategy would reduce mutant Huntingtin while leaving the wild-type mRNA intact. Unfortunately, targeting the disease causing CAG repeat expansion is difficult and risks targeting other CAG repeat containing genes.

We examined an alternative strategy, targeting single nucleotide polymorphisms (SNPs) in the Huntingtin mRNA. The ...


Molecular Mechanisms Of Endocytosis: Trafficking And Functional Requirements For The Transferrin Receptor, Small Interfering Rnas And Dopamine Transporter: A Dissertation, Deanna M. Navaroli Apr 2012

Molecular Mechanisms Of Endocytosis: Trafficking And Functional Requirements For The Transferrin Receptor, Small Interfering Rnas And Dopamine Transporter: A Dissertation, Deanna M. Navaroli

GSBS Dissertations and Theses

Endocytosis is an essential function of eukaryotic cells, providing crucial nutrients and playing key roles in interactions of the plasma membrane with the environment. The classical view of the endocytic pathway, where vesicles from the plasma membrane fuse with a homogenous population of early endosomes from which cargo is sorted, has recently been challenged by the finding of multiple subpopulations of endosomes. These subpopulations vary in their content of phosphatidylinositol 3- phosphate (PI3P) and Rab binding proteins. The role of these endosomal subpopulations is unclear, as is the role of multiple PI3P effectors, which are ubiquitously expressed and highly conserved ...


Molecular Mechanisms Of Pirna Biogenesis And Function In Drosophila: A Dissertation, Chengjian Li Apr 2011

Molecular Mechanisms Of Pirna Biogenesis And Function In Drosophila: A Dissertation, Chengjian Li

GSBS Dissertations and Theses

In the Drosophila germ line, PIWI-interacting RNAs (piRNAs) ensure genomic stability by silencing endogenous selfish genetic elements such as retrotransposons and repetitive sequences.

We examined the genetic requirements for the biogenesis and function of piRNAs in both female and male germ line. We found that piRNAs function through the PIWI, rather than the AGO, family Argonaute proteins, and the production of piRNAs requires neither microRNA (miRNA) nor small interfering RNA (siRNA) pathway machinery. These findings allowed the discovery of the third conserved small RNA silencing pathway, which is distinct from both the miRNA and RNAi pathways in its mechanisms of ...


Drosophila Pirna Function In Genome Maintenance, Telomere Protection And Genome Evolution: A Dissertation, Jaspreet S. Khurana Oct 2010

Drosophila Pirna Function In Genome Maintenance, Telomere Protection And Genome Evolution: A Dissertation, Jaspreet S. Khurana

GSBS Dissertations and Theses

Upon fertilization, the early embryo sustains most of the cellular processes using the maternally deposited reserves in the egg itself until the zygotic gene expression takes charge. Among the plethora of essential components provided by the mother are small non-coding RNAs called PIWI-interacting RNAs (piRNAs), which provide immunity to the zygote against transposon challenge. In this thesis, I have presented three different functions of piRNAs in Drosophila melanogaster- in maintenance of genomic integrity, telomere protection and their role as an adaptive immune system against genomic parasites.

In Chapter 2, I have described the phenotypic effects of the loss of piRNA ...


Roles Of Cellular Rna-Dependent Rna Polymerases In Endogenous Small Rna Pathways In Caenorhabditis Elegans: A Dissertation, Jessica J. Vasale Jun 2010

Roles Of Cellular Rna-Dependent Rna Polymerases In Endogenous Small Rna Pathways In Caenorhabditis Elegans: A Dissertation, Jessica J. Vasale

GSBS Dissertations and Theses

The RNA interference (RNAi) pathway in Caenorhabditis elegans is a two-step, small RNA-mediated silencing pathway. Unlike in other organisms, Dicer processing of double-stranded RNA into small interfering (si) RNAs is not sufficient in worms to induce gene silencing. The activity of cellular RNA-dependent RNA polymerase (RdRP) is necessary to synthesize a secondary pool of siRNAs, which interact with a unique class of Argonaute proteins to form the functional effector complexes that mediate silencing. The aims of this thesis were to: 1) characterize the role of RdRP family members in endogenous small RNA biogenesis; 2) identify the Argonaute proteins that interact ...


Role And Regulation Of Fat Specific Protein (Fsp27) In Lipolysis In 3t3-L1 Adipocytes: A Dissertation, Srijana Ranjit May 2010

Role And Regulation Of Fat Specific Protein (Fsp27) In Lipolysis In 3t3-L1 Adipocytes: A Dissertation, Srijana Ranjit

GSBS Dissertations and Theses

The alarming rate of increase in incidence and prevalence of the type 2 diabetes mellitus has prompted intense research on understanding the pathogenesis of the type 2 diabetes. It is observed that the development of type 2 diabetes is preceded by a state of insulin resistance and obesity. Previous studies have suggested that the obesity induced insulin resistance may be mediated by elevated levels of circulating free fatty acids (FFAs). The increase in circulating levels of FFAs may be contributed by the release of FFAs from stored triglycerides (TG) in adipocytes via lipolysis. It is hypothesized that the decrease in ...


Endogenous Small Rnas In The Drosophila Soma: A Dissertation, Megha Ghildiyal Mar 2010

Endogenous Small Rnas In The Drosophila Soma: A Dissertation, Megha Ghildiyal

GSBS Dissertations and Theses

Since the discovery in 1993 of the first small silencing RNA, a dizzying number of small RNAs have been identified, including microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). These classes differ in their biogenesis, modes of target regulation and in the biological pathways they regulate.

Historically, siRNAs were believed to arise only from exogenous double-stranded RNA triggers in organisms lacking RNA-dependent RNA polymerases. However, the discovery of endogenous siRNAs in flies expanded the biological significance of siRNAs beyond viral defense. By high throughput sequencing we identified Drosophila endosiRNAs as 21 nt small RNAs, bearing a 2´-O ...


Pirna Function And Biogenesis In The Drosophila Female Germline: A Dissertation, Carla Andrea Klattenhoff Nov 2008

Pirna Function And Biogenesis In The Drosophila Female Germline: A Dissertation, Carla Andrea Klattenhoff

GSBS Dissertations and Theses

The studies presented in this thesis addressed mainly two aspects of Piwi-interacting RNA (piRNA) biology in the Drosophilagermline.

We investigated the role of the piRNA pathway in embryonic axis specification. piRNAs mediate silencing of retrotransposons and the Stellate locus. Mutations in the Drosophila piRNA pathway genes armitage and aubergine disrupt embryonic axis specification, triggering defects in microtubule polarization and asymmetric localization of mRNA and protein determinants in the developing oocyte. Mutations in the ATR/Chk2 DNA damage signal transduction pathway dramatically suppress these axis specification defects, but do not restore retrotransposon or Stellatesilencing. Furthermore, piRNA pathway mutations lead ...


Role Of Map4k4 Signaling In Adipocyte And Macrophage Derived Inflammation: A Dissertation, Gregory J. Tesz Jul 2008

Role Of Map4k4 Signaling In Adipocyte And Macrophage Derived Inflammation: A Dissertation, Gregory J. Tesz

GSBS Dissertations and Theses

Human obesity is increasing globally at an impressive rate. The rise in obesity has led to an increase in diseases associated with obesity, such as type 2 diabetes. A major prerequisite for this disease is the development of insulin resistance in the muscle and adipose tissues. Interestingly, experiments in rodent models suggest that adipocytes and macrophages can profoundly influence the development of insulin resistance. Accordingly, the number of adipose tissue macrophages increases substantially during the development of obesity. Numerous research models have demonstrated that macrophages promote insulin resistance by secreting cytokines, like TNFα, which impair whole body insulin sensitivity and ...


Small Rna Sorting In Drosophila Produces Chemically Distinct Functional Rna-Protein Complexes: A Dissertation, Michael D. Horwich Jun 2008

Small Rna Sorting In Drosophila Produces Chemically Distinct Functional Rna-Protein Complexes: A Dissertation, Michael D. Horwich

GSBS Dissertations and Theses

Small interfering RNAs (siRNAs), microRNAs (miRNAs), and piRNAs (piRNA) are conserved classes of small single-stranded ~21-30 nucleotide (nt) RNA guides that repress eukaryotic gene expression using distinct RNA Induced Silencing Complexes (RISCs). At its core, RISC is composed of a single-stranded small RNA guide bound to a member of the Argonaute protein family, which together bind and repress complementary target RNA. miRNAs target protein coding mRNAs—a function essential for normal development and broadly involved in pathways of human disease; small interfering RNAs (siRNA) defend against viruses, but can also be engineered to direct experimental or therapeutic gene silencing; piwi ...


Understanding Assembly Of Ago2 Risc: The Rnai Enzyme: A Dissertation, Christian B. Matranga Sep 2007

Understanding Assembly Of Ago2 Risc: The Rnai Enzyme: A Dissertation, Christian B. Matranga

GSBS Dissertations and Theses

In 1990, Richard Jorgensen’s lab initiated a study to test if they could create a more vivid color petunia (Napoli et al. 1990). Their plan was to transform plants with the chalcone synthase transgene––the predicted rate limiting factor in the production of purple pigmentation. Much to their surprise, the transgenic plants, as well as their progeny, displayed a great reduction in pigmentation. This loss of endogenous function was termed “cosuppression” and it was thought that sequence-specific repression resulted from over-expression of the homologous transgene sequence. In 1998, Andrew Fire and Craig Mello described a phenomenon in which double ...


Interaction Of A Mammalian Virus With Host Rna Silencing Pathways: A Dissertation, Bradford Michael Stadler Mar 2007

Interaction Of A Mammalian Virus With Host Rna Silencing Pathways: A Dissertation, Bradford Michael Stadler

GSBS Dissertations and Theses

In the complex relationships of mammalian viruses with their hosts, it is currently unclear as to what role RNA silencing pathways play during the course of infection. RNA silencing-based immunity is the cornerstone of plant and invertebrate defense against viral pathogens, and examples of host defense mechanisms and numerous viral counterdefense mechanisms exist. Recent studies indicate that RNA silencing might also play an active role in the context of a mammalian virus infection. We show here that a mammalian virus, human adenovirus, interacts with RNA silencing pathways during infection, as the virus produces microRNAs (miRNAs) and regulates the expression of ...


Rna Silencing Pathways In Schizosaccharomyces Pombe And Drosophila Melanogaster: A Dissertation, Alla A. Sigova Nov 2006

Rna Silencing Pathways In Schizosaccharomyces Pombe And Drosophila Melanogaster: A Dissertation, Alla A. Sigova

GSBS Dissertations and Theses

RNA silencing is an evolutionary conserved sequence-specific mechanism of regulation of gene expression. RNA interference (RNAi), a type of RNA silencing in animals, is based on recognition and endonucleolytic cleavage of target mRNA complimentary in sequence to 21-nucleotide (nt) small RNA guides, called small interfering RNAs (siRNAs). Another class of 21-nt small RNAs, called micro RNAs (miRNAs), is endogenously encoded in eukaryotic genomes. Both production of siRNAs from long double-stranded RNA (dsRNA) and biogenesis of miRNAs from hairpin structures are governed by the ribonuclease III enzyme Dicer. Although produced as duplex molecules, siRNAs and miRNAs are assembled into effector complex ...


Biochemical Mechanism Of Rna Interference In Higher Organisms: A Dissertation, Dianne S. Schwarz Aug 2005

Biochemical Mechanism Of Rna Interference In Higher Organisms: A Dissertation, Dianne S. Schwarz

GSBS Dissertations and Theses

RNA interference (RNAi) is an evolutionarily conserved, sequence-specific gene silencing pathway found in eukaryotes, in which 21-nucleotide, small interfering RNAs (siRNAs) guide destruction of a corresponding target mRNA. RNAi is a natural mechanism for both genome surveillance and gene regulation. Moreover, siRNAs can be transfected into cultured mammalian cells, causing the sequence-specific ‘knock down’ of an mRNA. My work in the Zamore lab has centered around the Drosophilain vitro system and cultured mammalian cells to study the RNA interference (RNAi) pathway. small interfering RNAs (siRNAs) are incorporated into the RNA-induced silencing complex (RISC), which culminates in the cleavage of ...


Hiv-1 Gene Expression: Transcriptional Regulation And Rna Interference Studies: A Dissertation, Ya-Lin Chiu Jan 2003

Hiv-1 Gene Expression: Transcriptional Regulation And Rna Interference Studies: A Dissertation, Ya-Lin Chiu

GSBS Dissertations and Theses

Gene expression of human immunodeficiency virus type-1 (HIV-1), which causes Acquired Immunodeficiency Syndrome (AIDS), is regulated at the transcriptional level, where negative factors can block elongation that is overcome by HIV Tat protein and P-TEFb. P-TEFb, a positive elongation transcription factor with two subunits, CDK9 and Cyclin T1 (CycT1), catalyzes Tat-dependent phosphorylation of Ser-5 in the Pol II C-terminal domain (CTD), allowing production of longer mRNAs. Ser-5 phosphorylation enables the CTD to recruit mammalian mRNA capping enzyme (Mce1) and stimulate its guanylyltransferase activity. This dissertation demonstrates that stable binding of Mce1 and cap methyltransferase to template-engaged Pol II depends on ...


Analysis Of Rna Interference In C. Elegans: A Dissertation, Alla Grishok Sep 2001

Analysis Of Rna Interference In C. Elegans: A Dissertation, Alla Grishok

GSBS Dissertations and Theses

RNA interference (RNAi) in the nematode Caenorhabditis elegans is a type of homology-dependent post-transcriptional gene silencing induced by dsRNA. This dissertation describes the genetic analysis of the RNA interference pathway and inheritance properties associated with this phenomenon. We demonstrate that the RNAi effect can be observed in the progeny of the injected animal for at least two generations. Transmission of the interference effect occurs through a dominant extragenic agent. The wild-type activities of the RNAi pathway genes rde-l and rde-4 are required for the formation of this interfering agent but are not needed for interference thereafter. In contrast, the rde-2 ...