Elementos de transposição são sequências de DNA que possuem a habilidade de se transpor. Estes elementos são divididos em dois grupos de acordo com seu mecanismo de transposição. Aqueles que se transpõem diretamente a partir de moléculas de DNA são chamados de transposons e os que se transpõem através de um intermediário de RNA são chamados de retrotransposons. Os retrotransposons são encontrados nos mais diversos organismos e sua atividade é altamente regulada pelos seus hospedeiros, uma vez que no processo de transposição, efeitos deletérios decorrentes da inserção/inativação de genes podem resultar em eventos drásticos para o organismo. Foram descritos dois mecanismos de controle desses elementos. O primeiro consiste na inativação da transcrição do elemento (inibição transcricional), e o segundo consiste na degradação do mRNA pelo sistema de interferência de RNA (Inibição pós-transcricional). Apesar de serem regulados negativamente, foi verificado em plantas de fumo a indução da expressão do retrotransposon Tnt1 em situações de estresse como em injúrias e processo de regeneração. De forma geral a ativação destes é bastante intrigante e durante diversos anos estes elementos foram considerados “DNA lixo" ou “DNA egoísta". O presente trabalho procurou avaliar a função destes elementos na regeneração de plantas de fumo através de sua inibição nessa fase. A inibição foi realizada através de construções que induzem o sistema de inteferência de RNA, clivando o mRNA dos elementos provindos de qualquer cópia no genoma. Foram selecionados indivíduos geneticamente modificados que apresentaram deficiência de desenvolvimento, ausência de raízes e resposta de hipersensibilidade. / Transposable elements are DNA sequences that have the ability of transposition. These elements are divided in two groups according to their transposition mechanism. Those that transpose directly as a DNA molecule are called transposons and those that are dependent on a RNA intermediate are called retrotransposons. Retrotransposons are found in various organisms and are highly regulated by their hosts. In many cases of transposition, the retroelements may be inserted into other genes, causing deleterious effects and damages to the organism. Two control mechanisms have been described in previous studies. One consists of the element transcription inactivation (transcriptional inhibition), and the second consists of the mRNA degradation by the RNA interference system (post-transcriptional inhibition). Despite the negative regulation of these elements, the induction of expression of Tnt1 retrotransposon was described in tobacco plants under stress situations such as injuries and regeneration. The function of the retroelements, in general, is not yet known although they are usually considered as “junk DNA" or “selfish genes". Therefore, studies on the inactivation of these elements are particularly interesting. In this work, the function of these elements in tobacco plants was studied by the use of RNAi methods and analysis during the regeneration phase and plant development were evaluated. Phenotypes are described which are related to previous expression studies supporting a specific role for these elements not yet described.

Elementos de transposição são sequências de DNA que possuem a habilidade de se transpor. Estes elementos são divididos em dois grupos de acordo com seu mecanismo de transposição. Aqueles que se transpõem diretamente a partir de moléculas de DNA são chamados de transposons e os que se transpõem através de um intermediário de RNA são chamados de retrotransposons. Os retrotransposons são encontrados nos mais diversos organismos e sua atividade é altamente regulada pelos seus hospedeiros, uma vez que no processo de transposição, efeitos deletérios decorrentes da inserção/inativação de genes podem resultar em eventos drásticos para o organismo. Foram descritos dois mecanismos de controle desses elementos. O primeiro consiste na inativação da transcrição do elemento (inibição transcricional), e o segundo consiste na degradação do mRNA pelo sistema de interferência de RNA (Inibição pós-transcricional). Apesar de serem regulados negativamente, foi verificado em plantas de fumo a indução da expressão do retrotransposon Tnt1 em situações de estresse como em injúrias e processo de regeneração. De forma geral a ativação destes é bastante intrigante e durante diversos anos estes elementos foram considerados “DNA lixo” ou “DNA egoísta”. O presente trabalho procurou avaliar a função destes elementos na regeneração de plantas de fumo através de sua inibição nessa fase. A inibição foi realizada através de construções que induzem o sistema de inteferência de RNA, clivando o mRNA dos elementos provindos de qualquer cópia no genoma. Foram selecionados indivíduos geneticamente modificados que apresentaram deficiência de desenvolvimento, ausência de raízes e resposta de hipersensibilidade. / Transposable elements are DNA sequences that have the ability of transposition. These elements are divided in two groups according to their transposition mechanism. Those that transpose directly as a DNA molecule are called transposons and those that are dependent on a RNA intermediate are called retrotransposons. Retrotransposons are found in various organisms and are highly regulated by their hosts. In many cases of transposition, the retroelements may be inserted into other genes, causing deleterious effects and damages to the organism. Two control mechanisms have been described in previous studies. One consists of the element transcription inactivation (transcriptional inhibition), and the second consists of the mRNA degradation by the RNA interference system (post-transcriptional inhibition). Despite the negative regulation of these elements, the induction of expression of Tnt1 retrotransposon was described in tobacco plants under stress situations such as injuries and regeneration. The function of the retroelements, in general, is not yet known although they are usually considered as “junk DNA” or “selfish genes”. Therefore, studies on the inactivation of these elements are particularly interesting. In this work, the function of these elements in tobacco plants was studied by the use of RNAi methods and analysis during the regeneration phase and plant development were evaluated. Phenotypes are described which are related to previous expression studies supporting a specific role for these elements not yet described.

The sea lamprey (Petromyzon marinus) is an invasive pest in the Laurentian Great Lakes. Current pest control programs for sea lamprey are expensive and can produce off-target effects. RNA interference (RNAi) based technologies have the potential to augment existing sea lamprey control programs. In this study, sea lamprey embryos and larvae (10-100 mm) were treated with small interfering RNAs (siRNAs) targeting housekeeping genes (β actin, α actinin, calmodulin, elongation factor 1α, splicing factor 1 and γ tubulin) and gene expression was measured. Three of the siRNA embryo treatments (α-actinin, calmodulin, splicing factor 1) produced significant knockdown and increased mortality while treatment with tubulin siRNA produced only knockdown. Larval siRNA treatments produced knockdown of four genes (α-actinin, calmodulin, elongation factor 1α, splicing factor 1) and increased mortality with tubulin-siRNA treatments. Differential effects of siRNA treatment across life stage and gene target are discussed. These results suggest that siRNAs have potential uses as species-specific pesticides in sea lamprey.

The sea lamprey (Petromyzon marinus) is an invasive pest in the Laurentian Great Lakes. Current pest control programs for sea lamprey are expensive and can produce off-target effects. RNA interference (RNAi) based technologies have the potential to augment existing sea lamprey control programs. In this study, sea lamprey embryos and larvae (10-100 mm) were treated with small interfering RNAs (siRNAs) targeting housekeeping genes (β actin, α actinin, calmodulin, elongation factor 1α, splicing factor 1 and γ tubulin) and gene expression was measured. Three of the siRNA embryo treatments (α-actinin, calmodulin, splicing factor 1) produced significant knockdown and increased mortality while treatment with tubulin siRNA produced only knockdown. Larval siRNA treatments produced knockdown of four genes (α-actinin, calmodulin, elongation factor 1α, splicing factor 1) and increased mortality with tubulin-siRNA treatments. Differential effects of siRNA treatment across life stage and gene target are discussed. These results suggest that siRNAs have potential uses as species-specific pesticides in sea lamprey.

Die subakute sklerosierende Panenzephalitis (SSPE), eine durch das Masernvirus (MV) verursachte sogenannte „ slow virus “ Infektion des zentralen Nervensystems, ist eine progrediente chronische Erkrankung, die zum Tod führt und bisher medikamentös nicht heilbar ist. Da die RNAi-Strategien grundsätzlich zur Inhibition von Viren in Säugetierzellen geeignet sind, stellt die RNAi eine Möglichkeit dar, die Infektion auf molekularer Ebene anzugreifen. Dafür wurden verschiedene miRNA-Expressionskassetten, welche gegen zwei Sequenzen im MV- Hämagglutinin-Gen (H) und sechs Sequenzen im MV-Nukleokapsid-Gen (N) gerichtet sind, konstruiert und in MV infizierte Zellen eingebracht. Diese miRNAExpressionskassetten wurden auf zwei verschiedenen Wegen in die Zelle eingebracht: Zum einen wurden sie über ein miRNA-Expressionsplasmid (pmiR), welches in die Zellen transfiziert wird, transient exprimiert; zum anderen wurden sie durch virale Vektoren (HIV, SIV und MoMLV) stabil in die Zellen transduziert. Dies ermöglicht die Integration der miRNAExpressionskassette in das Genom der Zelle und dadurch die Expression der miRNAs für einige Wochen. In erster Linie zielt die Wirkung der RNA-Interferenz auf die Degradierung der spezifischen MV-mRNAs. Diese Degradierung konnte mit Hilfe quantitative Reverse Transkription real time-PCR (qRT-PCR) nachgewiesen werden. Die transiente Expression der verschiedenen miRNAs gegen das MV-N-Gen bzw. MV-H-Gen führte in jedem Fall zu einer Reduktion der viralen genspezifischen mRNAs. Die Reduktion der MV spezifischen mRNA betrug 99,8% für das MV-N-Gen und 91,2% für das MV-H-Gen. Die Wirkung der RNA-Interferenz zielt am Ende auf die Reduktion der neu gebildeten infektiösen Viruspartikel und ihrer Verbreitung in der Zellkultur, welche die spezifische miRNA gegen MV-N oder MV-H exprimiert. Dieser Effekt konnte nur durch Plaque-Assay überprüft werden. Die Plaque-Assays, die mit den Überständen der miRNA-behandelten Zelllinien durchgeführt wurden, zeigten ebenfalls eine Reduktion der neu gebildeten infektiösen MV-Partikeln von 97,6% für die miRNA gegen das MV-H-Gen und 99,0 % für die miRNA gegen das MV-N-Gen. Die intrazelluläre Expression der miRNAs führte zu einer Hemmung der Virus-Ausbreitung in MV-infizierten Zellen. Die Reduktion betrug hier durch die Expression der miRNA-N10 98,8% und durch die Expression der miRNA-H2 80,0%. Hier zeigte sich, dass die Inhibition der viralen Proteinsynthese durch den RNAi-Mechanismus auch die Verbreitung der MVInfektion durch Zell-Zell-Fusion behindern kann. Dies zeigte sich durch die verringerte Bildung von Plaques bzw. Synzytien in miRNA-behandelten Zelllinien. Die vorliegende Arbeit zeigte, dass RNAi effektive gegen MV-Infektion in Zellkultur eingesetzt werden kann. Als nächster Schritt sollte daher dieser RNAi-Effekt im etablierten Tiermodell ausgetestet werden.

Axon guidance is the developmental process where developing neurons navigate their processes based on attractive and repulsive cues. C. elegans has been an instrumental model in the study of neurobiology with one of the key benefits being a relatively simple nervous system which comprises of only 302 neurons. The Eph Receptors are a canonical class of axon guidance molecules and in C. elegans there is only Eph receptor VAB-1. To understand axon guidance it is useful to study the mechanosensory neurons, and in particular a pair of neurons called the PLM (Posterior Lateral Microtubule). In this thesis I undertook a series of projects involving new techniques, and identified gene products that may interact with VAB-1 in the PLMs. I demonstrate that the use of the PLM Length and PLM/Body length ratio at the L1 stage offers an improved way of detecting axon guidance phenotypes. I show proof of concept that use of a light induced cell ablation technique can help study the developing nervous system. Further, I show that with the use of a tissue specific RNAi technique the role of lethal genes in axon guidance can be analyzed. Finally I conducted a screen that identified new effectors of the VAB-1 signal transduction pathway. / Thesis (Master, Biology) -- Queen's University, 2012-10-29 11:31:54.764

Long double-stranded RNA (dsRNA) is a unique structure formed during viral replication or transcription of repetitive elements. Mammalian cells evolved several mechanisms how to respond to dsRNA. dsRNA can be engaged in one of three pathways: interferon response, RNA editing, and RNA interference (RNAi). RNAi is evolutionary conserved effect of dsRNA, which results in sequence-specific messenger RNA degradation. However, in mammals, RNAi is functional only in mouse oocytes, which express truncated version of Dicer (DicerO ). In somatic cells, dsRNA triggers sequence-independent interferon pathway. The main aim of this Master's thesis was to examine how specific double-stranded RNA-binding proteins (DRBPs) influence distribution of long dsRNA into RNAi and sequence-independent pathways. We used a luciferase-based reporter RNAi assay to monitor sequence-specific and sequence-independent effects of dsRNA co-expressed with selected DRBPs. Our results suggest that none of the tested DRBPs is sufficient to stimulate RNAi in somatic cells. Interestingly, the overexpression of either TARBP2 or PACT suppressed RNAi in cells expressing DicerO . Moreover, microRNA pathway, which employs the same protein factors as RNAi, is not inhibited by TARBP2 or PACT. Therefore, we propose that DRBPs overexpression...

... / The goal of the project was to establish knock down of mRNA in human mesenchymal stem cells. Since these cells are difficult to transfect, a viral approach is needed to achieve sufficient expression of e. g. shRNA in a high percentage of cells to allow for an efficient silencing of corresponding mRNAs. For this purpose for every gene product of interest, a number of shRNA clones have to be tested to detect an individual shRNA with sufficient efficacy. Lentiviral systems for shRNA approaches have recently become available. The principal advantage of the lentiviral system is that it allows gene silencing in nondividing cells and therefore expands the usefulness of the RNAi-based gene silencing system. Lentivirus-delivered shRNAs are capable of specific, highly stable and functional silencing of gene expression in a variety of cell types. Since the viral transfection of MSCs is a time consuming process that involves transfection of 293 FT cells plus transduction of target cells, for this thesis the following approach was chosen: genes of interest were checked for expression in 293FT cells by RT-PCR. These gene products can be silenced in 293FT cells simply by transfection of shRNA clones and efficacy was subsequently tested by RT-PCR. Beyond this thesis then the project can proceed with effective clones to transduce primary MSCs with individual shRNA clones identified as effective silencing tool in this thesis.

Thesis (Master, Biology) -- Queen's University, 2010-07-10 14:30:31.444 / Neuropeptides are found in the nervous systems of both invertebrates and vertebrates. The function of many of these neuropeptides and how they interact with their receptors is largely unknown. Through the use of genetically manipulatable organisms such as Drosophila, we are able to analyze the function and roles of these peptides to help us better understand their regulation of behavioural and physiological functions. To functionally analyze genes that encode neuropeptides and their receptors in D. melanogaster, we knocked down genes via RNAi in specific tissues using the UAS/Gal4 system. RNAi lines for FGLamide-allatostatins (ASTs) and their cognate receptors, Dar-1 and Dar-2 were examined for foraging defects, for transcript levels, and triglyceride content. In addition, RNAi lines for Drosophila PISCF-AST and its cognate receptors, Drostar-1 and Drostar-2 alongside sNPF receptor and a neuropeptide-Y like receptor were analyzed for their effect on juvenile hormone biosynthesis. Dar-1 and FGLamide-AST RNAi lines showed a significant foraging defect similar to that of the dweller phenotype seen in NPR-9 (AST receptor-like) mutants in C. elegans. These lines also show a decrease in for transcript levels suggesting that the foraging defect seen may also be tied into rover and sitter phenotypes. Unlike npr-9 mutants in the worm, larvae showing a foraging defect on food did not show an accumulation of intestinal lipids. Ring gland expression of FGLamide-AST, Dar-1, and Dar-2 RNAi did not show a significant effect on JH biosynthesis in either stage. Similar to these results, ubiquitous expression of PISCF-AST RNAi did not show an effect on JH. However, ubiquitous expression of Drostar-1 and Drostar-2 RNAi showed an inhibitory effect on JH biosynthesis in larvae, but a stimulatory effect in adult females. Also, Drm-sNPF-R RNAi showed a stimulatory effect in 7-day old adult females, while NepYr RNAi showed an inhibitory effect in both stages. Our data suggests that PISCF ASTs regulate JH biosynthesis in the fruit fly and the regulatory effect of these peptides on JH biosynthesis differs depending on the stage of the animal. The effect of sNPF and NPY receptors on JH suggests a link between JH regulation and the insulin signaling pathway. / Master

The ability to create transgenic livestock is a tremendous benefit in scientific research for many disciplines including functional genomics, pharmaceutical synthesis and development of enhanced production animals. Transgenes can either be stably or transiently expressed to alter gene function and obtain a specifically engineered phenotype. To create a transgenic bovine embryo, genetically altered somatic cells must be used in somatic cell nucleus transfer, or early 1-cell embryos (zygotes) must be microinjected with plasmid DNA or small interfering RNA (siRNA). Given the cost and skill associated with both methods, a preliminary investigation exploring alternative delivery techniques of siRNA (transient expression) into bovine zygotes with a nonhomologous Cy3 labeled siRNA (Cy3-siRNA) was first performed. It was discovered that zygotes injected with more than 50 Bmol L-1 of Cy3-siRNA fail to form a blastocoel and that, although bovine zygotes are not susceptible to chemical transfection, the trophectoderm cells of the blastocyst are. Based on this information, bovine E-cadherin gene expression was compared in day 9 blastocysts derived from either injected zygotes (day 1) or transfected blastocysts (day 7) with a Cy3 labeled E-cadherin specific siRNA (Cy3-siEcad) to determine 1) if gene suppression in zygotes injected with 25 Bmol L-1 Cy3-siEcad continues during embryo development up to hatching, and 2) if blastocysts transfected at a ratio of 9:6 with GeneJammer® truly experience gene knock down after siRNA transfection capable of maintaining suppression to day 9. Quantitative PCR indicated blastocysts transfected with Cy3-siEcad had a significant 15.3% decrease (P < 0.05) in E-cadherin mRNA at day 9 compared to the injected zygotes. Protein fluorescence analysis from immunocytochemistry of whole mounted day 9 blastocysts revealed injected zygotes accumulated significantly less E-cadherin protein (67.7%) than the transfected blastocysts (P < 0.05). From these data, it can be concluded that although siRNA injection may be capable of knocking down gene expression for the first 7 days of embryonic development, it does not persist to the hatching stage; however, blastocysts transfected at day 7 do express altered gene expression in the trophectoderm which can continue through embryonic hatching events.