S100A8 is a highly-expressed calcium-binding protein in neutrophils and activated macrophages, and has proposed roles in myeloid cell differentiation and host defense. Functions of S100A8 are not fully understood, partly because of difficulties in generating S100A8 knockout mice. Attempts to silence S100A8 gene expression in activated macrophages and fibroblasts using RNA interference (RNAi) technology were unsuccessful. Despite establishing validated small interfering RNA (siRNA) systems, enzymaticallysynthesized siRNA targeted to S100A8 suppressed mRNA levels by only 40% in fibroblasts activated with FGF-2+heparin, whereas chemically-synthesized siRNAs suppressed S100A8 driven by an S100A8-expression vector by ~75% in fibroblasts. Suppression of the gene in activated macrophages/fibroblasts was low, and some enzymatically-synthesized siRNAs to S100A8, and unrelated siRNA to GAPDH, induced/enhanced S100A8 expression in macrophages. This indicated that S100A8 may be upregulated by type-1 interferon (IFN). IFN-β enhanced expression, but did not directly induce S100A8. Poly (I:C), a synthetic dsRNA, directly induced S100A8 through IL-10 and IFN-dependent pathways. Induction by dsRNA was dependent on RNA-dependent protein kinase (PKR), but not cyclooxygenase-2, suggesting divergent pathways in LPS- and dsRNA-induced responses. New mechanisms of S100A8 gene regulation are presented, that suggest functions in anti-viral defense. S100A8 expression was confirmed in lungs from influenza virus-infected mice and from a patient with severe acute respiratory syndrome (SARS). Multiple pathways via mitochondria mediated S100A8 induction in LPS-activated macrophages; Generation of reactive oxygen species via the mitochondrial electron transport chain and de novo synthesis of ATP may be involved. This pathway also regulated IL-10 production, possibly via PKR. Extracellular ATP and its metabolites enhanced S100A8 induction. Results support involvement of cell stress, such as transfection, in S100A8 expression. A breast tumor cell line (MCF-7) in which the S100A8 gene was silenced, was established using micro RNA technology; S100A8 induction by oncostatin M was reduced by >90% in stably-transfected cells. This did not alter MCF-7 growth. The new approach to investigate the role of S100A8 in a human tumor cell line may assist in exploring its functions and lead to new studies concerning its role in cancer.

Thesis (Ph.D. in Molecular Biology) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 137-151). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 6 uppsatser.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Global food security is hampered by a variety of insects/pest and plant diseases. In wheat, the Russian wheat aphid (RWA) is a significant pest problem in many areas of the world. Wheat has developed defensive mechanisms against the RWA over time which are activated upon feeding. One such mechanism is the hypersensitive response (HR) which is effective against phloem-feeding insects i.e. D. noxia (Diuraphis noxia, Kurdjumov, RWA). In this study, two genes associated with the hypersensitive response i.e. ascorbate peroxidase (APX) and glutathione S transferase (GSTF6b) were investigated to elucidate their function in the defensive mechanism of wheat using a reverse genetic approach i.e. particle bombardment. This study has succeeded in the established of a tissue culture and transformation system which generated three genetically modified wheat plants with decreased resistance to RWA feeding due to gene silencing. The establishment of this system enabled to test the association of defensive related genes in wheat to RWA resistance. Expression analysis performed on obtained transgenics before and after RWA infestation reavealed that the silenced plants were more susceptible to RWA feeding. Chlorosis was observed in the Gamtoos-S-APX transgenic plant which is an indicator of oxidative damage to the photosynthetic machinery of the plant. Decreased GSTF6b transcripts was found in the transgenic Gamtoos-S-GSTF6b and transgenic Gamtoos-R-GSTF6b transgenic plants but no visible symptoms of infestation was observed in these two plants. Resistance breeding could be strengthened by developing broad spectrum resistance plants by incorporating wheat defensive related genes with known function into the breeding programs. The use of this transformation system will allow rapid identification and introduction of agronomically important genes by upregulating these genes to enhance bread wheat against aphid infestation.

abstract: Gold nanoparticles have emerged as promising nanomaterials for biosensing, imaging, photothermal treatment and therapeutic delivery for several diseases, including cancer. We have generated poly(amino ether)-functionalized gold nanorods (PAE-GNRs) using a layer-by-layer deposition approach. Sub-toxic concentrations of PAE-GNRs were employed to deliver plasmid DNA to prostate cancer cells in vitro. PAE-GNRs generated using 1,4C-1,4Bis, a cationic polymer from our laboratory demonstrated significantly higher transgene expression and exhibited lower cytotoxicities when compared to similar assemblies generated using 25 kDa poly(ethylene imine) (PEI25k-GNRs), a current standard for polymer-mediated gene delivery. Additionally, sub-toxic concentrations of 1,4C-1,4Bis-GNR nanoassemblies were employed to deliver expression vectors that express shRNA ('shRNA plasmid') against firefly luciferase gene in order to knock down expression of the protein constitutively expressed in prostate cancer cells. The roles of poly(amino ether) chemistry and zeta-potential in determining transgene expression efficacies of PAE-GNR assemblies were investigated. The theranostic potential of 1,4C-1,4Bis-GNR nanoassemblies was demonstrated using live cell two-photon induced luminescence bioimaging. The PAE class of polymers was also investigated for the one pot synthesis of both gold and silver nanoparticles using a small library poly(amino ethers) derived from linear-like polyamines. Efficient nanoparticle synthesis dependent on concentration of polymers as well as polymer chemical composition is demonstrated. Additionally, the application of poly(amino ether)-gold nanoparticles for transgene delivery is demonstrated in 22Rv1 and MB49 cancer cell lines. Base polymer, 1,4C-1,4Bis and 1,4C-1,4Bis templated and modified gold nanoparticles were compared for transgene delivery efficacies. Differences in morphology and physiochemical properties were investigated as they relate to differences in transgene delivery efficacy. There were found to be minimal differences suggestion that 1,4C-1,4Bis efficacy is not lost following use for nanoparticle modification. These results indicate that poly(amino ether)-gold nanoassemblies are a promising theranostic platform for delivery of therapeutic payloads capable of simultaneous gene silencing and bioimaging. / Dissertation/Thesis / Doctoral Dissertation Bioengineering 2014

Orientadores: Iscia Teresinha Lopes-Cendes, Ivan de Godoy Maia / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-04T19:04:59Z (GMT). No. of bitstreams: 1 Pereira_TiagoCampos_D.pdf: 3895694 bytes, checksum: d999bfc92e9a2e2c757db34bbfc7d7fa (MD5) Previous issue date: 2005 / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular

Orientadores: Michel Georges Albert Vincentz, Júlio Cézar de Mattos Cascardo / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-16T03:26:24Z (GMT). No. of bitstreams: 1 Santos_AnaCristinaCaribedos_D.pdf: 6201793 bytes, checksum: ec32c93bb2e8f1111e029a6a82d77abd (MD5) Previous issue date: 2010 / Resumo: O fungo Moniliophthora perniciosa, agente causal da doença "vassoura-de-bruxa" (VB) do cacaueiro (Theobroma cacao L.) é responsável pela diminuição da produção de cacau no Brasil e em outras regiões da América do Sul e Central. Esse decréscimo está diretamente associado a graves problemas sociais e ambientais, particularmente nas regiões amazônica e Sul da Bahia. A interação cacau-M. perniciosa envolve mecanismos genéticos complexos e pouco estudados em nível molecular. Portanto, este trabalho objetivou estabelecer uma metodologia de silenciamento gênico por RNA de interferência (RNAi) em M. perniciosa, visando análises funcionais de genes relacionados à sua patogenicidade. RNAi é uma eficiente ferramenta para reprimir a expressão gênica experimentalmente, portanto, uma alternativa tecnológica muito promissora. Devido ao fato de RNAi ser um mecanismo pós-transcricional que promove o silenciamento de genes com alta especificidade e de modo sistêmico é considerado uma ferramenta versátil para o estudo de fungos filamentosos heterocarióticos. O trabalho foi conduzido em três etapas integradas: 1) estabelecimento e avaliação das condições adequadas para eletroporação e regeneração de protoplastos de M. perniciosa, como método alternativo para a transfecção de DNA e de dsRNAs nos experimentos de transformação e de silenciamento do fungo, respectivamente; 2) produção de uma linhagem transgênica de M. perniciosa que expresse o gene heterólogo gfp e o estabelecimento da metodologia de silenciamento por RNAi, usando o gene gfp como modelo; 3) silenciamento mediado por dsRNAs de dois genes endógenos de M. perniciosa, vinculados a detoxificação de ROS e a produção de corpos de frutificação. Os parâmetros que promoveram a maior freqüência de regeneração dos protoplastos eletroporados foram a aplicação de um pulso de 1.5 kV. Em relação à transformação de M. perniciosa, o gene repórter gfp foi estavelmente introduzido no genoma do fungo e posteriormente silenciado por transfecção de gfpdsRNA sintetizado in vitro, comprovando a operacionalidade do mecanismo de RNAi em M. perniciosa. Em adição, os genes endógenos MpPRX1 e MpHYD que codificam para peroxiredoxina e hidrofobina, respectivamente, foram silenciados, usando dsRNAs específicos e apresentaram níveis reduzidos de mRNAs que variaram de 18% a 98% quando comparados aos controles. O silenciamento dos genes gfp e MpPRX1 foi corroborado pela redução da fluorescência de GFP e da atividade da peroxidase, bem como da sobrevivência do micélio submetido a H2O2, respectivamente. O silenciamento de gfp e de MpPRX1 persistiu por aproximadamente quatro meses, indicando silenciamento sistêmico. O estabelecimento da técnica de silenciamento gênico por RNAi em M. perniciosa abre novos caminhos para explorar funcionalmente o genoma deste fitopatógeno, bem como o desenvolvimento de mecanismos que bloqueiem ou diminuam a ação deste / Abstract: The fungus Moniliophthora perniciosa, causal agent of cacao (Theobroma cacao L.) witches' broom (WB) disease, is responsible for the decrease in cacao production in Brazil and other regions of South and Central America. This decrease is directly associated to severe social and ecological problems, particularly in the Amazon and Southern Bahia regions. The cacao-M. perniciosa interaction involves complex genetic mechanisms little studied at the molecular level. Therefore, this study aimed to establish a methodology of gene silencing by RNA interference (RNAi) in M. perniciosa, aiming functional analyses of genes related to his pathogenicity. RNAi is a powerful tool to experimentally suppress or regulate gene expression, therefore, a very promising technological alternative. Because RNAi is a post-transcriptional event that promotes gene silencing with high specificity and in a systemic way, it is considered a versatile tool for the study of heterokaryotic fungi. The work was conducted in three integrated steps: 1) establishment and evaluation of appropriate conditions for electroporation and regeneration of M. perniciosa protoplasts, as an alternative method for transfection of transgenic DNA and dsRNAs in transformation and fungus silencing experiments, respectively; 2) production of a transgenic strain of M. perniciosa that expresses the heterologous gene gfp and the establishment of methodology for silencing by RNAi, using the gfp gene as a model; 3) silencing mediated by dsRNAs of two endogenous M. perniciosa genes linked to detoxification of ROS and the production of fruiting bodies. The parameters that promoted the highest frequency of regeneration of electroporated protoplasts were the application of one pulse of 1.5 kV. Regarding M. perniciosa transformation, the gfp reporter gene was stably inserted into the genome of the fungus and subsequently silenced by transfection of gfpdsRNA synthesized in vitro, demonstrating the operationality of the RNAi mechanism in M. perniciosa. In addition, the endogenous genes MpPRX1 and MpHYD, coding for peroxiredoxin and hydrophobin, respectively, were silenced using specific dsRNAs showing reduced levels of mRNAs ranging from 18% to 98% when compared to controls. Silencing of gfp and MpPRX1 was validated by experiments that showed correlation between reduced levels of GFP fluorescence and peroxidase activity, as well as survival of mycelium subjected to H2O2, respectively. The silencing of gfp and MpPRX1 persisted for approximately four months, indicating systemic silencing. The establishment of the gene silencing technique by RNAi in M. perniciosa opens new paths to functionally explore the genome of this pathogen and the development of mechanisms that block or decrease his action / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular

Desde seu descobrimento, o fenômeno de silenciamento gênico por RNA (RNAi) rapidamente se tornou uma técnica amplamente estudada e utilizada nos mais diversos aspectos da biologia molecular. Uma destas possibilidades é sua aplicação no campo da entomologia agrícola, mais especificamente para o controle de insetos-praga como uma alternativa de alta eficiência, especificidade e com impacto ambiental reduzido. Por meio da geração de plantas transgênicas expressando RNAi para genes essenciais de insetos-praga específicos, a ingestão destas moléculas de RNAi pelo inseto mediante herbivoria pode resultar no silenciamento do respectivo gene, resultando em fenótipos que podem variar entre perda de apetite, infertilidade ou até a morte. Neste contexto, o presente trabalho teve como objetivo provar a viabilidade de aplicação desta técnica para a interação Tomateiro x Tuta absoluta, cultura de grande expressão econômica e social no mercado nacional e internacional e que é amplamente atacada por esta praga, com prejuizos que podem alcançar a ordem dos 100% da produção. Por meio da clonagem de genes ortólogos essenciais descritos na literatura e de genes altamente expressos nos primeiros estádios larvais, após a caracterização transcriptômica em escala do inseto, foram realizados ensaios de alimentação contendo moléculas de dsRNAs que possuíam estes genes como alvo. Também, foi realizado a transformação genética de tomateiro cultivar \"Micro-Tom\" com dois destes genes (V-ATPase A e Arginina kinase) para a realização de ensaios de herbivoria. Com os resultados obtidos nestes experimentos, foram mostradas sólidas evidências da viabilidade da técnica de RNAi para o controle de Tuta absoluta, evidenciado pelo silenciamento gênico específico observado no inseto e consequentemente os efeitos nocivos deste silenciamento. / Since their discovery, the phenomenon of gene silencing by RNA ( RNAi ) has rapidly become a widely studied and used technique in the molecular biological field. One of these possible applications is in the entomology field, more specifically for the control of insect pests, as a high efficiency, specificity and with reduced environmental impact alternative. Through the generation of transgenic plants expressing dsRNA targeting essential insect genes, their ingestion by the insect and consequently the uptake of the silencing RNA, may result in specific gene silencing, resulting in a variety of phenotypes that can range from loss of appetite, infertility to death. In this context, this study aimed to prove the feasibility of this technique to control tomato leaf miner (Tuta absoluta) in tomatoes plant, a major crop worldwide and seriously attacked by this pest, with losses that can reach 100%. For the present work, orthologous genes from successfully cases of insect gene silence described in the literature, was selected together with highly expressed genes in the early larval stages of T. absoluta, chosen after the insect molecular characterization and used in feeding assays with dsRNAs molecules to targeted these genes. Also, genetic transformation of the \"Micro-Tom\" tomato cultivar with two of these genes (V-ATPase and Arginine kinase) was conducted for testing in an herbivore assay. With these two approaches was possible to get solid evidences of the feasibility of the RNAi technique to control this insect, evidenced by specific gene silencing observed and its consequent effect on pest phenotype.

A cana-de-açúcar (Saccharum spp.) é uma importante cultura na produção de alimentos e energia. Várias espécies de insetos podem causar sérios prejuízos econômicos à cultura da cana-de-açúcar. A broca da cana-de-açúcar (Diatraea saccharalis) é a praga de maior relevância por estar amplamente distribuída nas regiões canavieiras. O silenciamento gênico por RNA de interferência (RNAi) se tornou uma técnica amplamente estudada e utilizada nos mais diversos aspectos da biologia. Uma de suas aplicações é no controle de insetos-praga como uma alternativa de alta eficiência, especificidade e reduzido impacto ambiental. A ingestão de moléculas de RNA dupla fita (dsRNA) com identidade a regiões de genes essenciais de insetos-praga pode resultar no silenciamento destes genes, levando a fenótipos deficientes. Neste contexto, o presente trabalho teve como objetivo buscar genes alvos para o silenciamento com potencial para impedir o desenvolvimento normal da D. saccharalis e estabelecer uma forma de entrega do dsRNA eficiente para o teste de genes, visando assim validar o uso da técnica para a espécie. Por meio da clonagem de regiões de genes ortólogos já utilizados como alvo de silencimento em outras espécies de insetos (V-ATPase A, Receptor de Ecdisona e Arginina Kinase), e de genes com função específica identificadas após a caracterização do transcritoma de D. saccharalis (Juvenile Hormone Epoxide Hydrolase, Neverland e Quitina Sintase) foram conduzidos ensaios de RNAi. Foram realizados ensaios de dose resposta para o gene V-ATPaseem lagartas neonatas, onde a concentração selecionada por causar melhor redução na expressão do gene alvo foi de 2,5 µg µL-1. Esta concentração foi então utilizada em ensaios de alimentação para os outros genes. Os genes V-ATPase A, receptor de Ecdisona, Arginina Kinase, Juvenile Hormone Epoxide Hydrolase e Quitina Sintase apresentaram redução significativa no número de transcritos em larvas, demonstrando a viabilidade do uso de RNAi em D. saccharalisneonatas. O gene Neverland não demonstrou redução no acúmulo de transcritos nas condições trabalhadas. O gene GFP inicialmente utilizado como controle negativo apresentou variação na expressão de genes alvo, sendo desconsiderado como bom controle para D. saccharalis. O silenciamento dos genes alvo requer quantidades elevadas de dsRNA, superiores aos obtidos por transcrição in vitro, o que limita a viabilidade de ensaios com maiores replicatas e para determinar efeitos biológico. Alternativas de produção de dsRNA devem ser avaliadas para viabilizar a seleção de genes alvo efetivos / Sugarcane (Saccharum spp.) is an important crop for the production of food and bioenergy. Many insect species can cause economic losses in sugarcane. The sugarcane borer (Diatraea saccharalis) is the most important sugarcane pest, because it occurs in all production regions. Gene silencing by RNA interference (RNAi) rapidly became a widely investigated approach, adopted in various aspects of biology. One of the potential applications of RNAi is agricultural pest control, as an alternative with high efficiency, specificity and reduced environmental impact. The ingestion of double-stranded RNA (dsRNA) molecules with identity to regions of essential genes of the insect-pest can result in the target gene knock-down and, consequently, to deficient phenotypes. In the present work, target genes with the potential to affect the normal development of D. saccharaliswere searched, together with an efficient dsRNA delivery approach to test the target-genes to validate the use of the RNAi in D. saccharalis. Based on degenerated primers, expressed orthologous genes previously tested in other insect species (V-ATPase A, Ecdisone Receptor, and Arginine Kinase) were cloned,whilegenes with specific function (Juvenile Hormone Epoxide Hydrolase, Neverland, and Chitin Synthase) were identified from an in-house assembled transcriptome of D. saccharalis and cloned. A dose-response assay was conducted using the V-ATPase gene region delivered by droplets to neonate larvae, and the 2.5 µg µL-1dsRNA concentration was selected for further tests. This concentration was then used to deliver the other genes. The dsRNA version from the genes V-ATPase A, Ecdisone Receptor, Arginine Kinase, Juvenile Hormone Epoxide Hydrolase and Chitin Synthaseexhibited a significant reduction in the accumulation of transcripts, indicating the viability of RNAi to D. saccharalis in 1st instar larvae. The Neverland gene was not silenced by RNAi in the used conditions. The dsRNA of the Green Fluorescent Protein gene, used as negative control appeared to affectother gene targets. Target gene silencing require large amounts of dsRNA, more than what is achievable by in vitro transcription, which limits the viability to conduct large assays with more replicates and to determine biological effects. Alternatives to produce dsRNA need to be evaluated to enable the selection of effective target genes