Made available in DSpace on 2015-07-22T20:50:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-11-24 / O ultrassom (US) vem sendo amplamente utilizado para melhorar a eficiência de transfecção de vetores não-virais. No entanto, o mecanismo pelo qual o US promove a entrega de DNA nas células ainda é pouco entendido. Este fenômeno é normalmente atribuído a sonoporação. Porém, com base em experimentos anteriores realizados em nosso laboratório, suspeitamos que outro mecanismo esteja envolvido no processo de captação de DNA. Para estudar o mecanismo de entrega, um vetor plasmideal expressando EGFP (pEGFP-N3, 4,7 kb) foi utilizado para transfectar células NIH3T3 com um aparelho de US terapêutico sem a adição de microbolhas. Em condições de insonação de 2 W/cm2, duty cycle de 20% por 30s o US promoveu cerca de 40% de eficiência de transfecção, mas com 1 W/cm2 resultou em níveis muito baixos de transfecção. Fixados esses parâmetros, também foi avaliada a produção de espécies reativas de oxigênio (ROS), o aumento da concentração intracelular de cálcio ([Ca2+]i) e as alterações no potencial de membrana através de microscopia confocal. A produção de ROS foi aumentada durante a insonação, sendo interrompida logo que o US foi desligado. A [Ca2+]i também foi aumentada durante a exposição ao US, mas seus níveis não retornaram ao basal durante os 3 minutos de observação. Porém, 1 W/cm2 não foi suficiente para mobilizar o cálcio durante a insonação, e o influxo de cálcio teve início apenas 12 segundos após o término do US. Quando expostas ao US, as células também apresentaram mudanças no potencial de membrana atingindo um estado de hiperpolarização, retornando ao estado normal logo que o US foi desligado. A alteração desses três parâmetros pelo US sugere que a entrega de DNA plasmideal deva ocorrer por endocitose. Por fim, utilizando DNA plasmideal fluorescente, mostramos que esta molécula entra na célula via endocitose mediada por clatrina. / Ultrasound (US) has been widely used to improve the efficiency of non-viral vector transfection. However, the mechanism that enables the uptake of plasmid DNA in cells by US insonation is poorly understood, but it is typically attributed to sonoporation. Based on our previous results, we hypothesized that other mechanisms, such as endocytosis, are involved in this process. To explore the mechanism of plasmid DNA uptake, a plasmid vector expressing EGFP (pEGFP-N3: 4.7 kb) was used to transfect NIH3T3 cells using a therapeutic US without microbubbles and was monitored in real-time using a confocal microscope. We achieved about 40% transfection efficiency when we applied 2 W/cm2 with 20% of duty-cycle for 30 s, but 1 W/cm2 resulted in a very low level of transfection. In these experiments, the production of reactive oxygen species was augmented during the insonation but was stopped soon after turning off the US. Calcium influx was also augmented during the insonation, but its level did not return to basal levels following the 3-min observation period. However, 1 W/cm2 was not sufficient to mobilize calcium influx during the insonation, and calcium influx began 12 s after turning off the US. US insonation also changed the cell membrane potential to promote a hyperpolarization state, which returned to the normal state soon after turning off the US. The alteration of these parameters by US indicates the uptake of plasmid DNA by endocytosis. Finally, using a fluorescently labeled plasmid, we showed that this molecule enters into cells via clathrin-mediated endocytosis, not via caveolin-1. / TEDE / BV UNIFESP: Teses e dissertações

Resumo: O fator de transcrição Oct-4 é bem conservado entre as espécies e é conhecido por ser expresso em embriões e células-tronco embrionárias (CTE), sendo um importante marcador da pluripotência. Recentemente, foi relatado que a combinação de Oct-4 com três outros fatores de transcrição Klf-4, c-Myc e Sox2 foram capazes de reprogramar células somáticas a um estado indiferenciado pluripotente, chamadas células-tronco pluripotentes induzidas ("células iPS"), as quais apresentam várias das mesmas propriedades das CTE incluindo a pluripotência, auto-renovação e proliferação. O objetivo desse estudo foi avaliar a funcionalidade do promotor Oct-4 de eqüino em CTE de murinos. Três vetores plasmidiais expressando GFP ("green fluorescent protein") sob o controle do promotor Oct-4 de equinos, camundongo e quatro vetores lentivirais, também contendo o gene reporter GFP e os promotores Oct-4 de equinos, camundongo e humanos, pLZ2-ecOCT-EGFP (meq) (sequência equivalente de camundongos), pLZ2-ecOCT-EGFP (heq) (sequência equivalente de humanos), pLZ2-mOCT-EGFP e pLZ2-hOCT-EGFP, respectivamente, foram construídos. Todos os vetores também contêm um sítio de resistência à blasticidina que permite a seleção das células estáveis e das células transduzidas. Essas construções plasmidiais foram verificadas se funcionavam eficientemente, bem como o efeito do promotor Oct-4 em transfectar transientes e estáveis CTE. As construções com promotor Oct-4 de camundongo, humano e eqüino (sequência análoga à de camundongo) produziram somente 6% de células GFP positivas com intensidade de fluorescência (IF) >1000 pela análise em citômetro de fluxo, enquanto que o plasmídeo contendo o promotor Oct-4 de eqüino (sequência equivalente à de humanos) produziu menos células GFP positivas (>3%) com IF >1000, quando... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The pluripotency transcription factor Oct-4 is well conserved among species and is known to be expressed in embryos and embryonic stem (ES) cells; it is being an important pluripotency marker. It was recently demonstrated that the combination of Oct-4 with three other factors Klf-4, c-myc and Sox2 were able to reprogram somatic cells to a pluripotent and undifferentiated state. These cells known as induced pluripotent stem (iPS) cells share several properties with ES cells including self-renewal, proliferation and pluripotency. The aim of this study was to assess the functionality of the horse Oct-4 promoter in mouse ES cells. Three plasmids vectors expressing GFP (green fluorescent protein) under the control of the horse, mouse and four lentivirus vectors also containing reporter gene GFP and horse, mouse and human promoters, pLZ2-ecOCT-EGFP (mouse sequence equivalent), pLZ2-ecOCT-EGFP (human sequence equivalent), pLZ2-mOCT-EGFP and pLZ2-hOCT-EGFP, respectively, were built. All these vectors also contain a blasticidin resistance cassette to allow selection of transfected stable cells and transduced cells. Afterwards, to assess the functionality of the Oct-4 promoter all plasmids were tranfected the into transient and stable mouse ES cells. Constructs with mouse, human and horse (mouse analog sequence) Oct-4 promoter produced only 6% GFP positive cells with fluorescence intensity (FI)>1000 by 20 FACs assay, while plasmid horse (human analog sequence) Oct-4 promoter produced less GFP positive cells (>3%) with FI>1000, when compared with the positive control and among groups. However, GFP expression was not present in stable cells, whereas there were Blasticidin-resistant colonies-forming from 6 days post-transfection. To optimize the system in mouse ES cells, pLZ2-mOCT-EGFP and pLZ2-hOCT-EGFP lentivectors, were tested as controls. It was used HIV-1-derived... (Complete abstract click electronic access below) / Orientadora: Gisele Zoccal Mingoti / Coorientador: Joaquim Mansano Garcia / Banca: César Roberto Esper / Banca: Flávio Vieira Meirelles / Banca: Áureo Evangelista Santana / Banca: Simone Cristina Méo Niciura / Doutor

Made available in DSpace on 2014-08-20T13:32:59Z (GMT). No. of bitstreams: 1 tese_vinicius_farias_campos.pdf: 556217 bytes, checksum: 4c9dc7811c567f171aaebfafce2d2254 (MD5) Previous issue date: 2011-07-29 / Nanobiotechnology has provided new scientific and technological knowledge in distinct areas making it a priority area of research in developed and developing countries. The sperm-mediated gene transfer (SMGT) technique may become more simple, efficient and cost-effective technique for the generation of transgenic animals. The development of nanocomposites able to carry foreign DNA into the nucleus of cells with greater efficiency allows techniques such as SMGT be improved. The NanoSMGT is a technique used to generate transgenic animals in which nanotechnology is used to enhance the ability of sperm to capture exogenous DNA. The objective of this study was to determine whether cationic nanopolymer or halloysite clay nanotubes are able to transfect the exogenous DNA to unsorted and sex-sorted bovine sperm then evaluate whether these sperm are able to transmit transgene to in vitro fertilized bovine embryos. Using real-time PCR, we found that the cationic nanopolymer is capable of introducing exogenous DNA into unsorted and sex-sorted bovine sperm without negative effects to sperm motility and viability. Was also demonstrated for the first time that cationic nanopolymer or halloysite clay nanotubes are able to increase both the sperm DNA transfection of as the transmission of the transgene to bovine embryos produced in vitro. These results demonstrate that NanoSMGT can be a viable technique for producing transgenic bovine embryos. / A nanobiotecnologia tem proporcionado novos avanços científicos e tecnológicos em diversas áreas do conhecimento tornando-se assim área de pesquisa prioritária em países desenvolvidos e em desenvolvimento. A transferência gênica mediada por espermatozóides (SMGT) poderá se tornar a técnica mais simples, eficiente e com melhor custo-benefício para a geração de animais transgênicos. O desenvolvimento de nanocompósitos capazes de carrear o DNA exógeno para o interior de células com maior eficiência permite que técnicas como a SMGT sejam aperfeiçoadas. A NanoSMGT é uma técnica utilizada para a geração de animais transgênicos onde a nanotecnologia é utilizada para incrementar a habilidade dos espermatozóides em capurar o DNA exógeno. O objetivo do presente trabalho foi de verificar se nanopolímero catiônico ou nanotubos de haloisita são capazes de transfectar o DNA exógeno para o interior de espermatozóides bovinos sexados e não sexados e em seguida verificar se estes espermatozóides transfectados são capazes de gerar embriões bovinos transgênicos. Utilizando PCR em tempo real, verificou-se que o nanopolímero catiônico é capaz de introduzir o DNA exógeno em espermatozóides bovinos sexados e não sexados sem danos para a motilidade e viabilidade espermática. Também foi demonstrado pela primeira vez que o nanopolímero catiônico ou os nanotubos de haloisita são capazes de incrementar tanto a transfecção de DNA em espermatozóides como a transmissão do transgene para embriões bovinos produzidos in vitro. Estes resultados demonstram que a NanoSMGT pode ser uma técnica viável para a produção de embriões bovinos transgênicos.

T lymphocytes can be rendered specific against a wide range of antigens by the genetic transfer of a chimeric receptor, a fusion between the antigen-binding domain of an antibody and the signalling domain of a T cell receptor. The use of such chimeric T lymphocytes has shown promising results for cancer therapy. Previous experiments in our laboratory have shown low rates of gene transfer using retroviral vectors. In this study, investigations have been done to increase the number of genetically modified cells. Different enhancers such as PLL and polybrene have previously been used in combination with retroviral transduction. The optimal retroviral protocol in this study showed to be the use of retrovectors produced with twice the normal concentration of the plasmids encoding env and gag-pol rather than the use of the enhancers. A 6-day pre stimulation of T lymphocytes prior transduction together with a centrifugation step increased the rate of modified cells even further. Alternative approaches of gene transfer were also investigated, including plasmid transfection and adenoviral transduction. While transfection protocols yielded low numbers of modified cells, adenoviral vectors showed the highest rate of gene transfer. / Cancer är den sjukdom som idag, efter hjärt-kärl-sjukdomar, kräver flest dödsfall i i-länder. Som en alternativ behandlingsmetod mot cancer pågår just nu forskning om genetiskt förbättrade immunceller, s.k. chimära T lymfocyter, skulle kunna användas för att döda tumörceller. De chimära cellerna är utrustade med en konstgjord receptor som är en fusion av en antikropp och en signalkedja. Det gör att cellerna kan riktas mot ett brett urval av cancertyper. Att få cellerna att ta upp generna som behövs för den konstgjorda receptorn har visats sig vara problematiskt. Den här studien har därför som mål att förbättra cellernas förmåga att ta upp gener. För detta har vi använt oss av retrovirus- och adenovirus-system tillsammans med försök att få cellerna att spontant ta upp generna, sk. plasmid-transfektion. Studien har visat att de båda virussystemen ger högst antal modifierade celler. Olika substanser som tidigare har visat sig förhöja graden av gentillförsel har testats, men vår studie har visat att tillverkningen av virusvektorerna har större påverkan på resultaten än någon av de olika hjälpmedlen.

SUMMARYThe diversity of cancer molecular origins associated with the genetic variability of patients has encouraged the development of chemotherapeutic treatments adapted not only to the target tumor, but also to a specific patient. This personalized strategy is based on cancer biomarkers allowing a better identification and characterization of each tumor where predictive biomarkers provide the distinction between various factors indicative of the response to the treatment. In this context, several studies highlighted the role of the solute carrier transporter family 22 (solute carriers 22 or SLC22) in the uptake of platinum anticancer drugs. This mechanism being not well understood, our work intends to establish the potential role of SLC22 member A18 (SLC22A18) as predictive biomarker in the aim to help to a better targeted chemotherapeutic strategy for each patient. We optimized a system overexpressing SLC22A18 stably or transiently in HeLa cancer cell line. SLC22A18 expression was confirmed by qRT-PCR, western blotting, microscopy and flow cytometry. The cell lines were treated with taxane, anthracyclin, vinca alkaloid and nitrosoureas anticancer drug families. We showed that doxorubicin, camptothecin, chloroquine, tetracycline and carmustin had no effect on the cell viability assays suggesting that they are not substrates of SLC22A18. Interestingly, the cell line was sensitized in the presence of antimitotic drug with a sensitivity factor of 2.7 in the presence of paclitaxel, 1.4 with docetaxel, 1.8 with vinblastin and 2.2 in the presence of vincristine. To confirm these results, we elaborated a SLC22A18 knockdown cell line in HS683 cells using siRNA technology. The downexpression of SLC22A18 was correlated to a tendency to resist to the accumulation of paclitaxel thereby confirming the previous results. Simultaneously, a knockout cell line was established using the transcription activator-like effectors nuclease (TALEN) technology in U373 cell line. Our studies constitute a robust base of knowledge for further investigation on SLC22A18 transporter as a predictive biomarker promoting antimitotic treatment in tumors where this transporter is detected. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Abstract Human prostatic acid phosphatase (hPAP) was the first laboratory parameter used for prostate cancer diagnosis, whereas the mechanisms behind the androgen regulation and tissue-specific expression of this prostate epithelium-specific differentiation antigen are not yet clear. In this study, a transient transfection model and transgenic animal model have been set up for functional analysis of the promoter and first intron region of the hPAP gene. The promoter constructs covering the region-734/+467 of the gene were functional in both prostatic and nonprostatic cells. Although hPAP constructs included two putative AREs with in vitro AR-binding ability at -178 and +336, androgen treatment had little effect on the promoter activity of the gene in transiently transfected cells. The hPAP fragment -734/+467 could trigger the expression of the CAT reporter gene and restrict the expression mainly in the prostates of transgenic mice. The DNA-binding site with the sequence GAAAATATGATA of a regulatory protein involved in prostate-specific and androgen receptor-dependent gene expression was identified from rPB promoter. The exact same 12 bp sequence was found in the first intron +1144/+1155 of the hPAP gene. Five homologous sequence, A, B, C, D and E, were located in the -734/+467 region of the hPAP gene, where site C and E could bind the regulatory protein in EMSA. Deletion of site C decreased the transcriptional activities significantly compared to those of corresponding wild-type constructs in LNCaP cells when androgens were present. Deletion of site E or both sites D and E increased the promoter activity in LNCaP when androgens were absent. In conclusion, androgens could not directly regulate hPAP expression via receptor-binding to the AREs in LNCaP cells. The promoter and first intron fragment -734/+467 of the hPAP gene could direct and restrict the gene expression mainly in prostate epithelium. A prostatic regulatory protein binds to multiple sites with the GAAAATATGATA or homologous sequences along the regulatory areas of the hPAP gene with different affinities, modulating the prostate-specific expression of the gene in a bidirectional manner, depending on the hormone status.

in Hradec Králové Student: Johana Urbanová Supervisor: PharmDr. Jana Mandíková, Ph.D. = 38.02 μM), lowest indometacin μM

Dentre as vantagens do sistema de expressão gênica em células de drosófila observa-se ainda o estabelecimento rápido de linhagens estáveis de células que secretam de forma eficiente a proteína recombinante. Temos estabelecidas populações de células co-transfectadas S2AcGPV e transfectadas S2AcGPVHy. O objetivo deste trabalho é a comparação da expressão de GPV em células S2 co-transfectadas com os vetores pAcGPV (vetor de expressão) e o pCoHygro (vetor de seleção) ou transfectadas com um único vetor pAcGPVHygro (contendo o vetor de expressão e seleção). As populações obtidas foram analisadas em relação à expressão de GPV em imunoensaios: teste ELISA, Dot Blot, géis de SDS-PAGE, Western Blot, citometria de fluxo (FACS) e microscopia confocal. Os ensaios de imunofluorescência em citometria fluxo (FACS) realizados demonstraram que as células transfectadas e co-transfectadas estão expressando a proteína GPV. Valores entre 0,3 e 4 mg/107 células foram obtidos. Além disso, anticorpos anti-GPV foram capazes de reconhecer a proteína GPV . / Among the advantages of the system of gene expression in cells drosófila there is still the rapid establishment of stable cell lines that secrete efficiently the recombinant protein. We have established populations of cells co-transfected S2AcGPV and transfected S2AcGPVHy. The objective of this study is a comparison of the expression of GPV in S2 cells co-transfected with vector pAcGPV (vector of expression) and pCoHygro (vector of selection) or transfected with a single vector pAcGPVHygro (containing the vector of expression and selection gene). The populations were analyzed in relation to the expression of GPV in immunoassays: ELISA test, Dot Blot, the SDS-PAGE gels, Western Blot, flow cytometry (FACS) and confocal microscopy. Tests of immunofluorescence in flow cytometry (FACS) have shown that cells co-transfected and transfected are expressing the protein GPV. Values between 0.3 and 4 mg/107células were obtained. Moreover, anti-GPV were able to recognize the protein GPV.

V této diplomové práci je popsána přechodná transfekce buněk 293 HEK adaptovaných na růst při suspenzní kultivaci bez přítomnosti séra za použití polyethyleniminů (PEI). Buňky byly transfekovány plasmidem pcDNA5/SEAP, který exprimuje sekretovanou formu lidské placentální alkalické fosfatázy. K porovnání účinnosti jednotlivých transfekcí byla měřena koncentrace exprimované fosfatázy v buněčném supernatantu. Cílem této práce bylo optimalizovat různé faktory ovlivňující účinnost transfekcí s důrazem na nalezení optimálního poměru DNA:PEI.

Mitochondria are an attractive target for the design of cancer therapy. One of the mechanisms by which chemotherapeutics destroy cancer cells is by inducing apoptosis through extrinsic or intrinsic apoptotic pathways. Extrinsic pathways target cell surface receptors whilst intrinsic pathways target mitochondria. Several studies have shown cancer cell destruction through the extrinsic pathways, which target cancer-specific overexpressed growth factor receptors on the cell membrane. Although the mitochondria dependent apoptotic process is well understood, its application in cancer therapy is still not well developed. Therefore, to design an effective cancer therapy targeting mitochondria, a good understanding in mitochondria dependent apoptotic process is required. Recent developments in nanotechnology have enabled live cell investigations and non-destructive methods to obtain cellular information. The availability of such information would assist to design methods of targeted apoptosis induction. In view of this, I report on studies towards development of cancer therapy where nanoparticles (NPs) were targeted to human cell mitochondria for two purposes: (a) development of cell-imaging tools to investigate the fundamental cell biological pathways inside cells and (b) induction of apoptosis by targeting nanoparticles to mitochondria. Current medical and biological fluorescent imaging methods are mainly based on dye markers, which are limited in light emission per molecule, as well as photostability. Consequently, NPs are gaining prominence for molecular imaging because of their strong and stable fluorescence. Additionally, in order to get insight of mitochondrial molecular information, I investigated the use of optical properties of gold nanoparticles (Au NPs) for surface enhanced Raman spectroscopy (SERS). In this study, two types of Au NPs - nanospheres (Au NS) and nanorods (Au NR) were investigated. Results from this study showed the enhancement effect of Au NPs in Raman spectra of mitochondria, especially in the region from 1500 to 1600 cm-1. In this region, normal Raman spectra of mitochondria showed the presence of some understated Raman peaks probably due to the excitation wavelength dependence. Au NRs showed a larger enhancement effect than Au NS with respect to the penetration depth of the plasmonic nearfield enhancement effect. Although, the details of the enhancement mechanism are beyond the current studies, Au NPs could be enhancing vibrations of aromatic residues in proteins. This study therefore showed that Au NPs could enhance Raman spectra of mitochondria and in addition the shape of the nanoparticles had a significant effect on SERS spectra. In living cells, I investigated some transfection methods and targeting of NPs to mitochondria or cytosolic actin subunits. I tested the performance of three transfection reagents to deliver nanodiamonds (NDs) into living cells. Antibody functionalized NDs were targeted to mitochondria or cytosolic actin subunits. Three transfection reagents were used: cationic liposomes PULSin™, the cell penetrating peptide protamine, and oligosaccharide modified polypropylene imine (PPI) dendrimers. Fluorescence imaging results revealed that dendrimers were the most efficient in delivering ND conjugates to targeted organelles. Protamine-mediated transfections appeared to target ND conjugates to intended organelles, although there was a tendency of unfunctionalized NDs to be directed to the nucleus. PULSin™-mediated transfection formed ND aggregates regardless of the functionalization moiety. This reflected the unsuitability of the cationic liposome to mediate ND transfections. Further, I investigated the potential use of Au NPs for cell imaging and photothermal lysis of mitochondria inside cells. Just as above, I also tested the performance of the three-transfection reagents mentioned above on transfection capacity of Au NPs into living cells. Using transmission electron microscopy (TEM), oligosaccharide modified dendrimers showed the best transfection of functionalized Au NPs. Further experiments explored the use of the nearfield enhancement effect of Au NPs in combination with low-level laser irradiation (LLLI) to induce apoptosis in living cells. Analysis of the apoptotic process using cytochrome c release showed that Au NPs induced apoptosis most probably through mechanical disruption of the outer mitochondrial membrane. However, apoptosis was significantly accelerated in cells with mitochondrially targeted Au NRs than in cells without Au NRs. This study showed successful targeting of Au NPs to mitochondria in living cells, and demonstrated the potential of using Au NPs in combination with laser irradiation to induce the mitochondria dependent apoptotic pathway. In conclusion, the potential use of Au NPs in SERS of mitochondria and the application of NDs for cell imaging of intracellular organelles were demonstrated. Lastly, Au NPs were targeted to mitochondria in living cells and could induce apoptosis due to mechanical disruption of the outer mitochondrial membrane. Consequently, application of low-level laser irradiation to Au NP transfected cells accelerated the apoptotic process.