Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-04-25T23:51:26Z No. of bitstreams: 1 TabataLoiseCunhaLima_DISSERT.pdf: 1403767 bytes, checksum: 7424c70f286f04743b45ac7531e6829b (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-04-28T20:23:59Z (GMT) No. of bitstreams: 1 TabataLoiseCunhaLima_DISSERT.pdf: 1403767 bytes, checksum: 7424c70f286f04743b45ac7531e6829b (MD5) / Made available in DSpace on 2016-04-28T20:23:59Z (GMT). No. of bitstreams: 1 TabataLoiseCunhaLima_DISSERT.pdf: 1403767 bytes, checksum: 7424c70f286f04743b45ac7531e6829b (MD5) Previous issue date: 2015-03-13 / A dengue ? uma doen?a de notifica??o compuls?ria e cerca de 50 a 100 milh?es de casos s?o registrados anualmente. Possui amplo espectro cl?nico e ? transmitida ao homem atrav?s da picada dos mosquitos do g?nero Aedes, tendo como principal vetor a esp?cie Aedes aegypti. O agente etiol?gico da doen?a ? o v?rus dengue (DENV) pertencente ao g?nero Flavivirus, fam?lia Flaviviridae e s?o conhecidos quatro sorotipos antigenicamente distintos (DENV-1, DENV-2, DENV-3 e DENV-4). Atualmente o tratamento da dengue ? apenas de suporte, feito atrav?s de intensa hidrata??o. Ainda n?o existe uma vacina comprovadamente eficaz ou tratamento espec?fico, o estudo de poss?veis antivirais que possam diminuir a viremia no paciente ? de alt?ssima relev?ncia, uma vez que a carga viral ? um dos fatores associado ao aparecimento das formas graves da doen?a (febre hemorr?gica da dengue e s?ndrome do choque da dengue). No presente estudo n?s avaliamos o potencial antiviral de extratos brutos obtidos a partir das folhas das plantas do Nordeste brasileiro Annona muricata (graviola) e Spondias mombin (caj?) contra o DENV-2 em cultura de c?lulas C6/36 e Vero. A avalia??o da a??o dos extratos brutos foi feita por meio da quantifica??o da carga viral atrav?s da PCR em Tempo Real (qRT-PCR) e pela t?cnica de contagem de unidades formadoras de placa (PFU). As concentra??es dos extratos de ambas as plantas utilizadas foram: 0,01, 0,1 e 1mg/mL. As culturas de c?lulas infectadas foram submetidas ao tratamento com os extratos durante os per?odos de 24-168h horas (7 dias). C?lulas Vero tratadas com o extrato da S. mombin n?o apresentaram redu??o na carga viral. Em contrapartida, quando estas c?lulas foram tratadas com o extrato da A. muricata, uma hora ap?s infec??o, observou-se uma redu??o significativa na carga viral nas primeiras horas (24h), quando comparadas com as c?lulas n?o tratadas utilizadas como controle positivo. Ao serem tratadas em intervalos de 24 horas apresentaram uma redu??o na carga viral nos dias subsequentes (at? o s?timo dia). N?o foi observada redu??o na carga viral em c?lulas C6/36 tratadas com ambos os extratos. De acordo com os nossos resultados, o extrato da planta A. muricata possui potencial antiviral promissor contra a infec??o pelo DENV-2 em cultura de c?lulas Vero. / Dengue is a reportable disease and about 50 to 100 million cases are reported annually. It has a wide clinical spectrum and is transmitted to humans through the bite of Aedes mosquitos, the main vector the Aedes aegypti species. The causative agent of disease is dengue virus (DENV) belonging to the genus Flavivirus, family Flaviviridae and are known four antigenically distinct serotypes (DENV-1, DENV-2, DENV-3 and DENV-4). Currently the treatment of dengue is supportive, made by intense hydration. Although there is no proven effective vaccine or specific treatment, the study of potential antiviral drugs that can reduce viremia in patients is very high importance, since the viral load is one of the factors associated with the development of severe forms of the disease (hemorrhagic fever dengue and dengue shock syndrome). In the present study we evaluated the antiviral potential of crude extracts obtained from the leaves of plants in Northeastern Brazil Annona muricata (soursop) and Spondias mombin (caja) against DENV-2 in cultured C6/36 and Vero. The evaluation of the activity of the crude extracts was performed by the quantification of viral load by RT-PCR (qRT-PCR) and counting technique of plaque forming units (PFU). The concentrations of extracts of both plants used were 0.01, 0.1 and 1 mg/mL. The infected cell cultures were subjected to treatment with the extracts during periods of 24-168h hours (7 days). Vero cells treated with the S. mombin extract showed no reduction in viral load. In contrast, when these cells were treated with the extract of A. muricata, one hour after infection, significant reductions in viral load in the first hour was observed (24 h) when compared to untreated cells used as positive control. When they are treated at 24 hour intervals showed a reduction in viral load in subsequent days (until day). There was no reduction in viral load in C6/36 cells treated with both extracts. According to our results, the plant extract has antiviral A. muricata promising potential against infection by DENV-2 in Vero cell culture.

The aim of this diploma thesis was to investigate the effect of surfactants and cosmetic polysaccharides on skin parameters and its microbiome. Three surfactants were tested to determine their effect: Sodium Dodecyl Sulfate (SDS), Cocamidopropyl Betaine (CAPB), Decylglucoside (DG). Distilled water was also used for comparison. For the next part of the experimental work were selected 6 polysaccharides: high molecular weight Hyaluronic Acid (HMW HA), very low molecular weight Hyaluronic Acid (VLMW HA), Sodium Caproyl Hyaluronate (CaproylHA), Sodium Carboxymethyl -Glucan (NaCMG), Schizophyllan and Glucomannan. For comparison, placebo and untreated control (only CAPB treatment) were also included in the tests. The first part of the work is a literature search on the assigned topic, which contains the following parts: skin anatomy and its biophysical properties, skin microbiome and its functions, description of used surfactants and polysaccharides. The experimental part is mainly focused on bioengineering methods for evaluation of skin parameters and qRT-PCR to determine the relative proportion of main bacterial species of skin microbiome. First, the effect on the CT gene of 16S rDNA was analysed, and Propionibacterium acnes and Staphylococcus epidermidis strains were selected for further analysis. In conclusion are presented an overview of all properties of selected substances and assessment of their application in cosmetics.

No description available.

Environmental stress has shown to affect both the exposed individuals and the development of their offspring. Generally, it is thought that the stressed organism responds to stress by trying to adapt to it. This thesis investigates possible evolutionary consequences of cross-generational transmissions of stress, where the parent has been stressed but the offspring has not. In two studies we have exposed chicken parents of different breeds to an unpredictable circadian light rhythm, to investigate the influence of genetic background on the transmission of behaviour and patterns of genome-wide gene expression across generations. In Paper I, we can show that the domesticated chicken, by means of epigenetic factors, transmit their behaviours as well as their gene expression profiles to their offspring to a higher extent than their wild ancestor, the red junglefowl. Furthermore, in Paper II, even though the offspring never experienced the stress or had any contact with their stressed parents, they seemed to have adapted to it, which suggests that the parents might have prepared (or pre-adapted) them for living in the unpredictable environment. Additionally, eggs of stressed hens showed increased levels of estradiol that might have affected gene expression of specific immune genes, which were up-regulated in the offspring of stressed parents. It is possible that the traditional distinction between stress responses and evolutionary adaptation may be reevaluated, since our results indicate that they could be parts of the same evolutionary event.

Les trichothécènes de Fusarium appartiennent au groupe des sesquiterpènes qui sont des inhibiteurs la synthèse des protéines des eucaryotes. Les trichothécènes causent d’une part de sérieux problèmes de santé aux humains et aux animaux qui ont consommé des aliments infectés par le champignon et de l’autre part, elles sont des facteurs importants de la virulence chez plantes. Dans cette étude, nous avons isolé et caractérisé seize isolats de Fusarium de la pomme de terre infectée naturellement dans un champs. Les tests de pathogénicité ont été réalisés pour évaluer la virulence des isolats sur la pomme de terre ainsi que leur capacité à produire des trichothécènes. Nous avons choisi F. sambucinum souche T5 comme un modèle pour cette étude parce qu’il était le plus agressif sur la pomme de terre en serre en induisant un flétrissement rapide, un jaunissement suivi de la mort des plantes. Cette souche produit le 4,15-diacétoxyscirpénol (4,15-DAS) lorsqu’elle est cultivée en milieu liquide. Nous avons amplifié et caractérisé cinq gènes de biosynthèse trichothécènes (TRI5, TRI4, TRI3, TRI11, et TRI101) impliqués dans la production du 4,15-DAS. La comparaison des séquences avec les bases de données a montré 98% et 97% d'identité de séquence avec les gènes de la biosynthèse des trichothécènes chez F. sporotrichioides et Gibberella zeae, respectivement. Nous avons confrenté F. sambucinum avec le champignon mycorhizien à arbuscule Glomus irregulare en culture in vitro. Les racines de carotte et F. sambucinum seul, ont été utilisés comme témoins. Nous avons observé que la croissance de F. sambucinum a été significativement réduite avec la présence de G. irregulare par rapport aux témoins. Nous avons remarqué que l'inhibition de la croissance F. sambucinum a été associée avec des changements morphologiques, qui ont été observés lorsque les hyphes de G. irregulare ont atteint le mycélium de F. sambucinum. Ceci suggère que G. irregulare pourrait produire des composés qui inhibent la croissance de F. sambucinum. Nous avons étudié les patrons d’expression des gènes de biosynthèse de trichothécènes de F. sambucinum en présence ou non de G. irregulare, en utilisant le PCR en temps-réel. Nous avons observé que TRI5 et TRI6 étaient sur-exprimés, tandis que TRI4, TRI13 et TRI101 étaient en sous-exprimés en présence de G. irregulare. Des analyses par chromatographie en phase-gazeuse (GC-MS) montrent clairement que la présence de G. irregulare réduit significativement la production des trichothécènes par F. sambucinum. Le dosage du 4,15-DAS a été réduit à 39 μg/ml milieu GYEP par G. irregulare, comparativement à 144 μg/ml milieu GYEP quand F. sambucinum est cultivé sans G. irregulare. Nous avons testé la capacité de G. irregulare à induire la défense des plants de pomme de terre contre l'infection de F. sambucinum. Des essais en chambre de croissance montrent que G. irregulare réduit significativement l’incidence de la maladie causée par F. sambucinum. Nous avons aussi observé que G. irregulare augmente la biomasse des racines, des feuilles et des tubercules. En utilisant le PCR en temps-réel, nous avons étudié les niveaux d’expression des gènes impliqué dans la défense des plants de pommes de terre tels que : chitinase class II (ChtA3), 1,3-β-glucanase (Glub), peroxidase (CEVI16), osmotin-like protéin (OSM-8e) et pathogenèses-related protein (PR-1). Nous avons observé que G. irregulare a induit une sur-expression de tous ces gènes dans les racines après 72 heures de l'infection avec F. sambucinum. Nous avons également trové que la baisse provoquée par F. sambucinum des gènes Glub et CEVI16 dans les feuilles pourrait etre bloquée par le traitement AMF. Ceci montre que l’inoculation avec G. irregulare constitut un bio-inducteur systémique même dans les parties non infectées par F. sambucinum. En conclusion, cette étude apporte de nouvelles connaissances importantes sur les interactions entre les plants et les microbes, d’une part sur les effets directs des champignons mycorhiziens sur l’inhibition de la croissance et la diminution de la production des mycotoxines chez Fusarium et d’autre part, l’atténuation de la sévérité de la maladie dans des plantes par stimulation leur défense. Les données présentées ouvrent de nouvelles perspectives de bio-contrôle contre les pathogènes mycotoxinogènes des plantes. / Fusarium trichothecenes are a large group of sesquiterpenes that are inhibitors of eukaryotic protein synthesis. They cause health problems for humans and animals that consume fungus-infected agricultural products. In addition some of Fusarium trichothecenes are virulence factors of plant pathogenesis. In this study, sixteen Fusarium strains were isolated and characterized from naturally infected potato plants. Pathogenicity tests were carried out to evaluate the virulence of these isolates on potato plants and their trichothecene production capacity. We chose F. sambucinum strain T5 as a model for this study because it was the most aggressive strain when tested on potato plants. It induces a rapid wilting and yellowing resulting in plant death. This strain produced 4,15-diacetoxyscirpenol (4,15-DAS) when grown in liquid culture. We amplified and characterized five trichothecene genes (TRI5, TRI4, TRI3, TRI11, and TRI101) involved in the production of 4,15-DAS. Nucleotide BLAST search showed 98% and 97% sequence identity with trichothecene biosynthetic genes of F. sporotrichioides and Gibberella zeae, respectively. We used F. sambucinum to determine if trichothecene gene expression was affected by the symbiotic arbuscular mycorrhizal fungus (AMF) Glomus irregulare. We found that the growth of F. sambucinum was significantly reduced in the presence of G. irregulare isolate DAOM-197198 compared with controls that consisted of carrot roots without G. irregulare or F. sambucinum alone. Furthermore, inhibition of the growth F. sambucinum was associated with morphological changes, which were observed when G. irregulare hyphae reached F. sambucinum mycelium, suggesting that G. irregulare may produce compounds that interfere with the growth of F. sambucinum. Using real-time qRT-PCR assays, we assessed the relative expression of trichothecene genes of F. sambucinum confronted or not with G. irregulare. When G. irregulare was confronted with F. sambucinum, TRI5 and TRI6 genes were up-regulated, while TRI4, TRI13 and TRI101 were down-regulated. We therefore used GC-MS analysis to determine whether G. irregulare affects trichothecene production by F. sambucinum. We found that the production of 4,15-DAS trichothecene was significantly reduced in the presence of G. irregulare compared with controls that consisted of carrot roots without G. irregulare or F. sambucinum alone. Interestingly, 4,15-DAS pattern was reduced to 39 μg/ml GYEP medium by G. irregulare compared to 144 μg/ml GYEP with F. sambucinum grown with carrot roots or F. sambucinum alone respectively. We tested the AMF capacity to induce defense responses of potato plants following infection with F. sambucinum. The response of AMF-colonized potatoes to F. sambucinum was investigated by tracking the expression of genes homologous with pathogenesis-related proteins chitinase class II (ChtA3), 1,3-β-glucanase (gluB), peroxidase (CEVI16), osmotin-like protein (OSM-8e) and pathogenesis-related protein (PR-1). We found that the AMF treatment up-regulated the expression of all defense genes in roots at 72 hours post-infection (hpi) with F. sambucinum. We also found that a decrease provoked by F. sambucinum in gluB and CEVI16 expression in shoots could be blocked by AMF treatment. Overall, a differential regulation of PR homologues genes in shoots indicates that AMF are a systemic bio-inducer and their effects could extend into non-infected parts. In conclusion, this study provides new insight into on the interactions between plants and microbes, in particular the effects of AMF on the growth and the reduction of mycotoxins in Fusarium. It also shows that AMF are able to reduce the disease severity in plants by stimulating their defense. The data presented provide new opportunities for bio-control against mycotoxin-producing pathogens in plants.

INTRODUÇÃO: A propensão do CaP em gerar metástases decorre de mecanismos moleculares específicos em um processo composto por múltiplas etapas, sendo que o remodelamento do meio extracelular através de ações de enzimas proteolíticas denominadas metaloproteinases da matriz (MMP) é uma etapa fundamental. As MMP degradam vários componentes da matriz extracelular, sendo que seu controle pode ser exercido por outras proteínas denominadas TIMPs. Em nível gênico, outro controle pode ser exercido por moléculas chamadas microRNAs. OBJETIVO: O objetivo deste estudo é avaliar a regulação da MMP-9 por miRNAs. A partir de dados da literatura identificamos que a MMP-9 pode sofrer influência do miR-21 e 338-3p. MÉTODOS: Para os experimentos in vitro, linhagens celulares de CaP (DU145, PC3 e LNCaP) foram transfectadas com os miRNAs de interesse e a expressão de MMP-9 foi avaliada por reação em cadeia de polimerase quantitativa com transcriptase reversa (qRT-PCR). O sobrenadante da transfecção foi usado para ensaios de invasão com matrigel, e ELISA. Nos experimentos in vivo, células da linhagem PC-3-luc foram implantadas no subcutâneo de camundongos Balb-c nude e tratadas com injeções de anti-miR-21, miR-338-3p ou a combinação de ambos. RESULTADOS: O miR-21 aumentou expressão de MMP-9 em 72% na PC3. Houve maior invasão celular tanto na PC3 como DU145. In vivo, o bloqueio do miR-21 reduziu em 10% a expressão de MMP-9 nos tumores implantados (p=0,04). O miR-338-3p reduziu a expressão de MMP-9 em 53% na PC3 (p=0,001), 31% na LnCaP (p=0,23) e 24% na DU145 (p=0,16). No ensaio de invasão, menor número de células e colônias foram capazes de invadir a membrana de matrigel. In vivo, houve redução de 27% na expressão de MMP-9 nos camundongos tratados com o miR-338-3p (p=0,07). A combinação anti-miR-21+miR-338-3p reduz a expressão de MMP-9 em maior intensidade tanto in vitro como in vivo. CONCLUSÕES: A expressão de MMP-9 pode ser regulada pelo miR-21 e miR-338-3p. O primeiro se comporta como um oncomiR ao passo que o segundo como um supressor tumoral. A combinação de miRNAs é uma estratégia plausível para ampliar o efeito sobre expressão de genes de interesse / INTRODUCTION: The propensity of CaP to generate metastases results from specific molecular mechanisms in a multiphase process and the remodeling of the extracellular medium through the actions of proteolytic enzymes called matrix metalloproteinases (MMP) is a fundamental step. MMPs degrade several components of the extracellular matrix, and their control can be exerted by other proteins called TIMPs. At the gene level, another control can be exerted by molecules called microRNAs. OBJECTIVE: The objective of this study is to evaluate the regulation of MMP-9 by miRNAs. From literature data we have identified that MMP-9 may be influenced by miR-21 and 338-3p. METHODS: For in vitro experiments, CaP cell lines (DU145, PC3 and LNCaP) were transfected with the miRNAs of interest and the expression of MMP-9 was assessed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The transfection supernatant was used for matrigel and ELISA invasion assays. For the in vivo experiments, PC3-luc cells were implanted into the subcutaneous Balb-c nude mice and treated with anti-miR-21, miR-338-3p injections or the combination of both. RESULTS: The miR-21 increased MMP-9 expression by 72% in PC3. There was greater cell invasion in both PC3 and DU145. In vivo, miR-21 blockade reduced MMP-9 expression by 10% in implanted tumors (p = 0.04). MiR-338-3p reduced MMP-9 expression by 53% in PC3 (p = 0.001), 31% in LNCaP (p = 0.23), and 24% in DU145 (p = 0.16). In the invasion assay, fewer cells and colonies were able to invade the matrigel membrane. In vivo, there was a 27% reduction in MMP-9 expression in mice treated with miR-338-3p (p = 0.07). The combination of anti-miR-21 + miR-338-3p reduces MMP-9 expression in greater intensity both in vitro and in vivo. CONCLUSIONS: MMP-9 expression can be regulated by miR-21 and miR-338-3p. The former behaves as an oncomyR while the second as a tumor suppressor. The combination of miRNAs is a plausible strategy to extend the effect on gene expression of interest

Les trichothécènes de Fusarium appartiennent au groupe des sesquiterpènes qui sont des inhibiteurs la synthèse des protéines des eucaryotes. Les trichothécènes causent d’une part de sérieux problèmes de santé aux humains et aux animaux qui ont consommé des aliments infectés par le champignon et de l’autre part, elles sont des facteurs importants de la virulence chez plantes. Dans cette étude, nous avons isolé et caractérisé seize isolats de Fusarium de la pomme de terre infectée naturellement dans un champs. Les tests de pathogénicité ont été réalisés pour évaluer la virulence des isolats sur la pomme de terre ainsi que leur capacité à produire des trichothécènes. Nous avons choisi F. sambucinum souche T5 comme un modèle pour cette étude parce qu’il était le plus agressif sur la pomme de terre en serre en induisant un flétrissement rapide, un jaunissement suivi de la mort des plantes. Cette souche produit le 4,15-diacétoxyscirpénol (4,15-DAS) lorsqu’elle est cultivée en milieu liquide. Nous avons amplifié et caractérisé cinq gènes de biosynthèse trichothécènes (TRI5, TRI4, TRI3, TRI11, et TRI101) impliqués dans la production du 4,15-DAS. La comparaison des séquences avec les bases de données a montré 98% et 97% d'identité de séquence avec les gènes de la biosynthèse des trichothécènes chez F. sporotrichioides et Gibberella zeae, respectivement. Nous avons confrenté F. sambucinum avec le champignon mycorhizien à arbuscule Glomus irregulare en culture in vitro. Les racines de carotte et F. sambucinum seul, ont été utilisés comme témoins. Nous avons observé que la croissance de F. sambucinum a été significativement réduite avec la présence de G. irregulare par rapport aux témoins. Nous avons remarqué que l'inhibition de la croissance F. sambucinum a été associée avec des changements morphologiques, qui ont été observés lorsque les hyphes de G. irregulare ont atteint le mycélium de F. sambucinum. Ceci suggère que G. irregulare pourrait produire des composés qui inhibent la croissance de F. sambucinum. Nous avons étudié les patrons d’expression des gènes de biosynthèse de trichothécènes de F. sambucinum en présence ou non de G. irregulare, en utilisant le PCR en temps-réel. Nous avons observé que TRI5 et TRI6 étaient sur-exprimés, tandis que TRI4, TRI13 et TRI101 étaient en sous-exprimés en présence de G. irregulare. Des analyses par chromatographie en phase-gazeuse (GC-MS) montrent clairement que la présence de G. irregulare réduit significativement la production des trichothécènes par F. sambucinum. Le dosage du 4,15-DAS a été réduit à 39 μg/ml milieu GYEP par G. irregulare, comparativement à 144 μg/ml milieu GYEP quand F. sambucinum est cultivé sans G. irregulare. Nous avons testé la capacité de G. irregulare à induire la défense des plants de pomme de terre contre l'infection de F. sambucinum. Des essais en chambre de croissance montrent que G. irregulare réduit significativement l’incidence de la maladie causée par F. sambucinum. Nous avons aussi observé que G. irregulare augmente la biomasse des racines, des feuilles et des tubercules. En utilisant le PCR en temps-réel, nous avons étudié les niveaux d’expression des gènes impliqué dans la défense des plants de pommes de terre tels que : chitinase class II (ChtA3), 1,3-β-glucanase (Glub), peroxidase (CEVI16), osmotin-like protéin (OSM-8e) et pathogenèses-related protein (PR-1). Nous avons observé que G. irregulare a induit une sur-expression de tous ces gènes dans les racines après 72 heures de l'infection avec F. sambucinum. Nous avons également trové que la baisse provoquée par F. sambucinum des gènes Glub et CEVI16 dans les feuilles pourrait etre bloquée par le traitement AMF. Ceci montre que l’inoculation avec G. irregulare constitut un bio-inducteur systémique même dans les parties non infectées par F. sambucinum. En conclusion, cette étude apporte de nouvelles connaissances importantes sur les interactions entre les plants et les microbes, d’une part sur les effets directs des champignons mycorhiziens sur l’inhibition de la croissance et la diminution de la production des mycotoxines chez Fusarium et d’autre part, l’atténuation de la sévérité de la maladie dans des plantes par stimulation leur défense. Les données présentées ouvrent de nouvelles perspectives de bio-contrôle contre les pathogènes mycotoxinogènes des plantes. / Fusarium trichothecenes are a large group of sesquiterpenes that are inhibitors of eukaryotic protein synthesis. They cause health problems for humans and animals that consume fungus-infected agricultural products. In addition some of Fusarium trichothecenes are virulence factors of plant pathogenesis. In this study, sixteen Fusarium strains were isolated and characterized from naturally infected potato plants. Pathogenicity tests were carried out to evaluate the virulence of these isolates on potato plants and their trichothecene production capacity. We chose F. sambucinum strain T5 as a model for this study because it was the most aggressive strain when tested on potato plants. It induces a rapid wilting and yellowing resulting in plant death. This strain produced 4,15-diacetoxyscirpenol (4,15-DAS) when grown in liquid culture. We amplified and characterized five trichothecene genes (TRI5, TRI4, TRI3, TRI11, and TRI101) involved in the production of 4,15-DAS. Nucleotide BLAST search showed 98% and 97% sequence identity with trichothecene biosynthetic genes of F. sporotrichioides and Gibberella zeae, respectively. We used F. sambucinum to determine if trichothecene gene expression was affected by the symbiotic arbuscular mycorrhizal fungus (AMF) Glomus irregulare. We found that the growth of F. sambucinum was significantly reduced in the presence of G. irregulare isolate DAOM-197198 compared with controls that consisted of carrot roots without G. irregulare or F. sambucinum alone. Furthermore, inhibition of the growth F. sambucinum was associated with morphological changes, which were observed when G. irregulare hyphae reached F. sambucinum mycelium, suggesting that G. irregulare may produce compounds that interfere with the growth of F. sambucinum. Using real-time qRT-PCR assays, we assessed the relative expression of trichothecene genes of F. sambucinum confronted or not with G. irregulare. When G. irregulare was confronted with F. sambucinum, TRI5 and TRI6 genes were up-regulated, while TRI4, TRI13 and TRI101 were down-regulated. We therefore used GC-MS analysis to determine whether G. irregulare affects trichothecene production by F. sambucinum. We found that the production of 4,15-DAS trichothecene was significantly reduced in the presence of G. irregulare compared with controls that consisted of carrot roots without G. irregulare or F. sambucinum alone. Interestingly, 4,15-DAS pattern was reduced to 39 μg/ml GYEP medium by G. irregulare compared to 144 μg/ml GYEP with F. sambucinum grown with carrot roots or F. sambucinum alone respectively. We tested the AMF capacity to induce defense responses of potato plants following infection with F. sambucinum. The response of AMF-colonized potatoes to F. sambucinum was investigated by tracking the expression of genes homologous with pathogenesis-related proteins chitinase class II (ChtA3), 1,3-β-glucanase (gluB), peroxidase (CEVI16), osmotin-like protein (OSM-8e) and pathogenesis-related protein (PR-1). We found that the AMF treatment up-regulated the expression of all defense genes in roots at 72 hours post-infection (hpi) with F. sambucinum. We also found that a decrease provoked by F. sambucinum in gluB and CEVI16 expression in shoots could be blocked by AMF treatment. Overall, a differential regulation of PR homologues genes in shoots indicates that AMF are a systemic bio-inducer and their effects could extend into non-infected parts. In conclusion, this study provides new insight into on the interactions between plants and microbes, in particular the effects of AMF on the growth and the reduction of mycotoxins in Fusarium. It also shows that AMF are able to reduce the disease severity in plants by stimulating their defense. The data presented provide new opportunities for bio-control against mycotoxin-producing pathogens in plants.

Arrière-plan: les cellules tumorales circulantes (CTC) sont détectables dans de nombreux cancers et peuvent être utiles cliniquement pour le pronostic de la maladie, pour mesurer la récidive et pour prédire la sensibilité aux medicaments chimiothérapeutiques. Au cours des dernières années, l’études des CTC dans de nombreux cancers tels que le cancer du sein, du poumon, du côlon et de la prostate a grandement évolué. Alternativement, il y peu d'études à ce sujet concernant le cancer du col de l’utérus (CCU). Objectifs: Notre objectif est d’optimiser le processus d'enrichissement des CTC dans le CCU et la détection moléculaire des biomarqueurs E6 et E7. Matériel et Méthodes: Dans l’optique de mimer la présence de CTC dans le sang, nous avons dilué des cellules cancéreuses CaSki VPH16-positif provenant d’un CCU dans du sang humain prélevé sur des volontaires sains. Les CaSki ont été collectées suite à une centrifugation par densité avec le Ficoll, la lyse des globules rouges (RBC) et la lyse des RBC combinée avec un enrichissement positif et négatif à l’aide de marqueurs de surface cellulaire. Les CTC ont été détectées par la mesure d’expression des oncogènes E6 et E7 du virus du papillome humain (VPH), de la cytokératine 19 (CK19) et de la cycline p16INK4 en utilisant la technique quantitative en temps réel de Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR). Pour valider notre méthode de détection des CTC in vivo, nous avons recruté dix patientes atteintes d’un CCU VPH16 positif et six contrôles sains. Résultats: Dans le modèle de dilutions de cellules CaSki, la lyse des RBC seule ou combinée avec l'enrichissement négatif ou positif suggèrent des limites de détection de 1 CTC par mL de sang pour tous les biomarqueurs moléculaires utilisés. La sensibilité de détection est accrue lors de l'utilisation de l’enrichissement positif et négatif en réduisant le bruit de fond causé par les monocytes sanguins. Contrairement aux oncogènes E6 et E7, les marqueurs CK19 et p16INK4A ont été détectés chez des individus sains, les niveaux d'expression de base appropriés doivent donc être déterminés avec précision par rapport aux patientes CCU. Le gradient de densité par Ficoll a une limite de détection de seulement environ 1000 cellules par mL de sang. Enfin, les CTC ont été détectées dans 2/10 patientes en utilisant le marqueur CK19. Cependant, ces patientes étaient négatives pour les oncogènes E6/E7. Le marqueur p16INK4A était exprimé au même niveau dans tous les échantillons (CCU et normaux). Conclusion: Notre étude suggère que les oncogènes E6 et E7 du VPH16 sont les marqueurs biologiques les plus sensibles et spécifiques en qRT-PCR pour détecter les CTC dans le modèle de dilution de cellules de CCU dans le sang. Chez les patientes atteintes d’un CCU de stade précoce, seulement CK19 a révélé la présence potentielle de CTC, ce qui suggère que ces cellules sont rares à ce stade de la maladie. Mots clés: cancer du col de l’utérus, cellules tumorales circulantes, RT-qPCR, E6 et E7, CK19, p16INK4A, enrichissement immunomagnétique, détection moléculaire. / Background: Circulating tumor cells (CTCs) have been detected in many cancers and are used in multiple clinical applications including disease prognosis, tumor recurrence prediction and prediction of tumor sensitivity to chemotherapeutic drugs. Studies in most major solid cancer(s) (breast, lung, colon and prostate) are progressing rapidly, but there has been very little progress concerning uterine cervical cancer (UCC).Objective: our aim is to optimize enrichment processes and the molecular biomarker-based detection of human circulating tumor cells (CTCs) in uterine cervical cancer (UCC). Material & Methods: To mimic CTCs in patients, we designed an experimental spiking model where the CaSki HPV16-positive UCC cell line was serially diluted and spiked into human blood collected from healthy volunteers. CaSki CTCs were enriched using either Ficoll density centrifugation, red blood cell (RBC) lysis or RBC lysis combined with cell surface markers negative or positive enrichment. CTCs were detected using real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) to measure the gene expression of human papillomavirus (HPV) viral oncogenes (E6 and E7), cytokeratin 19 (CK19), or the cyclin dependent kinase inhibitor p16INK4A. Finally, ten HPV16- positive UCC patients and six healthy controls were recruited to validate CTCs detection in vivo. Result: In the spiking model, RBC lysis alone or combined with negative or positive enrichment suggests detection limits close to 1 CTC per mL of blood for all molecular biomarkers used. The sensitivity of detection increased when using positive and negative enrichment probably by reducing the peripheral blood mononuclear cell-derived RNA background. Unlike HPV oncogenes, CK19 and p16INK4A were detected in normal individuals, thus appropriate basal expression levels need to be accurately determined compared to cancer patients. Alternatively, Ficoll density gradient had a detection limit of only about 1000 cells per mL of blood. Finally CTCs were detected in 2/10 patients using CK19. None of the patients had E6/E7 transcripts and p16INK4A was expressed at similar level across all samples (cancer and healthy). Conclusion: qRT-PCR of HPV16 E6 and E7 is the most sensitive and specific biomarker used to detect CTCs in the spiking model. In early disease UCC patients, only CK19 revealed the presence of CTCs suggesting that these cells are rare at that stage of the disease. Keywords: uterine cervical cancer, circulating tumor cells, qRT-PCR, E6 and E7 oncoprotein, CK19, p16INK4A, immune-magnetic enrichment, molecular detection.

Pancreatic cancer (PC) is extremely severe malignant disease with a five-year survival of less than 5%. Currently there is no reliable tool for the diagnosis of PC in its early stages. At the time of clinical symptoms most patients are in an advanced stage of the disease and the treatment does not usually have a significant effect. For these reasons emphasis is gradually shifting to the search for the suitable molecular markers for improvement of the diagnosis and assessment of the survival prognosis with respect to a possibility of surgical treatment. MiRNA represent one of the most promising markers, although, their examination in pancreatic tissue is a complicated process. One of the reasons is the very small amount of the source material coming from a fine needle biopsy. A second cause of problems is the subtle character of the pancreatic tissue resulting in significantly lower yields of molecular genetic analysis when compared to other epithelial tissues. An additional negative factor is heterogeneity of the tissue resulting in disproportionate representation of tumor cells within the sample. A suitable choice of procedures for isolation of nucleic acids (NA) and subsequent analysis including quantification of tumor cells is critical for accurate evaluation of the miRNA levels. This work is...

The main objective of this thesis dissertation is functionally characterizing the genes involved in biotic and abiotic stresses of plants at molecular level. Previously, upon pathogen attack Rad6 gene expression was found to be changed in wheat and barley plants. To functionally characterize the Rad6 gene, VIGS (Virus induced gene silencing) system was used. HR (Hypersensitive response) like symptoms was detected in every silenced barley and wheat plants. To figure out, transcriptomes and proteomes of Rad6 silenced plants were analyzed. 2-D PAGE analysis was also performed on silenced and control wheat plants. No pathogen growth was observed in Rad6 silenced barley lines. Additionally, the susceptible wild type Arabidopsis plants showed resistant phenotype when any of the Rad6 gene copies is mutated. This suggests that Rad6 gene has a negative regulatory role in plant disease resistance which was proved for the first time. Yeast two hybrid protein interaction study suggests that RAD6 carrying out its function by interacting with SGT1 protein and regulating resistance related genes. It has been first time reported in this thesis that E2 (Ubiquitin conjugating enzyme) takes role in plant disease resistance. Boron which is the other consideration in the scope of thesis as an abiotic stress factor at a very limited amount is necessary for the normal development of plants. This study is conducted on highly boron tolerant Gypsophila perfoliata L. collected from a location in the boron mining area. The plant samples were tested in the presence of high boron (35 mg/kg) concentrations. The transcriptomes of the plant samples treated with the excess levels of boron to that of the samples grown under normal concentration were compared using differential display PCR method. Thirty bands showing differential expression levels at varying time points were analyzed. 18 of them were confirmed via qRT-PCR.