Listeria monocytogenes em condições de simulação do sistema gastrointestinal e a infecção no modelo de Galleria mellonella

Dissertação de mest., Biologia Molecular e Microbiana, Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2011

Perkinsus Atlanticus – desenvolvimento de um método de diagnóstico para detecção de infecção em moluscos bivalves

Diferentes espécies do género Perkinsus, um protozoário parasita pertencente ao novo phylum Perkinsea, podem ser encontradas mundialmente e constituem uma séria ameaça aos moluscos bivalves como as ostras, amêijoas, abalones e vieiras, os quais têm um papel importante não só do ponto de vista comercial, mas também ecológico.

Envolvimento de Phytophthora cinnamomi no declínio de Quercus suber e Q. rotundifolia: estudo da influência de factores bióticos e abióticos na progressão da doença. Possibilidades de controlo químico do declínio

Tese dout., Ciências Agrárias, Universidade do Algarve, 2007

Study of transcripts and reactive oxygen species involved in the defence responses of Quercus suber against Phytophthora cinnamomi

The present work has the merit of exploring an insight into the activation of defence genes of Quercus suber during response to infection by Phytophthora cinnamomi. Thus, cDNA-AFLP methodology was used to identify gene fragments differentially present in the mRNA profiles of host cells of micropropagated Q. suber plantlets roots infected with zoospores of P. cinnamomi at different post challenge time points. Six candidate genes were selected based on their interesting cDNA-AFLP expression patterns and homology to genes known to play a role in defence. These six genes encode a cinnamyl alcohol dehydrogenase 2 (QsCAD2), a protein disulphide isomerase (QsPDI), a CC-NBS-LRR resistance protein (QsRPc), thaumatin-like protein (QsTLP), chitinase (QsCHI) and a 1,3-beta glucanase (QsGLU). The current work has been successful in evaluation of the expression of these genes by qRT-PCR. Data analysis revealed that transcript levels of QsRPc, QsCHI, QsCAD2 and QsPDI increased during the early hours of inoculation, while transcript profiles of thaumatin-like protein showed decreasing. No expression was detected for 1,3-beta-glucanase (QsGLU). Furthermore, the choice of suitable reference genes in any new experimental system is absolutely crucial in qRT-PCR; for this reason in this study and for the first time a set of potential reference genes were analyzed and validated for qRT-PCR normalization in the patho-system Phytophthora-Q. suber. Four candidate reference genes polimerase II (QsRPII), eukaryotic translation initiation factor 5A(QsEIF-5A), b-tubulin (QsTUB) and a medium subunit family protein of Clathrin adaptor complexes (QsCACs) were evaluated to determine the most stable internal references in Q. suber. Analysis of stability of genes was carried out using Genex software. Results indicated all these four potential reference genes assumed stable expression. Data analysis revealed that QsRPII and QsCACs were the two most stable genes, while genes QsTUB and QsEIF-5A were the third and the fourth most stable gene, respectively. In this study, a plasmid-based quantitative PCR method was developed to measure P. cinnamomi colonization during infection process of Q. suber. Plasmid-based detection of P. cinnamomi showed a gradual accumulation of the pathogen DNA in cork oak root tips up to 24 h post infection. The higher increase in P. cinnamomi/plasmid DNA ratio occurred between 18 and 24 h. One of the primary objectives of this research was to study the effect of cinnamomins (elicitins secreted by P. cinnamomin) on inducing defence mechanism against the pathogen, as recent histological and ultra-structural studies showed that P. cinnamomi was restricted to the outer cortex root fragments pre-treated with capsicien and cryptogein, suggesting that elicitins can stimulate plant defence reactions against P. cinnamomi. To complement these studies and to have a clear view of the nature of the interaction, the role of cinnamomins in the production of the oxidative burst [ROS and ROS scavenging enzymes such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD)] and in the defence responses was evaluated. Cork oak seedlings were pretreated with alpha-cinnamomin and then inoculated with P. cinnamomi mycelia. Results showed a significant higher production of reactive oxygen species (ROS) (H2O2 and O2•-) in elicitin and non-elicitin treated roots in interaction with P. cinnamomi in comparison to the corresponding control. The plant group inoculated with the pathogen after cinnamomin treatment showed an earlier increase in H2O2 production but this was lower as compared with that group inoculated with P. cinnamomi alone. Also, in elicitin pre-treated group generally, a lower level of O2•− production during infection was observed as compared with inoculated roots with P. cinnamomi alone without elicitin treatment. Furthermore, in this study, we evaluated activities of antioxidant enzymes upon challenge with P. cinnamomi, with and without pretreatment with alpha cinnamomin. Results indicated that the activities of defense enzymes POD, SOD and CAT increased after P. cinnamomi inoculation when compared with those in the control group. Also, in the group treated with alpha-cinnamomin followed by P. cinnamomi inoculation, a higher level of enzymatic activities was detected as compared with elicitin non-treated group, which suggest the protective effect of alpha-cinnamomin against the pathogen due to higher elevated levels of defense enzymes POD, SOD and CAT during the infection period. Furthermore, a sensitive qPCR method was applied to measure the pathogen biomass in elicited and non-elicited Q. suber roots challenged with P. cinnamomi to elucidate the effect of cinnamomins on the colonization of P. cinnamomi. Plasmid-based quantification of P. cinnamomi showed a significant decrease in accumulation of the pathogen DNA in cork oak roots after treatment with alpha and beta-cinnamomins which attest the role of cinnamomins in promoting defense responses in cork oak against P. cinnamomi invasion.

Testes com novos fármacos no tratamento do parasita de peixes Amyloodinium ocellatum

O Amyloodinium ocellatum é um parasita dinoflagelado que aparece em aquaculturas por todo o mundo, infectando as brânquias e o tegumento dos peixes. Esta parasita é particularmente relevante na aquacultura mediterrânica onde os surtos causam perdas consideráveis. Apesar de já terem sido experimentados vários tratamentos, nenhum deles se revelou efectivo na erradicação do parasita e ainda alguns dos químicos utilizados podem ter importantes efeitos nocivos sobre o ambiente. Deste modo, o objectivo deste estudo foi testar novos fármacos para impedir o desenvolvimento e/ou eliminar o parasita em peixes já infectados. Os fármacos utilizados neste estudo foram endoperóxidos, uma recente classe de antimaláricos. Na primeira parte deste estudo realizaram-se ensaios in vitro onde se testaram os efeitos de diferentes concentrações (0,1mM; 1 mM; 2mM e 2,5mM) dos fármacos NAD17, NAD19, LCD93 e LCD67A sobre a divisão dos tomontes. Às 24 e 48 horas de exposição a 0,1mM NAD19 e 1mM LCD93, os tomontes observados não tinham entrado em divisão. Na segunda parte realizou-se um ensaio in vivo no qual os fármacos foram administrados a juvenis de dourada (Sparus aurata) a uma concentração de 10μmol/kg através de tratamento oral. No ensaio in vivo avaliou-se o possível efeito profiláctico dos fármacos na infecção através da análise da abundância e prevalência do parasita, da eficácia dos fármacos e dos efeitos do parasita e do tratamento na fisiologia e no eixo do stress dos animais infectados através de análises enzimáticas e de parâmetros bioquímicos do sangue. As medições realizadas ao plasma sanguíneo e às brânquias de douradas com diferentes níveis de infecção de A. ocellatum revelaram que não existe diferença significativa entre os controlos e os endoperóxidos para a dose testada. Será necessário realizar mais ensaios para obter conclusões sobre o efeito dos endoperóxidos no controlo do parasita de peixes A. ocellatum.