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In the light of increasing pesticides resistance in agricultural pests and in insect vectors of human diseases, leading to the rise in occurrence of mosquito-borne diseases, new, efficient and environmentally friendly methods of pest control are needed. Sterile Insect Technique (SIT), relying on mass releases of radiation sterilised males to reduce reproductive potential of target pest populations, although not new, offers an alternative to the use of pesticides and is an environmentally non-polluting method of insect control. Many insect species, however, are not very amenable to classical SIT, due to detrimental side-effects of radiation treatment. We propose a new method, a genetically engineered modification of classical SIT, replacing radiation with genetically induced sterility. Based on conditional expression of male-germline targeted nucleases which introduce double strand breaks into the male germline DNA to render males sterile, this method emulates SIT mechanism, at the same time eliminating radiation and associated detrimental side-effects. Different variants of such a system were investigated in this project, eventually leading to the creation of functional conditional male-sterility systems in two model organisms – the Yellow fever mosquito, Aedes aegypti and the Mediterranean fruit fly, Ceratitis capitata. Both systems utilise chimeric nuclease composed of protamine and FokI cleavage domain fusion. The sperm-specificity and the conditionality of the sterile phenotype have been achieved through the use of tetracycline repressible expression system driven by the β2-tubulin promoter in Ceratitis capitata and by the Topi promoter in Aedes aegypti.

Rhynchosciara americana é estudada desde a década de 50 quando foi evidenciada a amplificação gênica em regiões de seus cromossomos politênicos. Os fenômenos de amplificação e a formação destes cromossomos gigantes se dão por meio da ocorrência de endociclos, regulados pela oscilação na atividade do complexo ciclinaE/CDK2. Nas glândulas salivares de nosso modelo, durante o desenvolvimento, há ocorrência de cromossomos politênicos. Foi seqüenciada uma mensagem de uma quinase dependente de ciclina (CDK10) que não apresenta qualquer ligação com os endociclos, descrita na literatura. Este transcrito teve sua sequencia revelada por experimentos de 5\'RACE. A sequência genômica foi parcialmente determinada. O perfil de expressão deste gene foi determinado nos ovários, glândulas salivares e corpo gorduroso. A presença da proteína CDK10 foi determinada por experimentos de western blot em glândulas salivares. A localização celular foi determinada nos ovários. Resultados sugerem a existência de duas isoformas deste gene nos tecidos analisados. / Rhynchosciara americana is studied since the 50s decade when gene amplifications was discovered in some regions of its polytene chromosomes. The phenomena of amplification and the formation of these giant chromosomes happens through the endocycles, regulated by an activity oscillation of the complex CyclinE/CDK2. It is known that, in the salivary glands of our model, during it is development, there is the occurrence of these polytene chromosomes, formed since the beginning of the larval life. It was sequenced a message of CDK10, a cyclin depended kinase that shows no participation with the endocycles, as described at the literature. This transcript was fully sequenced by 5\'RACE experiments. Its genomic sequence was partially determined. The relative expression pattern was determined for ovaries, salivary glands and fat body. The CDK10 protein was observed by western blot experiments in the salivary glands. Its cellular location was determined at the ovaries. Results suggest the existence of two isoforms of the RaCDK10 gene at the tissues analyzed.

Diss. (sammanfattning) Umeå : Sveriges lantbruksuniv., 2002. / Härtill 6 uppsatser.

Rhynchosciara americana é estudada desde a década de 50 quando foi evidenciada a amplificação gênica em regiões de seus cromossomos politênicos. Os fenômenos de amplificação e a formação destes cromossomos gigantes se dão por meio da ocorrência de endociclos, regulados pela oscilação na atividade do complexo ciclinaE/CDK2. Nas glândulas salivares de nosso modelo, durante o desenvolvimento, há ocorrência de cromossomos politênicos. Foi seqüenciada uma mensagem de uma quinase dependente de ciclina (CDK10) que não apresenta qualquer ligação com os endociclos, descrita na literatura. Este transcrito teve sua sequencia revelada por experimentos de 5\'RACE. A sequência genômica foi parcialmente determinada. O perfil de expressão deste gene foi determinado nos ovários, glândulas salivares e corpo gorduroso. A presença da proteína CDK10 foi determinada por experimentos de western blot em glândulas salivares. A localização celular foi determinada nos ovários. Resultados sugerem a existência de duas isoformas deste gene nos tecidos analisados. / Rhynchosciara americana is studied since the 50s decade when gene amplifications was discovered in some regions of its polytene chromosomes. The phenomena of amplification and the formation of these giant chromosomes happens through the endocycles, regulated by an activity oscillation of the complex CyclinE/CDK2. It is known that, in the salivary glands of our model, during it is development, there is the occurrence of these polytene chromosomes, formed since the beginning of the larval life. It was sequenced a message of CDK10, a cyclin depended kinase that shows no participation with the endocycles, as described at the literature. This transcript was fully sequenced by 5\'RACE experiments. Its genomic sequence was partially determined. The relative expression pattern was determined for ovaries, salivary glands and fat body. The CDK10 protein was observed by western blot experiments in the salivary glands. Its cellular location was determined at the ovaries. Results suggest the existence of two isoforms of the RaCDK10 gene at the tissues analyzed.