Desenvolvimento e validação do método LNA-FISH para deteção de Aeromonas

Dissertação de Mestrado Mestrado em BioengenhariaUm potencial problema em aquacultura são as infeções bacterianas que são facilmente transmissíveis podendo contaminar uma grande variedade de espécies de peixes, levando a grandes perdas económicas. O género Aeromonas compreende um grande número de espécies patogénicas para peixes, e representa uma das principais causas de perdas na aquacultura. Torna-se necessário a melhoria de métodos para o diagnóstico e controlo deste patógeneo de forma a minimizar o seu impacto. A utilização de técnicas moleculares tem sido mencionada como alternativa viável para detetar e diagnosticar surtos de doenças, prevenindo perdas económicas em aquacultura. O método de hibridação fluorescente in situ (FISH) tem sido muito explorado na área da microbiologia alimentar. É um método de deteção simples, rápido e altamente sensível que utiliza sondas marcadas por fluorescência cujo alvo são regiões específicas de ácido ribonucleico (RNA) bacteriano. Este método tem sofrido um desenvolvimento notável devido à aplicação de moléculas sintéticas na construção das sondas, que tornam o FISH mais rápido, robusto e sensível. Neste trabalho o principal objetivo foi o desenvolvimento e otimização de uma sonda para deteção de Aeromonas spp com recurso a uma molécula sintética, o LNA (locked nucleic acid). Uma completa otimização não foi conseguida, pois apesar dos vários parâmetros testados e da elevada afinidade da sonda, não foi possível eliminar completamente a hibridação cruzada com espécies não -Aeromonas. Foi possível verificar que a eficiência da sonda depende de uma relação muito forte entre o tipo/concentração de desnaturante e a concentração salina usada na solução de hibridação. Para melhor compreender este efeito a assim passar de uma otimização baseada numa abordagem tentativa-erro, para uma abordagem baseada em modelos da cinética de hibridação, usou-se a “response surface methodology” (RSM). RSM é uma ferramenta matemática e estatística que permite modelar e analisar problemas influenciados por inúmeras variáveis de forma a encontrar uma aproximação razoável do relacionamento real entre esse conjunto de variáveis. Foi possível perceber que, dos três desnaturantes testados, a Ureia foi a que permitiu obter um maior sinal de hibridação, previsto a ocorrer a uma temperatura de hibridação de 50ºC para 5M de NaCl numa concentração de Ureia de 4M.A potential problem in aquaculture are bacterial infections that can easily be transmitted and may contaminate a wide variety of fish species, leading to major economic losses. The genus Aeromonas comprises a large number of species pathogenic to fish, which can cause infections worldwide and is a major cause of losses in aquaculture. It is necessary to improve methods for rapid diagnosis and control of this pathogen in order to minimize its impact. The use of molecular techniques has been mentioned as a viable alternative to detect and diagnose disease outbreaks, preventing economic losses in aquaculture. The fluorescent in situ hybridization (FISH) method has long been explored in the field of food microbiology. It is a simple detection method that uses fast and highly sensitive fluorescence probes that target specific regions of bacterial ribonucleic acid (RNA). This method has been suffering a remarkable development due to the application of synthetic molecules in the construction of probes, which make FISH faster, more robust and sensitive. In this work, the main objective was the development and optimization of a probe for detection of Aeromonas spp using a synthetic molecule, LNA (locked nucleic acid). Despite the probe high affinity; a complete optimization was not achieved because cross-hybridization with non-Aeromonas species was not completely eliminated. It was possible to verify that probe efficiency is strongly dependent on a relationship between the type and concentration of denaturant and the salt concentration used in the hybridization solution. To better understand this effect and move from a trial-and–error optimization to an approach based on hybridization kinetics models, the "response surface methodology" (RSM) was used. RSM is a mathematical and statistical tool that allows to model and analyze behavious influenced by many variables in order to find a reasonable approximation of the actual relationship between this set of variables. It can be seen that, from the three denaturants tested, the urea was the one that allowed the best hybridization signal, predicted to occur at an hybridization temperature of 50 ° C for NaCl at a concentration of 5M and Urea at 4M

MAMMALS IN PORTUGAL : A data set of terrestrial, volant, and marine mammal occurrences in P ortugal

Mammals are threatened worldwide, with 26% of all species being includedin the IUCN threatened categories. This overall pattern is primarily associatedwith habitat loss or degradation, and human persecution for terrestrial mam-mals, and pollution, open net fishing, climate change, and prey depletion formarine mammals. Mammals play a key role in maintaining ecosystems func-tionality and resilience, and therefore information on their distribution is cru-cial to delineate and support conservation actions. MAMMALS INPORTUGAL is a publicly available data set compiling unpublishedgeoreferenced occurrence records of 92 terrestrial, volant, and marine mam-mals in mainland Portugal and archipelagos of the Azores and Madeira thatincludes 105,026 data entries between 1873 and 2021 (72% of the data occur-ring in 2000 and 2021). The methods used to collect the data were: live obser-vations/captures (43%), sign surveys (35%), camera trapping (16%),bioacoustics surveys (4%) and radiotracking, and inquiries that represent lessthan 1% of the records. The data set includes 13 types of records: (1) burrowsjsoil moundsjtunnel, (2) capture, (3) colony, (4) dead animaljhairjskullsjjaws, (5) genetic confirmation, (6) inquiries, (7) observation of live animal (8),observation in shelters, (9) photo trappingjvideo, (10) predators dietjpelletsjpine cones/nuts, (11) scatjtrackjditch, (12) telemetry and (13) vocalizationjecholocation. The spatial uncertainty of most records ranges between 0 and100 m (76%). Rodentia (n=31,573) has the highest number of records followedby Chiroptera (n=18,857), Carnivora (n=18,594), Lagomorpha (n=17,496),Cetartiodactyla (n=11,568) and Eulipotyphla (n=7008). The data setincludes records of species classified by the IUCN as threatened(e.g.,Oryctolagus cuniculus[n=12,159],Monachus monachus[n=1,512],andLynx pardinus[n=197]). We believe that this data set may stimulate thepublication of other European countries data sets that would certainly contrib-ute to ecology and conservation-related research, and therefore assisting onthe development of more accurate and tailored conservation managementstrategies for each species. There are no copyright restrictions; please cite thisdata paper when the data are used in publications.info:eu-repo/semantics/publishedVersio

Mammals in Portugal: a data set of terrestrial, volant, and marine mammal occurrences in Portugal

Mammals are threatened worldwide, with ~26% of all species being included in the IUCN threatened categories. This overall pattern is primarily associated with habitat loss or degradation, and human persecution for terrestrial mammals, and pollution, open net fishing, climate change, and prey depletion for marine mammals. Mammals play a key role in maintaining ecosystems functionality and resilience, and therefore information on their distribution is crucial to delineate and support conservation actions. MAMMALS IN PORTUGAL is a publicly available data set compiling unpublished georeferenced occurrence records of 92 terrestrial, volant, and marine mammals in mainland Portugal and archipelagos of the Azores and Madeira that includes 105,026 data entries between 1873 and 2021 (72% of the data occurring in 2000 and 2021). The methods used to collect the data were: live observations/captures (43%), sign surveys (35%), camera trapping (16%), bioacoustics surveys (4%) and radiotracking, and inquiries that represent less than 1% of the records. The data set includes 13 types of records: (1) burrows | soil mounds | tunnel, (2) capture, (3) colony, (4) dead animal | hair | skulls | jaws, (5) genetic confirmation, (6) inquiries, (7) observation of live animal (8), observation in shelters, (9) photo trapping | video, (10) predators diet | pellets | pine cones/nuts, (11) scat | track | ditch, (12) telemetry and (13) vocalization | echolocation. The spatial uncertainty of most records ranges between 0 and 100 m (76%). Rodentia (n =31,573) has the highest number of records followed by Chiroptera (n = 18,857), Carnivora (n = 18,594), Lagomorpha (n = 17,496), Cetartiodactyla (n = 11,568) and Eulipotyphla (n = 7008). The data set includes records of species classified by the IUCN as threatened (e.g., Oryctolagus cuniculus [n = 12,159], Monachus monachus [n = 1,512], and Lynx pardinus [n = 197]). We believe that this data set may stimulate the publication of other European countries data sets that would certainly contribute to ecology and conservation-related research, and therefore assisting on the development of more accurate and tailored conservation management strategies for each species. There are no copyright restrictions; please cite this data paper when the data are used in publications

Characterisation of microbial attack on archaeological bone

As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones. © 2003 Elsevier Science Ltd. All rights reserved