Agrobacterium are Alphaproteobacteria common in most soils that closely interact with plants in two respects. Firstly, they are rhizospheric bacteria saprophytically living in the rhizosphere of numerous plants and they are likely beneficial to plants. Secondly, when they harbor a dispensable Ti plasmid (i.e. tumor inducing plasmid), agrobacteria are plant pathogens able to cause the crown gall disease to most dicots and gymnosperms and some monocots. An epidemiological survey of crown gall thus also requires expert determination of the Agrobacterium taxonomy. In this thesis we evaluated the usefulness of MALDI-TOF MS technique as a high throughput tool to determine and classify agrobacteria. Then we set up a recA-based PCR method to accurately and exhaustively assess agrobacterial diversity either of isolated agrobacteria or directly in various biotopes. We applied standard biochemical, recA-based and Ti plasmid-based identification methods to study the prevalence of pathogenic and non-pathogenic agrobacteria at the country and local scales. Finally, we tested whether analyzing the internal composition of recA amplicons could be a way to directly assess the micro-diversity of agrobacterial populations using cloning sequencing or pyrosequencing approaches. The later methodology was applied to establish the actual field diversity of Agrobacterium and to evaluate whether plant genotypes differentially select agrobacteria in their root systems, providing first data upon biotic factors shaping the population structure of agrobacteria

Introdução: O gênero Acinetobacter sp apresenta considerável heterogeneidade possuindo inúmeras espécies. Atualmente, 23 espécies já foram nomeadas e nove outras espécies já foram descritas. Quatro destas espécies possuem contextos clínicos e epidemiológicos diferentes, no entanto são agrupadas em um complexo denominado Acinetobacter calcoaceticus-Acinetobacter baumannii (ABC) devido à sua similaridade genética e fenotípica. Diversos testes para diferenciação do complexo já foram descritos, porém a maioria não pode ser realizado na rotina laboratorial, pois são caros e laboriosos. Objetivos: Avaliar um método rápido e viável na rotina laboratorial, capaz de diferenciar as espécies do complexo ABC; Determinar a prevalência das diferentes espécies do complexo ABC; Avaliar o perfil de suscetibilidade aos antimicrobianos nas diferentes espécies. Métodos: Foram analisadas 118 amostras de dois hospitais de Porto Alegre-RS através do método Multiplex PCR para o gene gyrB e posteriormente confirmadas pelo padrão-ouro: sequenciamento do 16S-23S ITS. O perfil de suscetibilidade foi realizado através de microdiluição em caldo. Resultados: Das 118 amostras identificadas inicialmente como Acinetobacter sp., a grande maioria dos isolados (106 -89.9%) foram identificados como A. baumannii; mas doze isolados foram identificados como sendo das demais espécies do complexo ABC: 6 (5.1%) A. nosocomialis, 5 (4.2%) A. pittii, e 1 (0.8%) A. genoespécie 10, através da técnica de Multiplex PCR. Todos os resultados foram confirmados por sequenciamento. A. baumannii apresentou um elevado nível (72,6%) de resistência ao imipenem em comparação com as outras espécies seguido da espécie A. nosocomialis que apresentou metade de seus isolados resistentes. Todas as espécies apresentaram baixos índices (inferior a 7,5%) de resistência à Polimixina B e Tigeciclina. Conclusão: O Multiplex PCR para o gene gyrB apresentou resultados fidedignos quando comparados ao padrão-ouro, demonstrando, assim, ser um método confiável para a identificação das espécies do complexo ABC. Outras espécies, além de A. baumannii, ABC podem apresentar percentuais significativos de resistência ao imipenem. / Background: Introduction: The genus Acinetobacter sp presents considerable heterogeneity possessing numerous species. Currently, 23 species have been named and nine other species have been described. Four of these species have different clinical and epidemiological contexts, but are grouped in a complex called Acinetobacter calcoaceticus-Acinetobacter baumannii (ABC) due to their genetic and phenotypic similarity. Several tests for differentiation of the complex have been described, but most can not be performed routinely in the laboratory, they are expensive and laborious. Objectives: To evaluate a fast and feasible in routine laboratory able to differentiate the species of the complex ABC; determine the prevalence of different species of the complex ABC; evaluate the antimicrobial susceptibility profile of the different species. Methods: We analyzed 118 samples from two hospitals in Porto Alegre-RS by the method of Multiplex PCR for gene gyrB and subsequently confirmed by the gold standard: sequencing the 16S-23S ITS. The susceptibility profile was performed by microdilution. Results: Of the 118 samples initially identified as Acinetobacter sp. The great majority of isolates (106 -89.9%) were identified as A. baumannii, but twelve isolates were identified as being from other species of the complex ABC: 6 (5.1%) A. nosocomialis, 5 (4.2%) A. pittii, and 1 (0.8%) A. genospécie 10 by Multiplex PCR technique. All results were confirmed by sequencing. A. baumannii showed a high level (72.6%) of imipenem resistance in comparison with the other species followed by the species A. nosocomialis showed that half of his resistant isolates. All species showed low levels (less than 7.5%) of resistance to Polymyxin B and Tigecycline. Conclusion: Multiplex PCR for gene gyrB results presented totally reliable when compared to the gold standard, demonstrating thus be a safe method for the laboratory. Other species besides A. baumannii, ABC may have significant percentages of resistance to imipenem.

Introdução: O gênero Acinetobacter sp apresenta considerável heterogeneidade possuindo inúmeras espécies. Atualmente, 23 espécies já foram nomeadas e nove outras espécies já foram descritas. Quatro destas espécies possuem contextos clínicos e epidemiológicos diferentes, no entanto são agrupadas em um complexo denominado Acinetobacter calcoaceticus-Acinetobacter baumannii (ABC) devido à sua similaridade genética e fenotípica. Diversos testes para diferenciação do complexo já foram descritos, porém a maioria não pode ser realizado na rotina laboratorial, pois são caros e laboriosos. Objetivos: Avaliar um método rápido e viável na rotina laboratorial, capaz de diferenciar as espécies do complexo ABC; Determinar a prevalência das diferentes espécies do complexo ABC; Avaliar o perfil de suscetibilidade aos antimicrobianos nas diferentes espécies. Métodos: Foram analisadas 118 amostras de dois hospitais de Porto Alegre-RS através do método Multiplex PCR para o gene gyrB e posteriormente confirmadas pelo padrão-ouro: sequenciamento do 16S-23S ITS. O perfil de suscetibilidade foi realizado através de microdiluição em caldo. Resultados: Das 118 amostras identificadas inicialmente como Acinetobacter sp., a grande maioria dos isolados (106 -89.9%) foram identificados como A. baumannii; mas doze isolados foram identificados como sendo das demais espécies do complexo ABC: 6 (5.1%) A. nosocomialis, 5 (4.2%) A. pittii, e 1 (0.8%) A. genoespécie 10, através da técnica de Multiplex PCR. Todos os resultados foram confirmados por sequenciamento. A. baumannii apresentou um elevado nível (72,6%) de resistência ao imipenem em comparação com as outras espécies seguido da espécie A. nosocomialis que apresentou metade de seus isolados resistentes. Todas as espécies apresentaram baixos índices (inferior a 7,5%) de resistência à Polimixina B e Tigeciclina. Conclusão: O Multiplex PCR para o gene gyrB apresentou resultados fidedignos quando comparados ao padrão-ouro, demonstrando, assim, ser um método confiável para a identificação das espécies do complexo ABC. Outras espécies, além de A. baumannii, ABC podem apresentar percentuais significativos de resistência ao imipenem. / Background: Introduction: The genus Acinetobacter sp presents considerable heterogeneity possessing numerous species. Currently, 23 species have been named and nine other species have been described. Four of these species have different clinical and epidemiological contexts, but are grouped in a complex called Acinetobacter calcoaceticus-Acinetobacter baumannii (ABC) due to their genetic and phenotypic similarity. Several tests for differentiation of the complex have been described, but most can not be performed routinely in the laboratory, they are expensive and laborious. Objectives: To evaluate a fast and feasible in routine laboratory able to differentiate the species of the complex ABC; determine the prevalence of different species of the complex ABC; evaluate the antimicrobial susceptibility profile of the different species. Methods: We analyzed 118 samples from two hospitals in Porto Alegre-RS by the method of Multiplex PCR for gene gyrB and subsequently confirmed by the gold standard: sequencing the 16S-23S ITS. The susceptibility profile was performed by microdilution. Results: Of the 118 samples initially identified as Acinetobacter sp. The great majority of isolates (106 -89.9%) were identified as A. baumannii, but twelve isolates were identified as being from other species of the complex ABC: 6 (5.1%) A. nosocomialis, 5 (4.2%) A. pittii, and 1 (0.8%) A. genospécie 10 by Multiplex PCR technique. All results were confirmed by sequencing. A. baumannii showed a high level (72.6%) of imipenem resistance in comparison with the other species followed by the species A. nosocomialis showed that half of his resistant isolates. All species showed low levels (less than 7.5%) of resistance to Polymyxin B and Tigecycline. Conclusion: Multiplex PCR for gene gyrB results presented totally reliable when compared to the gold standard, demonstrating thus be a safe method for the laboratory. Other species besides A. baumannii, ABC may have significant percentages of resistance to imipenem.

As abelhas sem ferrão estão entre os animais mais importantes para o equilíbrio do meio ambiente, isso devido a fatores como sua enorme diversidade e principalmente por serem importantes polinizadores tanto de ecossistemas naturais como de agroecossistemas. A necessidade de identificação dos animais amostrados por especialistas é fundamental para estudos ecológicos. Neste trabalho foram feitas análises interespecíficas e intra-populacionais sobre os indivíduos do gênero Schwarziana a fim de aperfeiçoar a utilização da técnica de morfometria geométrica, que nos permite identificações baseadas apenas nos padrões de venação das asas desses insetos. Não obstante, foram realizadas também análises de sequenciamento de dois genes do DNA mitocondrial, o COI e o 16S. O gênero estudado foi Schwarziana, com duas espécies válidas, S. quadripunctata e S. mourei. Nas análises morfométricas, os testes realizados por indivíduos os testes de validação cruzada identificaram de forma correta 70% das amostras de um total de 10 localidades diferentes. Esta acurácia aumenta ainda mais à medida que novos grupos são formados, alcançando próximo de 85% quando separadas por regiões. Em todos os ensaios realizados com a morfometria geométrica (partial warps e coordenadas alinhadas) atingiu-se uma taxa de 100% de identificação correta entre as duas espécies e nos ensaios feitos com as médias das colônias esta taxa também foi atingida para a identificação de todas as populações amostradas. Os testes feitos com os partial warps mostraram-se mais eficazes em relação às coordenadas alinhadas, já que nestes últimos a tolerância mínima para a separação dos grupos não foi atingida em alguns ensaios. As análises moleculares apontaram 53 sítios polimórficos para o gene COI, com um índice de diversidade nucleotídica de 0,02180 e de diversidade haplotípica de 0,8854, separando as amostras em 9 haplótipos, porém a rede de haplótipos não foi suficientemente conclusiva. A diferenciação genética total medida pelo parâmetro Fst somente para as populações de S. quadripunctata foi de 0.9453 (P<0,05). Já para o gene 16S foram encontrados 14 sítios polimórfico e um índice de diversidade nucleotídica de 0,00649 e de diversidade haplotípica de 0,8419, com 7 haplótipos gerados. O parâmetro Fst para todas amostras foi de 0.9552 (P<0,05) e somente para as de S. quadripunctata foi de 0.8736 (P<0,05). Para ambos os genes os testes Fst par-a-par mostraram uma maior variação entre as populações dos estados, mostrando que estas populações estão estruturadas. Os testes de Mantel correlacionaram positivamente os dados morfométricos, geográficos e moleculares. Das duas metodologias aplicadas em nossa pesquisa, podemos afirmar que a morfometria mostrou-se extremamente eficiente na diferenciação das populações amostradas. As análises moleculares indicaram que estas populações estão estruturadas mesmo analisando poucas amostras. No entanto ao unirmos estas duas metodologias, combinamos a simplicidade e a rapidez da morfometria geométrica com a capacidade sempre inovadora de estudos moleculares, obtendo desta forma ferramentas eficazes para acessar a biodiversidade em Meliponini, inclusive para rastrear geograficamente espécimes a partir de estudos com indivíduos de sua área de distibuição natural. / Stingless bees are among the most important animals to the environmental balance, that due to their diversity and mainly because they are important pollinators of both natural and agro-ecosystems. The need for identification of sampled animals by experts is essential for ecological studies. In this work, intra and interspecific population analysis was performed in order to improve the technique of geometric morphometry, which allows us to access this biodiversity through identifications based on patterns of wing venation of these insects. We also sequenced two mitochondrial genes, the COI and 16S. Schwarziana Lepeletier 1836 was the gender studied with two valid species, S. quadripunctata and S. mourei. In morphometric analysis, cross-validation tests carried out by individuals correctly identified 70% of samples from a total of 10 different locations. This accuracy increases further as new groups are formed, according to geographic proximity, reaching around 85% when separated by regions. In all tests with geometric morphometry (partial warps and aligned coordinates) reached a rate of 100% correct identification between the two species and the tests made with the averages of the colonies that rate achieved the perfect identification of all populations sampled. Partial warps tests were more effective in relation to aligned coordinates ones, as these last a minimum tolerance for the separation of the groups was not achieved in some tests. Molecular analysis showed 53 polymorphic sites for the COI gene, with nucleotide diversity of 0.02180 and haplotype diversity of 0.8854, separating the samples into 9 haplotypes, but their network of haplotypes was not sufficiently conclusive. The total genetic differentiation measured by Fst parameter only for the populations of S. quadripunctata was 0.9453 (P <0.05). As for the gene 16S, we found 14 polymorphic sites and a nucleotide diversity of 0.00649 and haplotype diversity of 0.8419, with seven haplotypes generated. The parameter Fst for all samples was 0.9552 (P <0.05) and only for S.quadripunctata was 0.8736 (P <0.05). For both genes Fst pairwise tests showed greater variation among populations of the states, showing these groups as a genetic structure. Mantel tests were positively correlated for morphometric, geographical and molecular data. Of the two methodologies in our research, we can affirm that the morphometry proved to be extremely efficient in the differentiation of populations. Molecular analysis showed that populations are consistent, even that a low sample size is available. However combining these two methodologies, we have joined the simplicity and speed of geometric morphometric with the always innovative ability of molecular studies, thus achieving effective tools for accessing biodiversity in Meliponini, including a geographically trace for specimens by studying individuals from their natural range.

Orientador: Aricio Xavier Linhares / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-04T02:54:55Z (GMT). No. of bitstreams: 1 Thyssen_PatriciaJacqueline_D.pdf: 2658711 bytes, checksum: 6ae5d4aeb98843b0a14d74606d3f57c1 (MD5) Previous issue date: 2005 / Resumo: A correta identificação e avaliação da idade de insetos envolvidos com a decomposição de cadáveres é de suma importância para a estimativa do intervalo pós-morte (IPM) na área das ciências forenses, particularmente quando o IPM é baseado em informações sobre o ciclo de vida de insetos necrófagos. Entretanto, a análise destes parâmetros em insetos, especialmente quando se encontram em seus estágios imaturos, é difícil mesmo para taxonomistas bem treinados. Além das minúsculas diferenças morfológicas que há entre várias espécies, algumas variáveis tais como temperatura e substâncias tóxicas podem afetar o seu tempo de desenvolvimento gerando um erro no cálculo do IPM. Entre os insetos envolvidos neste processo, as larvas de dípteros da família Calliphoridae são freqüentemente as mais predominantes consumidoras de carcaça e estão presentes em todos os estágios de decomposição. Assim, este estudo teve como objetivo caracterizar morfologicamente e avaliar o tempo de desenvolvimento e as exigências térmicas das formas imaturas de duas espécies de dípteros em diferentes temperaturas: Hemilucilia segmentaria (Fabricius) e Hemilucilia semidiaphana (Rondani) (Calliphoridae). Todos os experimentos foram realizados em câmaras climáticas com temperaturas controladas em 10, 15, 20, 25, 30 e 35ºC, com fotoperíodo de 12 horas e umidade relativa de 70%. Dieta artificial própria para larvas foi oferecida para que estas completassem seu desenvolvimento. Neste estudo, além da descrição e caracterização morfológica tradicional, também foram utilizadas as técnicas da reação em cadeia da polimerase, associada ao polimorfismo baseado no comprimento do fragmento de restrição (PCR-RFLP), para a identificação das duas espécies / Abstract: The correct identification and age determination of insect species involved in cadaver decomposition is of particular importance in estimating the post-mortem interval (PMI) in forensic sciences, particularly since the PMI is based on information on the life cycle of necrophagous insects. However, the correct identification of several insects species, especially in their immature stages, is difficult even for experienced taxonomists. In addition to the minuscule morphological differences between several species, there are some variables such as temperature and toxic substances that may affect the developmental time of insects, generating errors in the estimate of the PMI. Among the insects that are involved in cadaver decomposition, maggots of blowflies (Calliphoridae) are often the most important consumers of carrion and are present in all stages of decomposition. Thus, this study aimed to characterize morphologically and to evaluate the developmental time and the thermal requirements of the immature stages of two species of blowflies reared in different temperatures: Hemilucilia segmentaria (Fabricius) e Hemilucilia semidiaphana (Rondani) (Calliphoridae). All experiments were done in growth chambers with temperatures set at 10, 15, 20, 25, 30 and 35ºC, photophase of 12 hours and relative humidity at 70%. The maggots were reared using an artificial diet for their complete development. In addition to traditional morphological description and characterization of the immatures, the usefulness of the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to identify the two species mentioned above was also assessed in this study / Doutorado / Parasitologia / Doutor em Parasitologia

Herbal medicines (HMs) have been used to supplement, maintain, and treat health conditions, and have inspired the development of many Western pharmaceuticals. Migrant South Asian (SA) communities in the UK have brought with them their own traditional forms of medicine, yet little is known about their current use of HMs in the UK. Consuming HMs alongside conventional Western medicines could affect pharmacological treatment and lead to herb-drug interactions; hence, healthcare professionals (HCPs) should be aware of their patients’ use of HMs. The import of HMs to the UK raises concerns over the quality, safety and regulation of HMs. Deoxyribonucleic acid (DNA) barcoding can be used to discriminate between different species, and identify contaminants and adulterants, thus can be used for the authentication of HMs. The South Asian Traditional Medicines (SATMED) questionnaire explored the knowledge and use of HMs by diasporic SA communities in the UK. It uncovered a vast range of HMs which were used by participants, where ingredients were sourced from, the concurrent use of herbal and Western medicines, and how minor ailments were treated. An online survey designed to investigate UK based practitioners’ views of HMs revealed that HCPs claimed to lack sufficient knowledge of HMs. HCPs said they needed more training on HMs to help them make better informed decisions. Tulsi (Ocimum tenuiflorum L.) was identified as a culturally and commercially valuable plant, which was used for molecular analysis. A variety of tulsi samples were collected for authentication: community samples from SA families in the UK, commercial samples, and referenced specimens. Both ITS and trnH-psbA regions were successfully used to distinguish between several Ocimum species, and identify a potential species substitution. This research represents the first time that DNA based methods have been used to authenticate medicinal plants species used by migrant SA communities living in the UK. The results of this multi-disciplinary study provide a unique contribution to the evolving discipline of ethnopharmacology.

Background: Critically ill patients are at particular risk of developing bloodstream infection. Such infections are associated with the development of sepsis, leading to a marked increase in mortality rate. Early detection of the causative organism and appropriate antibiotic treatment are therefore critical for optimum outcome of patients with nosocomial infection. Current infection diagnosis is based on standard blood culture techniques. However, microbiological culture has a number of limitations, not least that it takes several days to confirm infection and is therefore not useful in directing the early treatment with antibiotics. New techniques based on the detection of pathogen DNA using real-time polymerase chain reaction (PCR) technology have the potential to address these limitations but their clinical utility is still to be proved. Objectives: Develop and evaluate novel PCR-based approaches to bloodstream infection diagnosis in critical illness based on detection and identification of bacterial and fungal DNA in blood. Methods: A range of commercial and 'in-house' PCR-based assays for detection of bacterial and fungal DNA were developed and/or optimised for use in clinical blood samples. These included LightCycler SeptiFast, a CE-marked multi-pathogen assay for common bloodstream pathogens, BactScreen and GramScreen, broad spectrum bacterial assays based on 16S rRNA gene and real-time PCR assays developed to detect a range of clinically important fungal pathogens. Novel approaches to speciation of pathogen DNA using melting temperature (Tm) profiling and high resolution melting analysis (HRMA) were developed. Clinical evaluation of assays was either on blinded clinical isolates or blood samples from critically ill patients with clinical suspicion of bloodstream infection against conventional microbiological culture. Several techniques aimed at improving extraction of pathogen DNA from blood were also investigated. Results: The CE-marked commercial assay SeptiFast showed analytical sensitivity and specificity of 79% and 83% respectively. Concordance with positive culture results was good but high levels of 'false positives' were detected possibly attributed to detection of free pathogen DNA not associated with viable pathogens. The predictive value of a negative SeptiFast test was 98% suggesting that absence of pathogen DNA is a strong indicator of absence of infection. Further studies were aimed at detailed optimisation and validation of 16S rRNA gene real-time PCR assays for bacterial DNA. BactScreen and GramScreen were able to detect a broad range of clinically important bacteria down to <50 CFU/ml blood. A preliminary comparative evaluation against SeptiFast showed BactScreen gave excellent concordance with blood culture results with minimal false positive results compared to SeptiFast. Efficient extraction of pathogen DNA was shown to be a key factor in determining analytical sensitivity and several protocols were evaluated. Low cost approaches to speciation of bacterial DNA were developed by combining broad range real-time PCR with HRMA. A novel HRMA method based on Tm profiling was shown to identify 89% and 96% of blinded clinical isolates at species or genus level respectively. Real-time PCR/HRMA approaches were also successfully developed for detection and identification of fungal pathogens including a range of Candida and Aspergillus species associated with bloodstream fungal infection. Conclusions: These studies have highlighted some of the key factors that need to be considered when developing and validating PCR based assays for pathogen DNA detection in blood. A set of novel tools have been developed for rapid detection and identification of bacterial and fungal pathogens that could address the challenges of infection diagnosis based on pathogen DNA detection. Further work is required, not least in development of more efficient pathogen DNA extraction and detailed clinical validation but the tools described here have the potential to provide cost effective solutions to aid infection diagnosis that would be complementary to current culture-based methods. The provision of time critical information could have a positive impact on clinical decision-making leading to more effective management and treatment of patients with suspected bloodstream infection.

Most methods for analysis of genomic data work with symbolic sequences. Numerically represented genomic sequences can be analyzed by signal processing methods. A new method of numerical representation of DNA sequences, nucleotide density vectors, is proposed in this thesis. Usability of this method for purposes of molecular species identification is tested on DNA barcoding sequences. DNA barcoding is modern and popular methodology based on comparison of short mitochondrial DNA sequences. Beside species identification by proposed method based on nucleotide density vectors, higher taxa rank identification (e.g. families) was also tested. Furthermore, dendrograms were constructed from standardly used evolutionary distances and distances between nucleotide density vectors and the dendrograms were compared.

>Magister Scientiae - MSc / Aims DNA metabarcoding has recently emerged as a valuable supplementary tool to ensure food authenticity within the global food market. However, it is widely known that highly processed food samples are one of DNA metabarcoding’s greatest shortfalls due to high DNA degradation, presence of PCR inhibitors and the incomplete removal of several undesirable compounds (such as polysaccharides) that makes the amplification of desired DNA challenging. This project has two main aims, the first of which was to determine and develop a cost and time effective DNA metabarcoding system that could successfully describe to species level the ingredient composition of highly processed vegetarian food products. The DNA metabarcoding system was thoroughly evaluated and tested by combining well-researched primers with varying concentrations into a multiplex reaction. The combination of plant and animal primers selected that yielded the best results were used to determine the species composition in the samples. The second aim is to determine the possible presence of meat contaminants within the highly processed vegetarian food samples. Numerous studies have shown that food adulteration is a wide-spread phenomenon throughout the world due to the economic gains it can provide. Animal primers were introduced into the multiplex reaction to aid in the identification of any meat products that could have been inserted into the vegetarian products to lower the overall cost to company. Methodology Thirty-two highly processed vegetarian food samples were collected in the Cape Town area from local and franchised supermarkets. DNA was extracted using the Chloroform/Isoamyl alcohol method best suited for plant-based samples followed by amplification of the following mini-barcoding regions: the mitochondrial 16S ribosomal rRNA, cytochrome B, tRNALeu – trnL – UAA intron and the ribosomal internal transcribed spacer region – ITS2 for plant and fungi identification. The PCR products were purified using the Qiaquick kit and library preparation and building was conducted using the TruSeq DNA PCR-free Library kit. Final purification was completed using AMPure XP kit and the pooled libraries were sequenced on an Illumina Miseq using 300bp paired-end run. Statistical and bioinformatic analysis on the NGS raw sequence reads was performed in R version 3.6.3. Results The results of the data analysis showed that the cytochrome B primer couldn’t detect any animal DNA in the vegetarian samples, however animal-derived sequences were detected in the positives present, validating the efficacy of the multiplex reaction. Mitochondrial 16S ribosomal rRNA was only able to detect plant-based DNA due to the structural homology between chloroplast and mitochondrial DNA. The fungal ribosomal internal transcribed spacer region – ITS2 detected sequences deriving from “Viridiplantae”. This result could have been due to the fungal and plant ribosomal internal transcribed spacer region – ITS2 sharing a reverse primer during amplification. The trnL region was able to detect the presence of undeclared coriander, mustard and wheat in 8 (29%), 6 (21%) and 5 (18%) samples respectively. Additionally, trnL was able to detect the presence of tobacco in 11 (35%) samples. This could have been due to cross-contamination between samples being co-extracted and amplified at the same time for separate studies. The PITS2 region was able to detect the presence of undeclared barley, mustard and wheat in 8 (25%), 4 (14%) and 4 (14%) samples respectively. Our results show the possibility of DNA metabarcoding for the authentication of a wide range of species present in highly processed vegetarian samples using a single assay. However, further optimization of the technique for the identification of both plant and animal species within vegetarian samples needs to be performed before the wide-spread implementation of this technology would be both feasible and viable. Eliminating primer biases, decreasing the risk of homology between different primers in the same assay as well as preventing the amplification of sequencing of undesirable DNA need to be further explored and ultimately mitigated before DNA metabarcoding can be widely seen as an effective and cost-effective method for authentication and food control.

De nos jours, l’identification automatique des espèces de plantes par l’analyse d’images, devient incontournable pour faire perdurer, standardiser voire approfondir les connaissances relatives à la communauté végétale. Cette thèse aborde le problème d’identification automatique des espèces de plantes en utilisant les images de feuilles. Elle s’attaque à deux principaux challenges: Le premier challenge est le grand nombre et la large variabilité de la morphologie foliaire des espèces et le deuxième challenge est la variabilité intra-espèces qui se manifeste localement au niveau de régions particulières des feuilles. Pour pallier à ces deux problèmes, un retour à la botanique et notamment aux concepts botaniques foliaires a été établi pour définir une structuration automatique des feuilles à deux niveaux: Le premier niveau concerne un schéma de catégorisation selon les deux concepts botaniques “arrangement” et “lobation”. Le deuxième niveau consiste à définir les parties sémantiques qui composent la feuille. L’approche de la thèse s’articule autour de deux principaux volets: Dans le premier volet, nous nous intéressons à mettre en place cette structuration guidée par la sémantique botanique en définissant des propriétés géométriques simples corrélées avec les définitions et les observations botaniques. Dans le deuxième volet, nous étudions la faisabilité et la pertinence d’intégrer cette structuration dans la chaîne d’identification. Particulièrement, nous établissons des recherches ciblées dans les catégories et nous définissons des modèles de parties à significations botaniques. Nous établissons notre évaluation sur les deux bases d’images de Scans de feuilles ImageCLEF 2011 et ImageCLEF 2012. Nous comparons notre approche par rapport à un schéma d’identification de référence, appliqué sur la totalité de la base et en utilisant l’image entière, et par rapport à plusieurs méthodes référencées dans la littérature. / Nowadays, automatic identification of plant species, by image analysis, has become crucial to maintain, standardize or deepen knowledge about the plant community. This thesis focus on the problem of automatic identification of plant species using leaf images. It addresses two main challenges: The first challenge is the large number and the high variability in foliar morphology across species. The second challenge is the intra-species variability which occurs locally at particular regions of leaves. To overcome these two problems, a return to botany and especially to leaf botanical concepts is established in order to define an automatic structuring of leaves at two levels: The first level concerns a categorisation scheme according to the botanical concepts “arrangement” and “lobation". The second level consists in decomposing leaves into semantic parts. The approach of the thesis is based on two key parts: In the first part, we focus on establishing this botanical-based structuring process by defining simple geometric properties correlated with botanical definitions and observations. In the second part, we investigate the feasibility and opportunities to integrate this structuring process in the identification scheme. Particularly, we make targeted researches in categories and we define specific part-based models.Experiments are conducted using the ImageCLEF 2011 and 2012 Scan images leaf databases. We compare our approach with respect to the reference identification scheme, applied on the whole databaseand using the entire images, and with respect to several methods referenced in the literature.