Conselho Nacional de Desenvolvimento Científico e Tecnológico / A Mata Atlântica brasileira concentra uma das maiores diversidades biológicas da Terra com cerca de 7% das espécies animais e vegetais do planeta. Esse bioma abriga atualmente mais de 50% das espécies de anuros do Brasil (c.a. 500 espécies), mas sofre intensa perda e fragmentação de habitat. Um dos principais fragmentos da Mata Atlântica, a Reserva Ecológica de Guapiaçu (REGUA) abriga vasta anurofauna, com cerca de 71 espécies já descritas. Acredita-se, porém, que muitas ainda precisam ser identificadas e estudadas. A identificação de espécies baseada em caracteres moleculares vem se mostrando uma alternativa para dar suporte à identificação morfológica, e dentro deste contexto os genes de DNA mitocondrial, como o 16S, são utilizados para a realização de barcode. O objetivo deste estudo foi testar a metodologia de identificação molecular de espécies (DNA barcode) na comunidade de anfíbios anuros da REGUA utilizando o gene mitocondrial 16S. Para isso, foram coletados tecidos de 99 indivíduos, entre adultos e girinos de 23 espécies, agrupados em seis famílias distintas. Desses 99 indivíduos, 88 foram amplificados corretamente para o gene em referência e foram realizadas, com sucesso, a determinação de espécies de 84 anuros (95,45%) da REGUA. As espécies de Scinax albicans, Scinax flavoguttatus e Hylodes charadranaetes, cujas identificações haviam sido determinadas com base em critérios morfológicos, agruparam em clados de mesmo gênero, porém de espécies diferentes quando analisadas pelas metodologias neighbor-joining e maximum-likelihood. Além de altos valores de distância intraespecífica (2,18%, 3,49% e 3,77%, respectivamente) e distâncias interespecíficas nulas (0%) temos a indicação de possíveis equívocos em determinações de espécies feitas exclusivamente por critérios morfológicos. Nesse caso, as discordâncias morfológica e molecular são exclusivamente de girinos, demonstrando a dificuldade na identificação morfológica e a escassez de chaves de identificação dessas espécies em estágio larval. Os resultados mostram que o gene mitocondrial 16S teve seu uso na identificação de anuros da REGUA confirmada e apontam que, em casos de estudos com indivíduos em estágios larvais, como em girinos, a metodologia de barcode, quando complementada a identificação morfológica, suporta a correta identificação das espécies de anfíbios anuros. / The Brazilian Atlantic Forest focuses one of the greatest biological diversity of the Earth with about 7% of the planet's animal and plant species. This biome is currently home to more than 50% of anurans species from Brazil (c.a. 500 species), but it suffers severe loss and fragmentation of habitat. One of the main fragments of the Atlantic Forest, the Reserva Ecológica de Guapiaçu (REGUA) houses a wide anuran fauna, with about 71 species had already described. It is believed, however, that lots of them still need to be identified and studied. The identification of these species based on molecular characters has proven to be an alternative to support a morphological identification, and in this context the mitochondrial DNA genes, such as 16S are used to perform barcode. The goal of this study was to test the methodology for molecular identification (DNA barcode) in anurans of REGUA community using 16S mitochondrial gene. For this, tissues of 99 individuals, including adults and tadpoles of 23 species, grouped into six different families were collected. Of these 99 individuals, 88 were amplified correctly to the reference gene and were successful determination of 84 species of anurans (95.45%) of the REGUA. Scinax albicans, Scinax flavoguttatus and Hylodes charadranaetes species whose identifications had been determined based on morphological criteria, grouped into clades of the same gender, but different species when analyzed by methodologies neighbor-joining and maximum-likelihood. In addition to high intraspecific distances (2,18%, 3,49% and 3,77% respectively) and interspecific distances to nil (0%), we have an indication of possible mistakes of species determinations made by a morphological criterion. In this case, the morphological and molecular disagreements are exclusively on tadpoles, demonstrating the difficulty of morphological identification and the shortage of identification of these species larval stage. The results show that the 16S mitochondrial gene had its use in identifying the anurans REGUA confirmed and indicate that the case studies with individuals in larval stages, as in tadpoles , the methodology of the barcode when complemented morphological identification, supports the correct identification of species of anurans amphibians .

Conselho Nacional de Desenvolvimento Científico e Tecnológico / A Mata Atlântica brasileira concentra uma das maiores diversidades biológicas da Terra com cerca de 7% das espécies animais e vegetais do planeta. Esse bioma abriga atualmente mais de 50% das espécies de anuros do Brasil (c.a. 500 espécies), mas sofre intensa perda e fragmentação de habitat. Um dos principais fragmentos da Mata Atlântica, a Reserva Ecológica de Guapiaçu (REGUA) abriga vasta anurofauna, com cerca de 71 espécies já descritas. Acredita-se, porém, que muitas ainda precisam ser identificadas e estudadas. A identificação de espécies baseada em caracteres moleculares vem se mostrando uma alternativa para dar suporte à identificação morfológica, e dentro deste contexto os genes de DNA mitocondrial, como o 16S, são utilizados para a realização de barcode. O objetivo deste estudo foi testar a metodologia de identificação molecular de espécies (DNA barcode) na comunidade de anfíbios anuros da REGUA utilizando o gene mitocondrial 16S. Para isso, foram coletados tecidos de 99 indivíduos, entre adultos e girinos de 23 espécies, agrupados em seis famílias distintas. Desses 99 indivíduos, 88 foram amplificados corretamente para o gene em referência e foram realizadas, com sucesso, a determinação de espécies de 84 anuros (95,45%) da REGUA. As espécies de Scinax albicans, Scinax flavoguttatus e Hylodes charadranaetes, cujas identificações haviam sido determinadas com base em critérios morfológicos, agruparam em clados de mesmo gênero, porém de espécies diferentes quando analisadas pelas metodologias neighbor-joining e maximum-likelihood. Além de altos valores de distância intraespecífica (2,18%, 3,49% e 3,77%, respectivamente) e distâncias interespecíficas nulas (0%) temos a indicação de possíveis equívocos em determinações de espécies feitas exclusivamente por critérios morfológicos. Nesse caso, as discordâncias morfológica e molecular são exclusivamente de girinos, demonstrando a dificuldade na identificação morfológica e a escassez de chaves de identificação dessas espécies em estágio larval. Os resultados mostram que o gene mitocondrial 16S teve seu uso na identificação de anuros da REGUA confirmada e apontam que, em casos de estudos com indivíduos em estágios larvais, como em girinos, a metodologia de barcode, quando complementada a identificação morfológica, suporta a correta identificação das espécies de anfíbios anuros. / The Brazilian Atlantic Forest focuses one of the greatest biological diversity of the Earth with about 7% of the planet's animal and plant species. This biome is currently home to more than 50% of anurans species from Brazil (c.a. 500 species), but it suffers severe loss and fragmentation of habitat. One of the main fragments of the Atlantic Forest, the Reserva Ecológica de Guapiaçu (REGUA) houses a wide anuran fauna, with about 71 species had already described. It is believed, however, that lots of them still need to be identified and studied. The identification of these species based on molecular characters has proven to be an alternative to support a morphological identification, and in this context the mitochondrial DNA genes, such as 16S are used to perform barcode. The goal of this study was to test the methodology for molecular identification (DNA barcode) in anurans of REGUA community using 16S mitochondrial gene. For this, tissues of 99 individuals, including adults and tadpoles of 23 species, grouped into six different families were collected. Of these 99 individuals, 88 were amplified correctly to the reference gene and were successful determination of 84 species of anurans (95.45%) of the REGUA. Scinax albicans, Scinax flavoguttatus and Hylodes charadranaetes species whose identifications had been determined based on morphological criteria, grouped into clades of the same gender, but different species when analyzed by methodologies neighbor-joining and maximum-likelihood. In addition to high intraspecific distances (2,18%, 3,49% and 3,77% respectively) and interspecific distances to nil (0%), we have an indication of possible mistakes of species determinations made by a morphological criterion. In this case, the morphological and molecular disagreements are exclusively on tadpoles, demonstrating the difficulty of morphological identification and the shortage of identification of these species larval stage. The results show that the 16S mitochondrial gene had its use in identifying the anurans REGUA confirmed and indicate that the case studies with individuals in larval stages, as in tadpoles , the methodology of the barcode when complemented morphological identification, supports the correct identification of species of anurans amphibians .

Culex coronator Dyar & Knab e Culex usquatus Dyar são duas espécies irmãs, que fazem parte do Complexo Coronator, composto por mais quatro espécies (Culex usquatissimos Dyar, Culex ousqua Dyar, Culex camposi Dyar, Culex covagarciai Forattini). As fêmeas deste grupo são indistinguíveis por caracteres morfológicos, sendo a identificação possível somente através da distribuição e tamanho das cerdas apicais que ornamentam o gonocoxito da genitália masculina. Cx. coronator, é a espécie com maior distribuição geográfica, ocupando as Américas do Norte, Central e Sul. Já Cx. usquatus só foi registrado nas Américas Central e do Sul, ocorrendo em simpatria no Brasil com Cx. coronator. Apesar da semelhança morfológica das fêmeas das duas espécies, até o momento, somente Cx. coronator foi encontrado naturalmente infectado por diversas arboviroses. Considerando que estudos populacionais são importantes para compreender a evolução e a dinâmica de populações de pontencias vetores, e que a correta identificação de fêmeas é fundamental para estudos de competência vetorial, os objetivos deste trabalho foram: (1) distinguir fêmeas adultas de Cx. coronator de Cx. usquatus (2) obter conhecimento da estrutura populacional de Culex coronator nas regiões Sul e Sudeste (3) examinar a possível existência de outras espécies não descritas e/ou incorretamente identificadas sob o epíteto de Cx. coronator. Para tanto foram utilizadas duas ferramentas: uma morfológica (morfometria de asa), e outra genética (4 loci de microssatélites e sequenciamento do fragmento barcode do gene COI). As análises dos três marcadores mostraram que as populações do Sudeste são geneticamente e morfologicamente diversas, mas não apresentam estrutura populacional, enquanto as populações do Sul são mais homogêneas, e diferentes das do Sudeste. Assim, tanto os microssatélites como a morfometria geométrica, mostraram alguma estruturação populacional em relação às macrorregiões Sul e Sudeste. A análise da morfometria da asa distinguiu as espécies de Cx. coronator e Cx. usquatus, enquanto a análise do COI barcode apresentou uma politomia das duas espécies. Nenhum marcador indicou a existência de um complexo de espécies sob o espíteto Culex coronator. / Culex coronator and Culex usquatus are sibling species belonging to Coronator Group, which comprises five other species (Culex usquatissimos Dyar, Culex ousqua Dyar, Culex camposi Dyar, Culex covagarciai Forattini). Except by Cx. yojae, the females of this group are indistinguishable, being the identification only possible by the analysis of the arrangement and number of appendicles on the apical lobe of the gonocoxite of the male genitalia. Cx. coronator is the most widely distributed species in the complex, occupying North, Central and South America, while Cx. usquatus was recorded only in Central and South America. Therefore, these species are sympatric in Brazil. Despite the morphological similarity of the females of both species, only Cx. coronator has epidemiological importance, being found infected with many arboviruses. Since studies focusing in population structure are important to understand the evolution and dynamics of potencial vectors and that the correct female identification is critical for development of vectorial competence studies, the aims of this study were to: (1) distinguish adult females of Cx. coronator from Cx. usquatus (2) obtain knowledge of the population structure of Culex coronator in Southern and Southeastern areas (3) examine the presence of undescribed and/or incorrectly identified species under Cx. coronator. Thereby, a survey was carried out using morphological (wing geometry) and genetic (4 microsatellite loci and barcode region of the Cytochrome c oxidase subunit I gene) markers. Results showed genetic and morphological diversity of southeastern populations, with low population structure, while southern populations were more homogenous but different from those of Southeast Brazil. Thus, analysis of microsatellite and wing morphometry showed some populational structure according to South and Southeast macro-regions. Only wing morphometric analysis distinguished Cx. coronator from Cx. usquatus, while the COI barcode analysis showed a polytomy of the two species. No marker indicated Cx. coronator s.s as a group of cryptic species.

The goal of this thesis is to describe barcodes used in real world applications and to create application for real time detecting and decoding of chosen barcodes using computer vision. Normal linear barcode is first introduced, and then two dimensional matrix barcode is described with focus on its origin, usage and structure. Especially the process of localization, encoding and decoding of QR Code, which is chosen for future use in work, is described mainly. Also briefly description of computer vision and OpenCV library is available. Next the concept and implementation of application for localization and recognition of QR Codes is described. Finally description of testing, evaluation of thesis and ways of improvements are discussed.

This thesis deals with an optimization of goods flow in a store. In detail, the work provides information about technology of the automatic goods identification using barcode and RFID, and comparison the barcode and RFID readers.

In a supply chain, a warehouse is a crucial component for linking all chain parties. Automatic identification and data capture (auto-ID) technology, e.g. RFID and barcodes are among the essential technologies in the 21st century knowledge-based economy. Selecting an auto-ID technology is a long term investment and it contributes to improving operational efficiency, achieving cost savings and creating opportunities for higher revenues. The interest in auto-ID research for warehouse management is rather stagnant and relatively small in comparison to other research domains such as transport, logistics and supply chain. However, although there are some previous studies that explored factors for the auto-ID selection decision in a warehouse environment, those factors (e.g., operational factors) have been examined separately and researchers have paid no attention to all key factors that may potentially affect this decision. In fact, yet there is no comprehensive framework in the literature that comprehensively investigates the critical factors influencing the auto-ID selection decision and how the factors should be combined to produce a successful auto-ID selection process in warehouse management. Therefore, the main aim of this research is to investigate empirically the auto-ID technology-selection process and to determine the key factors that influence decision makers when selecting auto-ID technology in the warehouse environment. This research is preceded by a comprehensive and systematic review of the relevant literature to identify the set of factors that may affect the technology selection decision. The Technology-Organisation-Environment (TOE) framework has been used as lens to categorise the identified factors (Tornatzky & Fleischer, 1990). Data were collected by conducting first a modified (mixed-method) two-round Delphi study with a worldwide panel of experts (107) including academics, industry practitioners and consultants in auto-ID technologies. The results of the Delphi study were then verified via follow-up interviews, both face-to-face and telephone, carried out with 19 experts across the world. This research in nature is positivist, exploratory/descriptive, deductive/inductive and quantitative/qualitative. The quantitative data were analysed using the statistical package for social sciences, SPSS V.18, while the qualitative data of the Delphi study and the interviews were analysed manually using quantitative content analysis approach and thematic content analysis approach respectively. The findings of this research are reported on the motivations/reasons of warehouses in seeking to use auto-ID technologies, the challenges in making an auto-ID decision, the recommendations to address the challenges, the key steps that should be followed in making auto-ID selection decision, the key factors and their relative importance that influence auto-ID selection decision in a warehouse. The results of the Delphi study show that the six major factors affecting the auto-ID selection decision in warehouse management are: organisational, operational, structural, resources, external environmental and technological factors (in decreasing order of importance). In addition, 54 key sub-factors have been identified from the list of each of the major factors and ranked in decreasing order of the importance mean scores. However, the importance of these factors depends on the objectives and strategic motivations of warehouse; size of warehouse; type of business; nature of business environment; sectors; market types; products and countries. Based on the Delphi study and the interviews findings, a comprehensive multi-stage framework for auto-ID technology selection process has been developed. This research indicates that the selection process is complex and needs support and closer collaboration from all participants involved in the process such as the IT team, top management, warehouse manager, functional managers, experts, stockholders and vendors. Moreover, warehouse managers should have this process for collaboration before adopting the technology in order to reduce the high risks involved and achieve successful implementation. This research makes several contributions for both academic and practitioners with auto-ID selection in a warehouse environment. Academically, it provides a holistic multi-stage framework that explains the critical issues within the decision making process of auto-ID technology in warehouse management. Moreover, it contributes to the body of auto-ID and warehouse management literature by synthesising the literature on key dimensions of auto-ID (RFID/barcode) selection decision in the warehouse field. This research also provides a theoretical basis upon which future research on auto-ID selection and implementation can be built. Practically, the findings provide valuable insights for warehouse managers and executives associated with auto-ID selection and advance their understanding of the issues involved in the technology selection process that need to be considered.

La famille des Phytoseiidae comprend des espèces d'intérêt agronomique dont certaines sont utilisées pour lutter contre des acariens ravageurs appartenant principalement aux familles des Tetranychidae et Eriophyidae. L'identification et la description des espèces de Phytoseiidae sont essentiellement basées sur des caractères morphologiques. Cependant, plusieurs problèmes d'identification sont rencontrés du fait : (1) de leur petite taille (faible nombre de caractères observables), (2) du manque de recul sur la valeur diagnostique de certains caractères dits discriminants, (3) et enfin du diagnostic fiable uniquement au stade femelle. Dans ce contexte, ce travail de thèse a pour objectif de déterminer dans quelle mesure l'approche moléculaire peut assister le diagnostic traditionnel. Pour cela, nous avons étudié l'étendue des variations intra- et interspécifiques (moléculaires et morphologiques) de sept espèces de Phytoseiidae d'intérêt agronomique, ceci pour comparer la capacité discriminante de quatre marqueurs moléculaires (12S ARNr, Cytb, COI et ITS). Dans ce travail, nous mettons en évidence l'utilité de ces marqueurs pour le diagnostic de tous les stades de développement et montrons l'importance de la forme du calyx de la spermathèque pour différencier deux espèces du genre Euseius, ce qui nous a permis de décrire une nouvelle espèce. Nous montrons également que les marqueurs mitochondriaux et nucléaires peuvent différencier des espèces morphologiquement proches. Cependant, il faut rester prudent quand aux conclusions tirées (espèces différentes ou populations différentes au sein d'une même espèce) sur la base des résultats obtenus avec les marqueurs mitochondriaux. Enfin, nous montrons que les règles de décision (valeurs seuils) sont difficiles à (1) établir avec ces mêmes marqueurs et (2) surtout à généraliser pour la différenciation des espèces appartenant aux deux sous-familles étudiées. Pour conclure, ce travail met en évidence de façon claire la nécessité d'une approche taxonomique intégrative, incluant un va-et-vient entre données moléculaires, morphologiques et écologiques, pour la garantie d'un diagnostic fiable des espèces de Phytoseiidae. / The family Phytoseiidae includes numerous species of huge interest for biological control of mite pests, essentially belonging to the families Tetranychidae and Eriophyidae. Identification and description of Phytoseiidae species are mainly based on morphological characters. However, taxonomists have to face several problems for species diagnostic because of : (1) their small size (low number of visible characters), (2) little information on the diagnostic value of some characters considered as discriminant, and at last (3) diagnostic only reliable at female stage. In this context, the present works aims to determine how molecular approaches can assist traditionnal diagnostic. For this, we studied the intra- and interspecific variations (morphological and molecular) of seven agronomic important species, in order to compare the ability of four molecular markers (12S rRNA, Cytb mtDNA, COI mtDNA et ITS) to discriminate between species. We first show that molecular markers could be of great help for differentiating species at all life stages. Moreover, we show the importance of the spermatheca calyx shape for separating two species of the genus Euseius, and a new specie was then described. We also emphasize that both mitochondrial and nuclear markers differentiate morphological close species. However, some cautions have to be paid on the conclusions developped (different species or different populations within a same species) when only mitochondrial markers are used. At last, we underline that decision rules (treshold cutt-off values) are difficult (1) to establish with these latter markers and also (2) to generalise for the accurate differentiation of the Phytoseiidae species belonging to the two sub-families considered. To conclude, this work clearly emphasizes the importance of integrative taxonomy, including an « up-and-down » analysis of molecular, morphological and ecological data, to secure the specific diagnostic among the family Phytoseiidae

This thesis presents the design and development of a hand-held electronic reader, designed to decode conductive patterns printed on a paper substrate. Data read from the patterns, by the reader, is used to trigger events in the digital domain. The reader and associated conductive patterns are devices for linking paper documents with the digital world. The patterns are formed by masking conductive-coated paper with a non-conductive, printed lacquer. The reader is a low cost and ergonomic device, capable of transmitting the embedded data from the conductive paper to the computer. The first reader designed and developed was tethered to a computer by data cable, using the USB communication protocol. The second design was developed further, with transmission of data achieved by replacing the cable with short-range Bluetooth wireless technology. Both devices were designed and developed using embedded systems and low cost electronic components. Additional work was undertaken to optimise the device's mechanical structure, ergonomics and integration of hardware. Alongside the development of the reader, test and development work was carried out to optimise the printed media, in materials and design. User trials demonstrated that the complete printed and reading system was functional, with varied rates of success among participants. Further work is required to improve the conductivity of the coated paper, and the accuracy of the decoding algorithm.

Duikers are a species rich subfamily of threatened African antelope whose recent origin poses a challenge to the molecular identification of taxa and estimation of their phylogeny. I test the ability of DNA barcodes to identify all taxa within this group. I then use mitochondrial and nuclear genes to estimate a multi-locus species tree and to date divergence times. DNA barcodes are unable to distinguish many sister taxa, calling into question the utility of barcodes for the regulation of duiker trade or in identification of field-collected feces. The multi-locus phylogeny provides support for the relationships among major duiker lineages and placement of two problematic taxa, but challenges the validity of the savanna genus and identifies hybridization between taxa. This study reveals that most duikers diverged during the Pleistocene, meriting further inquiry into the role that Pleistocene glacial cycling played in the diversification and population structuring of duikers.

The repeated emergence of drug resistance in Plasmodium falciparum underscores the importance of understanding the genetic architecture of current resistance pathways, as well as any associated fitness costs. Why resistance emerges in particular regions of the world has been linked to particular genetic backgrounds that better tolerate resistance-associated polymorphisms; this is likely to play a key role in driving the epidemiology of drug resistance, however is infrequently studied at a large scale in a laboratory setting. The first results chapter establishes a barcoding approach for P. falciparum with the aim of tracking parasite growth in vitro. The strategy used was adapted for P. falciparum by using a pseudogene (PfRh3) as a safe harbour to insert unique molecular barcodes. These libraries of barcoded P. falciparum vectors were also used as a readout of transfection efficiency. The second chapter establishes a proof of principle for phenotyping by barcode sequencing, using a panel of barcoded parasites generated in different genetic backgrounds that comprise sufficient genetic diversity to pilot the method. These were grown in the presence and absence of antimalarial compounds, and growth phenotypes were measured in parallel using BarSeq. The third results chapter studies the contribution of mutations in Pfkelch13, a molecular marker of artemisinin resistance, to parasite fitness. Combining CRISPR/Cas9-based genome editing and high throughput sequencing, the impact of Pfkelch13 alleles on fitness in the context of particular strain backgrounds is revealed. In particular, the impact of genetic background in the emergence and spread of drug-resistant lineages (referred to as KEL1) in Southeast Asia carrying a Y580 Pfkelch13 allele. Overall, given the current pace of genome sequencing of pathogenic organisms such as P. falciparum, it will be important to increase the scale of experimental genetics, in order to tackle in real-time natural variation that might be under constant selection from drugs, thus anticipating the emergence of drug resistance in changing parasite populations. Through this work, tools were developed to facilitate parallel phenotyping by measuring in vitro growth using high-throughput sequencing. The work also develops novel approaches to address the importance of genetic background and a potential role for positive epistasis in a lineage responsible for the recent outbreak of drug-resistant malaria in Southeast Asia.