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Palynology or the study of pollen, is essential understand the relationship between plants and their pollinators. Traditionally, pollen grains are identified by microscopy. The method has several shortcomings, such as being time-consuming and having low taxonomic resolution. DNA-barcoding-based sequencing can identify pollen at the genus and species levels without specialized paleontological expertise. Aim of this study is to assess which molecular approach can be the most effective tool and is the most cost-effective for the identification of pollen from mixed pollen samples. A DNA metabarcoding study was conducted using the rbcL barcode gene for pollen identification using two sequencing techniques: Sanger and MinION. DNA metabarcoding produced taxonomic data easily. For the analysis of Sanger and MinION sequencing data, BLAST and KRAKEN2 were used respectively. Pavian and KRONA were later used to visualize the MinION sequencing data. Various plant species native to Sweden were identified with this metabarcoding approach. However, the reference database failed to identify a few of them, thus indicating the need to expand the reference database.

Many species have evolved to live in groups. Gregarious behavior was believed to be adaptive for whole-population survival and predator evasion until selfish-herd theory was introduced. It proposed that individuals congregate not as a method of protection for the entire population, but instead to better the individual's chance of survival, thereby using the group to benefit itself (i.e., "selfishness"). Selfish behavior is a common part of mutualisms, which are complex, dynamic interactions that often change with biotic or abiotic circumstance. Here, I investigate potential selfish behavior within the mixed-species spawning aggregations hosted by bluehead chub (Nocomis leptocephalus). I hypothesized that the host benefits more directly from the aggregation it supports than previous research suggests – specifically, by using nest associates to decrease its own risk of predation (creating a selfish-herd) and simultaneously forcing associate embryos into marginal nest locations while locating its own embryo in the safer locations within the nest (creating an "embryonic selfish-herd"). In Chapter 1, I investigated the adult spawning aggregation for possible selfishness by monitoring the chub's spatial location within the aggregation and vigilance behavior under varying associate abundances. I found that the host occupied the central location within the aggregation and was less vigilant when associate abundance was high. In Chapter 2, I examined the "embryonic herd" contained within Nocomis nests for possible embryonic selfishness orchestrated by the chub host, leading to increased embryonic survival for chub young and elevated fitness for chub parents. I found that deeper nest sections support higher embryonic survival than shallower sections, and that chub embryo make up a disproportionate percentage of embryos found in those deeper sections. I also conducted a preliminary study investigating embryo-predation by host on associate embryo which produced evidence for embryo-predation by both host and two associates on each other's offspring. Cohesively, my results support the identification of the host as a selfish participant and confirm the presence of both an adult and embryonic selfish-herd in this mutualism. This is the first study to demonstrate selfish behavior on the part of the host in this system and the first to prove the existence of an embryonic selfish-herd. / Master of Science / A fish has three goals: to survive, to grow, and to reproduce. Mutualisms – when all participants experience a net benefit from interacting – arise when two or more species rely on each other to meet these objectives. In many mutualisms, however, not all participants benefit equally. Selfish-herd theory identifies those that maximize their own benefit while minimizing or negating any cost as "selfish" individuals. I examined the mutualistic mixed-species spawning aggregations hosted by a freshwater minnow, bluehead chub (Nocomis leptocephalus), for possible selfish behavior. I found that bluehead chubs disproportionately benefit from the aggregations they host and use the aggregation to improve their own survival, improve their offspring's survival, and obtain food (by eating others' offspring). Thus, I identify bluehead chub as a selfish participant and provide evidence for the existence of two selfish herds in this mutualism – one among the adult fishes and one among their young – both orchestrated by and benefiting the bluehead chub. This study is the first to identify the bluehead chub as such and rewrites our previous understanding of this mutualism, which largely denied bluehead chub as a direct beneficiary. This work contributes to the global discussion of mutualisms by attesting the complexity of these relationships and offers support for re-examination of the classification of many known interspecific interactions, such as those cursorily termed nest parasitisms.

Le contrôle des ravageurs est l'un des principaux services éco-systémiques rendus par la biodiversité à l'agriculture. Les communautés d'insectes auxiliaires, hébergées par les espaces agricoles présentent des niveaux de diversité, spécifique et fonctionnelle, élevés et leurs biologie et traits de vie sont bien décrits et étudiés. Ces communautés constituent donc un excellent modèle pour aborder des questions d'intérêt à la fois fondamental et appliqué sur les mécanismes à l'origine de la biodiversité et sur l'impact de la biodiversité sur l'approvisionnement en services éco-systémiques. Dans cette thèse, nous développons une combinaison originale d'approches classiques et d'approches moléculaires de pointe pour élucider les relations trophiques au sein de la communauté de coléoptères carabiques en milieu agricole. Les coléoptères carabiques peuvent contribuer de façon significative au service de contrôle biologique mais les mécanismes généraux conditionnant cette contribution restent encore difficiles à évaluer par manque d'approche systémique pour l'analyse de leurs réseaux d'interactions.L'étude sans a priori du régime alimentaire de la communauté de carabes dans un paysage agricole typique révèle un partitionnement de la ressource entre groupes d'espèces. L'étude expérimentale des interactions entre espèces avec un régime alimentaire similaire montre une différentiation des activités spatio-temporelles à fine échelle. D'un point de vue fondamental, ces résultats semblent démontrer la prépondérance des processus déterministes (partage de niche) par rapport aux processus neutres (stochasticité environnementale) pour expliquer la coexistence des espèces. D'un point de vue appliqué, l'importance de la ressource dans la structuration des communautés de carabes fournit un levier d'action potentiel pour l'élaboration de stratégies de gestion afin d’optimiser leur fonction de régulateurs naturels. / Biological control is one of the main ecosystem services provided by biodiversity in agroecosystems. Communities of beneficial insects, hosted by agricultural areas exhibit high levels of species and functional diversity, and their biology and life history traits are well described today. These communities are therefore an excellent model for addressing issues of fundamental and applied interest about mechanisms at the origin of biodiversity and its impacts on the supply of ecosystem services. In this thesis, we develop an original combination of advanced molecular approaches and more traditional methods in order to elucidate trophic interaction network within the community of carabid beetles in agricultural areas. The carabid beetles can significantly contribute to the service of biological control, but their contribution and beneficial conditions are difficult to assess because of their opportunistic and plastic feeding behavior. A without a priori investigation of carabid diet at community level in a typical agricultural landscape reveals a resource partitioning between groups of species. Additional experimental studies in laboratory conditions indicate that interspecific competition could be the mechanism generating this partitioning. From a fundamental point of view, these results suggest a preponderance of deterministic processes (niche partitioning) compared to neutral processes (environmental stochasticity) to explain the coexistence of species. From an applied point of view, the importance of the resource in structuring carabid communities provides a potential lever of action for the development of efficient management strategies optimizing carabid function as crop auxiliaries.

Mon travail de thèse porte sur l'identification d'un arbre médicinal africain par le DNA barcoding et l'étude de composés à activité anti-VIH de cet arbre. Une première analyse de la séquence du marqueur ITS2 déterminée à partir d'ADN extrait de copeaux de bois a suggéré que la plante pourrait appartenir au genre Cassia ou au genre apparenté Senna. En analysant la séquence de ce marqueur ITS2 et aussi celle du trnHpsbA spacer d'une cinquantaine d'espèces des genres Cassia et Senna j'ai pu identifier la plante comme étant Cassia abbreviata. L'alignement de ces séquences m'a permis d'identifier, pour les deux marqueurs,des structures particulières spécifiques aux espèces du genre Cassia, permettant donc de les différencier des espèces du genre Senna. J'ai utilisé ces alignements pour effectuer une étude phylogenetique qui illustre que,pour les deux marqueurs, les Cassia forment en effet un clade bien séparé du clade des Senna qui peut être divisé en plusieurs sous-clades. Dans un deuxième temps j'ai étudié les effets anti-VIH de l'extrait alcoolique ainsi que de 57 composés purifiés obtenus au laboratoire. L'extrait brut ainsi qu'un des composés purifiés, le piceatannol, ont montré un grand spectre d’activités antivirales pour le VIH et le virus de l’herpès. Ils inhibent,à un stade précoce, l'infection par le VIH de lignées cellulaires de référence et d'isolats cliniques, ceci indépendamment de l'utilisation du co-récepteur (IC50: 10.47-40.77 μg/ml, CC50>1000 μg/ml; IC50: 8.04-47.46 μM, CC50>300 μM, respectivement). Ni l'un ni l'autre n'a d'effet sur CD4 et CCR5/CXCR4. L'extrait brut,mais pas le piceatannol, bloque l'interaction CD4-gp120, suggérant que l'extrait brut cible gp120 alors que le piceatannol agit comme un inhibiteur non-spécifique d'attachement du virus. Aussi, dans un modèle in vitro de tract génital femelle, le piceatannol inhibe l'infection de cellules cibles TZM-Bl par le VIH et n'active pas les cellules PBMCs, suggérant qu'il pourrait être un bon candidat comme microbicide. D'autres composés à activité anti-VIH dans l'extrait comportent l'acide oléanolique, l'acide palmitique, la taxifoline, ainsi que troiscomposés dont la structure est en train d'être élucidée. / My thesis project deals with the identification, by DNA barcoding, of an African medicinal plant and the study of anti-HIV compounds from this plant. A first analysis of the ITS2 marker sequence determined from DNA extracted from the wood suggested that the plant could belong to the Cassia or the related Senna genus. A further analysis of ITS2 as well as of trnH-psbA spacer sequences from about 50 species of the two genera allowed me to identify the plant as Cassia abbreviata. The sequence alignments, which reveal unique features present in the Cassia but not the Senna sequences, were used to construct phylogenetic trees showing the clear separation of the species of the Cassia and the Senna genus. The two markers therefore allow a quick discrimination between the species of the Cassia and the Senna genus and appear to be excellent barcode markers for identification of these species. Following the identification of the plant I have tested the crude ethanol extract as well as 57 purified compounds from the plant for an anti-HIV activity. The extract, as well as one of the compounds, namely piceatannol, showed a broad spectrum of antiviral activities for HIV and HSV. They inhibited HIV-1 infection at the early stage against various reference strains and resistant clinical isolates independent of the co-receptor usage (IC50: 10.47-40.77 μg/ml, CC50>1000 μg/ml; IC50: 8.04-47.46 μM,CC50>300 μM, respectively). Neither the crude extract nor piceatannol had an effect on CD4 and CCR5/CXCR4. The crude extract blocked CD4-gp120 interaction while piceatannol did not, indicating that CE may target gp120 and piceatannol may act as a non-specific viral attachment inhibitor. Moreover, piceatannol inhibited HIV infection of TZM-Bl target cells in an in vitro female genital tract model and did not activate PBMCs, suggesting that it may represent a good candidate as microbicide. Other anti-HIV compounds found in Cassia abbreviata include oleanolic acid, palmitic acid, taxifolin and three other compounds the structure of which is presently being elucidated.

Les tumeurs sont généralement constituées de différentes sous-populations de cellules cancéreuses génétiquement hétérogènes, responsables en grande partie de la capacité de la tumeur à évoluer rapidement et à s’adapter aux conditions environnementales. Cette diversité génétique a des conséquences majeures pour le patient, notamment au cours de la progression tumorale et pour l’acquisition d’une résistance aux traitements.Nous avons développé une nouvelle stratégie basée sur la technologie CRISPR/Cas9 qui consiste à introduire, en plus d’une altération de séquence voulue d’un gène d’intérêt, une série de mutations silencieuses, constituant une sorte d’étiquette génétique qui peut être détectée par PCR quantitative ou séquençage de nouvelle génération. En parallèle, un code-barres constitué exclusivement de mutations silencieuses est utilisé comme contrôle interne pour les effets non spécifiques potentiels qui peuvent être engendrés suite au clivage hors-cible par le système CRISPR/Cas9. Cette approche, que nous avons appelée CRISPR-barcoding, permet de générer et de suivre l’émergence d’un petit groupe de cellules cancéreuses contenant une mutation voulue au sein d’une population de cellules non modifiées, représentant ainsi un nouveau modèle expérimental d’hétérogénéité génétique intratumorale. Grâce à une série de preuves de concept, nous avons montré que CRISPR-barcoding est une nouvelle approche qui permet d’étudier de façon simple et rapide les conséquences fonctionnelles de différents types de modifications génétiques apportées directement au niveau de la séquence génomique.Dans la deuxième partie de ma thèse, nous avons utilisé cette nouvelle approche pour l'étude de la résistance du cancer bronchique non à petites cellules (CBNPC) à la thérapie ciblée. Les patients de CBNPC dont la tumeur présente une mutation activatrice de l'epidermal growth factor receptor (EGFR) sont généralement traités avec des inhibiteurs de ce récepteur. Malheureusement, malgré une réponse initiale, presque invariablement ces patients rechutent, suite au développement d’une résistance causée, dans la majorité des cas, par l’apparition de mutations secondaires ou tertiaire de l'EGFR. Grâce à un criblage réalisé en utilisant un modèle de CBNPC basé sur la stratégie CRISPR-barcoding, nous avons pu identifier l’inhibiteur multikinase sorafenib pour sa capacité à prévenir la résistance de ces cellules aux inhibiteurs d’EGFR. Ce composé présente un mécanisme d’action original, impliquant une diminution précoce du niveau de phosphorylation de STAT3, suivie par une baisse considérable de l’expression de l’EGFR, aboutissant à une inhibition des voies intracellulaires en aval de ce récepteur, telles que RAS-MAPK et PI3K-AKT-mTOR. Ces données ont été confirmées in vivo en utilisant un modèle de xénogreffe de cellules de CBNPC modifiées par CRISPR-barcoding.En conclusion, l’ensemble de nos résultats montre que le sorafenib peut prévenir l’émergence de cellules de CBNPC résistantes aux inhibiteurs d’EGFR, indiquant que ce composé pourrait représenter une nouvelle stratégie thérapeutique pour le traitement de ce type de tumeur. / Individual tumors are composed of multiple and genetically distinct subpopulations of transformed cells that can adapt and evolve in a different way based on environmental conditions. This genetic diversity has major consequences for the patient, particularly during tumor progression and for cancer treatment.We devised a new strategy based on CRISPR/Cas9 technology in which a potentially functional modification in the sequence of a gene of interest is coupled with a series of silent point mutations, functioning as a genetic label for cell tracing. In parallel, a second barcode consisting of distinct silent mutations is inserted in the same cell population and used as a control for CRISPR/Cas9 off-target cleavage. This approach, that we named CRISPR barcoding, enables detection of cells containing the mutation of interest within a mass population of unmodified cells using real-time quantitative PCR or deep sequencing. Through a series of proof-of-concept studies, we demonstrated that CRISPR-barcoding is a fast and highly flexible strategy to investigate the functional consequences of a specific genetic modification in a broad range of assays.In the second part of my thesis, we used CRISPR-barcoding to investigate non-small cell lung cancer (NSCLC) resistance to targeted therapy. Some NSCLCs harbor activating mutations of the epidermal growth factor receptor (EGFR) and are addicted to this signaling pathway. These tumors initially show a good response to EGFR inhibitors (EGFRi), but they almost invariably relapse, due to the acquisition of a resistance, as a result of additional genetic alterations, including secondary and tertiary EGFR mutations. Using a CRISPR-barcoding model, we identified the multikinase inhibitor sorafenib for its ability to prevent EGFRi resistance in NSCLC cells. This compound acts through an original mechanism that involves early reduction of STAT3 phosphorylation and late down-regulation of EGFR, resulting in the inhibition of different downstream pathways activated by this receptor, including, RAS-MAPK and PI3K-AKT-mTOR. These results were confirmed in vivo, using a CRISPR-barcoding xenograft model for NSCLC.Altogether, our data indicate that sorafenib can prevent NSCLC resistance to EGFRi through a novel mechanism, thus providing a new potential therapeutic strategy for the treatment of this type of cancer.

The alarming rate at which we see the decline in biodiversity due to human activity has raised concerns about the well-being of our planet. Butterflies which serve as pollinators are an essential part of many ecosystems and sensitive indicators of environmental changes and can provide valuable insight into how ecosystems function and evolve. This thesis aims to create phylogenetic trees based on DNA sequences from butterflies and compare different nucleotide substitution models and methods in order to better understand butterflies' evolution and genetic relationships. Our approach was to use Markov theory to investigate how the four nucleotides are evolving. In regard to the comparison of models, the General Time Reversible model with more degrees of freedom was found to be better than the K80 model. Although the Maximum Likelihood and Pairwise Distance methods were found to have different transition rate matrices, the tree reconstructions had no registered differences. Interestingly, the Q matrix was found to be similar across butterfly families. These findings can suggest that it is possible to have a standard Q matrix when estimating or inferring evolutionary relationships among butterflies, and probably other animal groups. This should improve the accuracy of estimations within phylogenetics when dealing with small data sets. The information helps with reconstructing evolutionary relationships and species, therefore contributing to preserving biodiversity and thereby the ecosystems to whom they belong - and in addition humankind. / Den oroväckande takten med vilken vi ser en minskning i biologisk mångfald på grund av mänsklig aktivitet har väckt rädsla för vår planets fortsatta välbefinnande. Fjärilar som är en väsentlig del av många ekosystem fungerar som pollinatörer och indikatorer för miljöförändringar, vilket ger värdefull insikt om ekosystemens funktion och utveckling. Fjärilar fungerar som pollinatörer och är en viktig del av många ekosystem. Därmed är de känsliga indikatorer på miljöförändringar och kan ge värdefull insikt om hur ekosystem fungerar samt utvecklas. Detta kandidatexamensarbete syftar till att skapa fylogenetiska träd baserade fjärilars DNA-sekvenser och jämföra olika modeller för nukleotid substitution och metoder för att bättre förstå fjärilars utveckling och genetiska relationer. Vårt tillvägagångssätt var att använda Markovs teori för att undersöka hur de fyra nukleotiderna utvecklas. När det gäller jämförelsen av modeller visade sig General Time Reversible-modellen med fler frihetsgrader vara bättre än K80-modellen. Fastän Maximum Likelihood och Pairwise Distance metoderna visade sig ha olika övergångsmatriser fanns det inga registrerade skillnader i trädrekonstruktionerna. Ytterligare ett intressant resultat var att Q-matrisen var liknande oberoende av fjärilsfamilj. Detta kan tyda på att det är möjligt att ha en standard Q-matris när man uppskattar eller drar slutsatser om evolutionära samband mellan fjärilar och förmodligen andra djurgrupper. Vidare studier behövs men detta skulle förbättra noggrannheten av uppskattningar inom fylogenetiken när man hanterar små datamängder. Sammanfattningsvis hjälper nämnda insikter till att rekonstruera evolutionära relationer bland arter och bidrar därför till att bevara den biologiska mångfalden, tillhörande ekosystem och dessutom mänskligheten.

Despite the existence of various molecular marker systems there are still limitations in distinguishing between closely related species based on molecular divergence, especially when hybridization events have occurred in the past. The characterisation of plant small nucleolar RNA (snoRNA) genes and their organisation into multigene clusters provides a potential nuclear marker system which could help in resolving the phylogenetic history of plants and might be applicable in DNA barcoding. Using closely and distantly related Senecio species, I investigated a combination of fragment length and sequence variation of snoRNA genes/snoRNA gene clusters to assess the utility of this marker system for barcoding and resolving species relationships. SnoRNA gene and gene cluster sequences identified in Arabidopsis thaliana were used to find homologues in other species and subsequently used for the design of universal primers. Most of the universal primer pairs designed were successful in amplifying snoRNA fragments in most Senecio species and fragment length variation between and within species could be detected. Furthermore, the combination of some fragment length datasets produced by different primer pairs enabled the separation of species and the detection of reticulate evolution indicating a high potential of snoRNA gene/gene cluster fragment length polymorphisms (SRFLPs) for phylogenetic reconstructions in Senecio and other plant genera. Most of the examined gene clusters showed a similar gene order in Senecio and Arabidopsis. However, the majority of these clusters appeared to exhibit more copies in Senecio, some of which were distinguishable by a combined sequencing/fragment profiling approach, and shown to be putative single copy regions with the potential to be used as co-dominant markers. However, a high number of paralogues and possible differences in copy number between species excludes these regions from being used in DNA barcoding. This is because specific primers would have to be developed for specific copies which would preclude development of a universal application for barcoding. None of the regions showed enough sequence variation to delimit distinctly closely related Senecio species and were therefore also considered to be unsuitable for DNA barcoding. Although most snoRNA genes and gene clusters might be inapplicable for DNA barcoding, they are likely to be valuable for phylogenetic studies of species groups, genera and families. On this scale, specific primers might act universally and the number of paralogous copies is likely to be equal across the species group of interest.

Symbiosis brought important evolutionary novelties to life on Earth. Lichens, the symbiotic entities formed by fungi, photosynthetic organisms and bacteria, represent an example of a successful adaptation in surviving hostile environments. Yet many aspects of the lichen symbiosis remain unexplored. This thesis aims at bringing insights into lichen biology and the importance of symbiosis in adaptation. I am using as model system a successful colonizer of tundra and alpine environments, the worm lichens Thamnolia, which seem to only reproduce vegetatively through symbiotic propagules. When the genetic architecture of the mating locus of the symbiotic fungal partner was analyzed with genomic and transcriptomic data, a sexual self-incompatible life style was revealed. However, a screen of the mating types ratios across natural populations detected only one of the mating types, suggesting that Thamnolia has no potential for sexual reproduction because of lack of mating partners. Genetic data based on molecular markers revealed the existence of three morphologically cryptic Thamnolia lineages. One lineage had a clear recombination structure and was found in the tundra region of Siberia, shorelines of Scandinavia, and Aleutian Islands. The other lineage was allopatric with the previous, and was highly clonal; only two haplotypes were found across the alpine region of central and southeastern Europe. However, the third lineage was sympatric with the other two, had a worldwide distribution, and although highly clonal, showed a recombinant population structure. Our data could not reveal whether the signs of recombination resulted from rare recombination events due to the extreme low frequency of the other mating type or ancestral variation before the loss of sexual reproduction. However, investigation of Thamnolia’s green algal population showed that in different localities, different algal genotypes were associated with the same fungal genotype. Furthermore, data suggest that Thamnolia carried several algal genotypes within its thalli and shared them with other distantly related but ecologically similar fungal species.