Carcinogênese é um fenômeno de múltiplas fases. A literatura recente aceita, baseada em evidências epidemiológicas, que a progressão neoplásica em direção ao carcinoma invasor da mama inicia-se em estados hiperplásicos do epitélio ductal, passando pelo carcinoma in situ. Questões abertas na literatura no que concerne à relação existente entre o carcinoma intraductal e o carcinoma ductal invasivo referem-se aos fatores envolvidos nos processos que levam à invasão tumoral. Quatro modelos de progressão de doença foram propostos baseados na morfologia dessas lesões. Três deles descreveriam diferentes vias de progressão direta do carcinoma intraductal para o carcinoma invasor. O quarto modelo propõe que a evolução do carcinoma intraductal e do carcinoma invasor se faz independentemente, \"em paralelo\", a partir de uma terceira lesão precursora comum que, na verdade, gera um tumor de colisão in situ e invasor. O presente trabalho estudou retrospectivamente o tecido tumoral da mama de 46 pacientes do sexo feminino que continham carcinoma intraductal associado ao componente invasor na mesma lesão. Foram determinados o conteúdo de DNA nuclear por citometria estática e o grau nuclear de ambas as lesões, assim como o padrão arquitetural do componente intraductal e o índice de formação de túbulos do componente invasor. Os resultados mostraram relação estatisticamente significante (kappa=0,713; p<0,001) para o grau nuclear entre os grupos, o mesmo acontecendo com o conteúdo de DNA nuclear (kappa=0,5018 p=0,0002). Ao compararmos o aspecto arquitetural do componente intraductal com o índice de formação de túbulos do componente invasor, não encontramos correlação estatisticamente significante (p=0,1033). Os resultados permitem especular que as características nucleares de um dado carcinoma invasor de ductos mamários são possivelmente determinados em fases pré-invasivas, enquanto que alterações arquiteturais provavelmente se desenvolvem em estágios avançados, após a célula tumoral adquirir o fenótipo invasor. / Carcinogenesis is a multiple stage phenomenon. Epidemiologic evidence indicates that progression to invasive breast cancer begins in hyperplastic states of the epithelium passing by in situ carcinoma. The actual relationship that exists between ductal carcinoma in situ and invasive ductal carcinoma of the breast and the factors that lead to the tumor invasion process are questions that remain unanswered in the literature. Four models have been developed to explain the progression of the neoplastic disease in the breast tissue. Three of them propose that there is a direct pathway of progression from ductal carcinoma in situ to invasive carcinoma. The fourth one suggests that there is a common precursor lesion and both carcinoma in situ and invasive carcinoma progress in an independent fashion with no relationship between each other. The present work retrospectively studied 46 tumor tissues of the female breast with coexisting ductal carcinoma in situ and ductal invasive carcinoma. DNA ploidy as well as nuclear were determined in both lesions. The architectural pattern of the in situ component and the degree of tubule formation of the invasive component were determined also. Our results showed that there are no statistically significant differences in both nuclear parameters studied (DNA ploidy, k=0,713,p<0,001; nuclear grade, k=0,5018, p=0,0002). When we compared the architectural features of ductal carcinoma in situ with the degree of tubule formation in the invasive component we found a statistically significant different between the groups (p=0,1033). The results lead us to conclude that the nuclear features of a given invasive breast cancer are possibly determined in a preinvasive level, while architectural changes probably develop in a late stage, after the acquisition of the invasive phenotype.

<p>Tolerance to herbivory is the ability of plants to maintain fitness in spite of damage. The goal of this thesis is to investigate the genetic variation and expression of tolerance within species, determine whether and in what conditions tolerance has negative side-effects, and how tolerance is affected by different ecological factors. Tolerance is investigated with special focus on the effects of different damage types, competitive regimes, history of herbivory, and polyploidization in plants. Studies are conducted as a literature review and three experiments on two cruciferous species Raphanus raphanistrum and Cardamine pratensis.</p><p>In the tolerance experiments, plants are subjected to artificial damage solely, or in a combination with natural damage. A literature review was conducted in order to investigate the effects of damage method. We found that traits related to tolerance, such as growth and fitness were not as sensitive in regard to damage method as measures of induced chemical traits, or measures of secondary herbivory.</p><p>Genetic variation of tolerance was demonstrated within populations of R. raphanistrum and between subspecies of C. pratensis. In R. raphanistrum, traits involved in floral display and male fitness were positively associated with plant tolerance to herbivore damage. A potential cost of tolerance was demonstrated as a negative correlation between levels of tolerance in high and low competitive regimes. I found no evidence of other proposed costs of tolerance in terms of highly tolerant plants suffering of reduced fitness in the absence of herbivores or trade-offs in terms of a negative association between tolerance to apical and leaf damage, or between tolerance and competitive ability. In C. pratensis, higher ploidy level in plants involved higher levels of tolerance measured as clonal reproduction. Furthermore, populations exposed to higher levels of herbivory had better tolerance than populations exposed to lower levels of herbivory. In this thesis, I demonstrate evidence of different components for the evolution of tolerance in plants: genotypic variation, selective factors in terms of costs and ploidization, and selective agents in terms of changing environment or herbivore pressure.</p>

Tolerance to herbivory is the ability of plants to maintain fitness in spite of damage. The goal of this thesis is to investigate the genetic variation and expression of tolerance within species, determine whether and in what conditions tolerance has negative side-effects, and how tolerance is affected by different ecological factors. Tolerance is investigated with special focus on the effects of different damage types, competitive regimes, history of herbivory, and polyploidization in plants. Studies are conducted as a literature review and three experiments on two cruciferous species Raphanus raphanistrum and Cardamine pratensis. In the tolerance experiments, plants are subjected to artificial damage solely, or in a combination with natural damage. A literature review was conducted in order to investigate the effects of damage method. We found that traits related to tolerance, such as growth and fitness were not as sensitive in regard to damage method as measures of induced chemical traits, or measures of secondary herbivory. Genetic variation of tolerance was demonstrated within populations of R. raphanistrum and between subspecies of C. pratensis. In R. raphanistrum, traits involved in floral display and male fitness were positively associated with plant tolerance to herbivore damage. A potential cost of tolerance was demonstrated as a negative correlation between levels of tolerance in high and low competitive regimes. I found no evidence of other proposed costs of tolerance in terms of highly tolerant plants suffering of reduced fitness in the absence of herbivores or trade-offs in terms of a negative association between tolerance to apical and leaf damage, or between tolerance and competitive ability. In C. pratensis, higher ploidy level in plants involved higher levels of tolerance measured as clonal reproduction. Furthermore, populations exposed to higher levels of herbivory had better tolerance than populations exposed to lower levels of herbivory. In this thesis, I demonstrate evidence of different components for the evolution of tolerance in plants: genotypic variation, selective factors in terms of costs and ploidization, and selective agents in terms of changing environment or herbivore pressure.

ABSTRACT Three different genotypes of sugar beet (diploid, triploid and tetraploid) / 4 varieties from diploid and triploid genotypes Soraya (KWS8123) and Leila (diploid), Visa (H68121) and Kassandra (triploid) and 2 lines from tetraploid genotype &Ccedil / BM315 and EA2075 (tetraploid) were used for investigating the effects of ploidy level on plant regeneration. Within three sugar beet genotypes, with respect to the treatments, triploids or tetraploids were found to respond to treatments significantly different when compared with diploids. The responses of polyploids were superior over the responses of diploids. Moreover, varieties from same genotype responded differently to treatments. Two types of calli were obtained / one white and friable with regenerative capacity and the other green and compact with no regenerative capacity. Concentration of sucrose on callus development was observed to be important. High concentration of sucrose (30 g/L) was found to cause discoloration and irresponsiveness of formed calli at callus enlargement and subsequent shoot regeneration stages. Therefore, low concentration (10 g/L) is advised to be used at these stages / although this caused less callus induction. Although initially used for the prevention of tissue discoloration, L-ascorbic acid inclusion into the medium was found to be positively affecting the regeneration capacity. When used at 20 mg/100 mL concentration, the only two spontaneous shoots from the tetraploid EA2075 line were obtained. Subsequently, these shoots were successfully rooted and whole plants were obtained. The effect of silver nitrate, in combination with L-ascorbic acid, on the prevention of sugar beet tissue discoloration was investigated. Unfortunately, the symptoms of discoloration did not diminish. Moreover, callus formation was reduced and the subsequent shoot recovery could not be achieved. Since a total of 3456 explants were used during this study, and only 2 whole plants were regenerated, the efficiency of plant recovery was calculated as a rather low value of 0.058 %.

Highly profitable black caviar market and the depletion of wild sturgeon stocks warrant improvements in sturgeon aquaculture. Therefore, chromosomal manipulations, particularly gynogenesis, are focused on for increasing the ratio of females over males in progeny. The present study focused on optimizing chromosomal manipulations in sturgeons, particularly gynogenesis. The reasons of low survival rates were analyzed and the critical steps of gynogenesis induction processes were optimized. In addition, alternative ways of DNA inactivation in sperms were investigated, as well as the influence of native light-dependent DNA repair mechanisms on gynogenesis induction. Methods of interspecific gynogenesis usage for simplifying gynogenetic progeny separation were also proposed. Spectrophotometry analysis was used to investigate the ability of UV light, as the most common DNA inactivating agent, to penetrate into sperm. In addition, investigation of UV-irradiated sperm motility and results of partial gynogenesis induction showed that low transparency of sperms for UV-light can cause significant heterogeneity of UV-irradiation. As a result, a proper dilution of sperm was suggested as a critical step for homogeneous UV-irradiation of samples. Gynogenesis in sterlet was induced with chemical agents that damage sperm DNA, as an alternative to UV irradiation for applied in large-scale production of gynogenotes. All tested substances showed ability to inactivate DNA in spermatozoa, and thus producing gynogenotes. Negative impact of treatments with chemical agents on the sperm motility was observed. Subsequently, these treatments had a low efficiency of gynogenesis induction. The highest percentage of produced gynogenetic larvae 19.8 ? 8.9% was obtained by treatment with aminomethyl-4,5?,8-trimethylpsoralen (AMT) at 50 ?M followed by UV-A (360 nm) irradiation at dose of 900 J/m2. Therefore, this treatment could be used as a substitute for commonly used UV-C irradiation, e.g., in the case of large volumes of sperm. Detailed investigation of photoreactivation in sturgeon sperm revealed a significant level of light-dependent DNA restoration in sperms irradiated with high doses of UV-C light. Induction of gynogenesis with UV-C irradiation followed by exposure to visible light resulted in significant deviations from the typical Hertwig effect. In contrast, the red light with a wavelength of more than 600 nm did not result in decreased DNA damage, instead a moderate increase in damage was observed, i.e., it did not induce photoreactivation. Therefore, the use of infrared light to illuminate work stations during the induction of gynogenesis is suggested. The use of interspecific gynogenesis, particularly gametes of sturgeon species with different ploidy levels, was suggested as a way to simplify the separation of gynogenotes. In addition, application of this method allowed studying the effectiveness of DNA-inactivation and ploidy restoration treatments separately, as well as evaluation of fitness parameters and survival rates in each group of progeny without the physical separation of fish. Finally, the protocol for tetraploidization in sterlet was optimized for the prospective using tetraploid individuals for the induction of gynogenesis and androgenesis with diploid eggs and sperm. In conclusion, the described methods and protocols allowed gynogenesis induction in sturgeons with a survival rate sufficient for aquaculture, taking into consideration their high fertility, although further studies of the consequences of this treatment on fish is required.

Genome duplication plays a significant role in plant evolution. Formation of new polyploids is generally considered to be rare. Nevertheless, under natural conditions mixed-ploidy populations occur in relatively large numbers. Only the observations in the cytotype contact zone can identify all the factors affecting the stability or instability of the population. Number of research focusing on study of cytotype coexistence in natural mixed-ploidy populations is still low. As a model system for the study of mechanisms governing cytotype coexistence was chosen the Tripleurospermum inodorum. Research focused on natural mixed-ploidy populations and also on planted mixed-ploidy populations. Permanent plots were located in south, west, northwest and north Bohemia. Field observations were supplemented by cultivation experiments carried out in the greenhouse. Several phenomena were discovered at the level of whole populations. Cytotype distribution in the plot was random. The spatial structure of natural populations was quickly changing even within a single season. Even between single plot evaluations the cytotype ratios varied. Three percent of all plants were triploid hybrids. Most often detected cytotype in soil seed bank was diploid cytotype. The study of population dynamics shows, that tetraploids are...

Une protéine prion peut adopter deux conformations distinctes, l’une cellulaire et l’autre prion. La conformation prion est le résultat de son agrégation en fibre amyloïde. Cette fibre est le support de l’information prion à partir duquel les isoformes cellulaires sont convertis en forme prion de façon autocatalytique. La transmission de l’information prion repose donc sur la transmission de cette fibre au cours des divisions cellulaires, qui est réalisée par de petits polymères. Ceux-ci sont le résultat d’un équilibre entre la fragmentation et la polymérisation de la fibre. Une perturbation de cet équilibre provoque une agrégation massive de la protéine prion, menant à la perte de l’information prion.L’objectif de ma thèse était de comprendre ce qui définit in vivo la transmission du prion. Mon modèle d’étude est la protéine Ure2p propageant le prion [URE3] dans la levure S. cerevisiae. J’ai montré que la concentration cellulaire d’Ure2p détermine la vitesse d’agrégation de la protéine prion et donc son efficacité de transmission. En effet, de trop fortes concentrations cellulaires sont incompatibles avec la propagation du prion. La concentration cellulaire d’Ure2p définit également la diversité des souches prions. Un crible génétique m’a permit de mettre en évidence que la présence de séquences centromériques surnuméraires dans la cellule interfère avec la transmission du prion [URE3]. Le même phénomène est observé avec une augmentation du niveau de ploïdie de la cellule. Dans les deux cas, la surexpression du chaperon Hsp104 restaure une propagation normale du prion. / A prion protein can adopt two distinct conformations, one cellular and one prion. Prion conformation is the result of its aggregation into amyloid fibers. This fiber is the support of the prion information from which the cellular isoforms are converted into prion form by autocatalytic manner. The prion information transmission is therefore based on the transmission of this fiber during cell division, which is done by small polymers. These are the result of a balance between fragmentation and polymerization of the fiber. A disturbance of this balance causes a massive aggregation of the prion protein, leading to the prion information loss.The objective of my thesis was to understand what defined in vivo the prion transmission. My studying model was the Ure2p protein propagating the [URE3] prion in S. cerevisiae yeast. I showed that the Ure2p cellular concentration determined the aggregation speed of the prion protein and thus its transmission efficiency. Indeed, too high cellular concentrations are incompatible with the prion propagation. The cellular concentration of Ure2p also defines the prion strains diversity. A genetic screen allowed me to highlight that the presence of centrometric supernumerary sequences in the cell interferes with the [URE3] prion transmission. The same phenomenon is observed with an increase in the cell ploidy. In both cases, overexpression of the Hsp104 chaperone restores normal prion propagation.

The genus Rhodotorula includes basidiomycetous oleaginous yeast species. Rhodotorula babjevae can produce compounds of biotechnological interest such as lipids, carotenoids, and biosurfactants from low value substrates such as lignocellulose hydrolysate. High-quality genome assemblies are needed to develop genetic tools and to understand fungal evolution and genetics. Here, we combined short- and long-read sequencing to resolve the genomes of two R. babjevae strains, CBS 7808 (type strain) and DBVPG 8058, at chromosomal level. Both genomes are 21 Mbp in size and have a GC content of 68.2%. Allele frequency analysis indicates that both strains are tetraploid. The genomes consist of a maximum of 21 chromosomes with a size of 0.4 to 2.4 Mbp. In both assemblies, the mitochondrial genome was recovered in a single contig, that shared 97% pairwise identity. Pairwise identity between most chromosomes ranges from 82 to 87%. We also found indications for strain-specific extrachromosomal endogenous DNA. A total of 7591 and 7481 protein-coding genes were annotated in CBS 7808 and DBVPG 8058, respectively. CBS 7808 accumulated a higher number of tandem duplications than DBVPG 8058. We identified large translocation events between putative chromosomes. Genome divergence values between the two strains indicate that they may belong to different species.

Freshwater sport fisheries contribute substantially to the economies of England and Wales. However, many trout fisheries rely partly or entirely on stocking farmed trout to maintain catches within freshwater fisheries. Farmed trout often differ genetically from their wild counterparts and wild trout could be at risk of reduced fitness due to interbreeding or competition with farmed fish. Therefore, to protect remaining wild brown trout (Salmo trutta L) populations and as a conservation measure, stocking policy has changed. Legislation introduced by the Environment Agency (EA, 2009) will now only give consent to stocking of rivers and some stillwaters with sterile, all-female triploid brown trout. There are reliable triploidy induction protocols for some other commercially important salmonid species however; there is limited knowledge on triploid induction in brown trout. Previously, triploid brown trout have been produced by heat shocks although reduced survivals were obtained suggesting that an optimised heat shock had not been identified, or that heat shock gives less consistent success than hydrostatic pressure shock (HP), which is now recognised as a more reliable technique to produce triploid fish. Thus the overall aim of this thesis was to conduct novel research to support the aquaculture and freshwater fisheries sector within the United Kingdom by optimising the production and furthering the knowledge of triploid brown trout. Firstly, this PhD project investigated an optimised triploidy induction protocol using hydrostatic pressure (Chapter 2). In order to produce an optimised hydrostatic pressure induction protocol three experiments were conducted to (1) determine the optimal timing of HP shock application post-fertilisation, (2) define optimal pressure intensity and duration of the HP shock and (3) study the effect of temperature (6-12 °C) on triploid yields. Results indicated high survival to yolk sac absorption stage (69.2 - 93.6 %) and high triploid yields (82.5 - 100 %) from the range of treatments applied. Furthermore, no significant differences in triploid rates were shown when shock timings and durations were adjusted according to the temperature used. In all treatments deformity prevalence remained low during incubation (<1.8 %) up to yolk sac absorption (~550 degree days post hatch). Overall, this study indicated that the optimised pressure shock for the induction of triploidy in brown trout delivering high survival and 100 % triploid rate (a prerequisite to brown trout restocking) is a shock with a magnitude of 689 Bar applied at 300 Centigrade Temperature Minutes (CTM) for 50 CTM duration. Regarding the assessment of triploid status, the second experimental chapter tested the accuracy and efficacy of three ploidy verification techniques (Chapter 3). Techniques studied were erythrocyte nuclei measurements (Image analysis), flow cytometry (Becton Dickinson Facscalibur flow cytometer) and DNA profiling (22 polymorphic microsatellite loci) to assess the effectiveness of triploidy induction in brown trout. Results indicated the validity of using erythrocyte indices major nuclear axis measurements, flow cytometric DNA distributions expressed as relative fluorescence (FL2-Area), and polymorphic microsatellite loci (Ssa410UOS, SSa197, Str2 and SsaD48) for assessing ploidy status in brown trout. Accuracy of each technique was assessed and indicated that all techniques correctly identified ploidy level indicating 100 % triploid rate for that commercial batch of brown trout. These techniques may be utilised within aquaculture and freshwater fisheries to ensure compliance with the legislation introduced by the EA. As a result of the legislation introduced by the Environment Agency triploid brown trout will freely interact with diploid trout therefore there is a need to assess feeding response and behavioural differences between diploid and triploid trout prior to release. Therefore, in the third experimental chapter (Chapter 4) diploid and triploid brown trout were acclimated for six weeks on two feeding regimes (floating/sinking pellet). Thereafter, aggression and surface feeding response was compared between pairs of all diploid, diploid and triploid and all triploid brown trout in a semi natural stream (flume). In each pairwise matching, fish of similar size were placed in allopatry and rank determined by the total number of aggressive interactions initiated. Dominant individuals initiated more aggression than subordinates, spent more time defending a territory and positioned themselves closer to the food source (Gammarus pulex) whereas subordinates occupied the peripheries. When ploidy was considered, diploid trout were more aggressive than triploid, and dominated their siblings when placed in pairwise matchings. However, surface feeding did not differ statistically between ploidy irrespective of feeding regime. Triploids adopted a sneak feeding strategy while diploids expended more time defending a territory. In addition, an assessment of whether triploids exhibited a similar social dominance to diploids when placed in allopatry was conducted. Although aggression was lower in triploid pairs than in the diploid/triploid pairs, a dominance hierarchy was observed between individuals of the same ploidy. Dominant triploid fish were more aggressive and consumed more feed items than subordinate individuals. Subordinate fish displayed a darker colour index than dominant fish suggesting increased stress levels. However, dominant triploid fish seemed more tolerant of subordinate individuals and did not display the same degree of invasive aggression as observed in the diploid/diploid or diploid/triploid matchings. These novel findings suggest that sterile triploid brown trout feed similarly but are less aggressive than diploid trout and therefore may provide freshwater fishery managers an alternative to stocking diploid brown trout. In addition to research at the applied level in triploid brown trout, this thesis also examined the fundamental physiological effects of ploidy in response to temperature regime. Triploid salmonids have been shown to differ in their tolerance to environmental temperature. Therefore the fourth experimental chapter (Chapter 5) investigated whether temperature tolerance affected feed intake and exercise recovery. Diploid and triploid brown trout were exposed to an incremental temperature challenge (10 and 19 °C) and subsequent survival and feed intake rates were monitored. Triploids took longer to acclimate to the increase in temperature however feed intake were significantly greater in triploids at high temperature. In a follow on study, we investigated post-exercise recovery processes under each temperature regime (10 and 19 °C). Exhaustion was induced by 10 minutes of forced swimming, with subsequent haematological responses measured to determine the magnitude of recovery from exercise. Plasma parameters (alkaline phosphatase, aspartate aminotransferase, calcium, cholesterol, triglycerides, phosphorous, total protein, lactate, glucose, pH, magnesium, osmolality, potassium, sodium, chloride, lactate dehydrogenase) were measured for each ploidy. Basal samples were taken prior to exercise and then at: 1; 4, and 24 hours post-exercise. Contrary to previous studies, there was no triploid mortality during or after the exercise at either temperature. Although diploid and triploid brown trout responded metabolically to the exercise, the magnitude of the response was affected by ploidy and temperature. In particular, triploids had higher levels of plasma lactate, osmolality, and lower pH than diploids at 1 hour post exhaustive exercise. By 4 hours post-exercise plasma parameters analysed had returned to near basal levels. It was evident that the magnitude of the physiological disturbance post-exercise was greater in triploids than diploids at 19 °C. This may have implications where catch and release is practiced on freshwater fisheries. Overall, this work aimed to develop and/or refine current industry induction and assessment protocols while better understand the behaviour and physiology of diploid and triploid brown trout. The knowledge gained from this work provides aquaculture and freshwater fisheries with an optimised protocol, which delivers 100 % triploid rates and profitability without compromising farmed trout welfare, thus ultimately leading towards a more sustainable brown trout industry within the United Kingdom.

La conservation au niveau population est extrêmement nécessaire pour limiter la perte de biodiversité au sein d'une espèce ou d'un complexe d'espèces. Ainsi, l'évaluation de la variabilité inter-populationnelle dans le complexe est reconnue comme première étape importante pour bien définir les plans de conservation des espèces menacées. Arenaria grandiflora form un complexe d'espèces herbacées pérennes à courte durée de vie (4 ans en moyenne) menacé dans certains sites de ses zones de distribution en Europe. A ce jour, sa taxonomie n'est pas bien résolue, ce qui entraîne des problèmes potentiels pour mettre en oeuvre une conservation efficace de ce taxon. Une variation inter-populationnelle du complexe d'espèces A. grandiflora est présentée dans cette étude aux niveaux génétiques, cytogénétiques et morphométriques. Quatre méthodes ont été utilisées : des marqueurs microsatellites nucléaires, une approche cytogénétique, la cytométrie en flux, et enfin la morphométrie sur les feuilles. De plus, les études phénotypiques de variation de taux de germination entre stocks de graines ont été développées. Une différenciation significative entre les profils de variations moléculaires, cytogénétiques et phénotypiques a été détectée dans le complexe d'espèces. Deux cytotypes (diploïdes 2n=2x=22 et tétraploïdes 2n = 4x = 44) ont été mis en évidence en utilisant à la fois des méthodes classiques et des méthodes plus récentes (marqueurs microsatellites, nombres chromosomiques et cytométrie de flux). Le complexe d'espèces d'A; grandiflora présente une forte variation de la valeur de l'ADN 2C, la taille du génome varie de 2.11 ± 0.74 pg à 2.70 ± 0.11 pg pour les populations diploïdes et de 4.30 ± 1.51 pg à 5.27 ± 0.14 pg pour les populations de tétraploïdes. En outre, les grains de tétraploïdes germent significativement mieux que les graines des diploïdes. Les feuilles diffèrent considérablement entre les diploïdes (aciculaires et linéaires) et les tétraploïdes (lancéolées). Cette étude peut être considérée comme préliminaire pour une révision taxonomique de ce complexe d'espèces. D'autre part, grâce à l'ensemble des résultats obtenus, il est également possible de revisiter le concept d'unités évolutives significatives (ESUs) dans le complexe d'espèces A. grandiflora et donc de définir les groupes de populations devant faire l'objet de mesures distinctes. Ainsi, il est possible d'évaluer la pertinence de plans déjà entrepris et de proposer de nouveaux plans de restauration efficaces pour l'avenir de ce complexe d'espèces. / Population-level conservation is being extremely required to restrain the biodiversity loss within a species. So, the assessment of the variability within the species complex is being renowned as an important first step to well implement the future conservation settings for threatened species. The species complex of Arenaria grandiflora is a short-lived perennial herbaceous and a threatened taxon in certain of sites of its distribution areas in Europe, with unresolved gentics and taxonomy, which lead to potential problems in the conservation and utilization of the resource. A differenciation among populations of the species complex of A. grandiflora is presented in this study based on the genetic, cytogenetic and phenotypic patterns. Intraspecific ploidy level varaition is an important aspect of numerous species, so, the present study explores this phenomenon within the A. grandiflora species complex in some type of populations (27 natural populations). To infer the intraspecific genetic and cytogenetic patterns of variability among the studied natural populations of the investigated species complex (A. grandiflora), three methods were used : nuclear microsatellite markers, cytogenetic and flow cytometry approaches. Moreover, the phenotypic patterns of variation among both the stock of seeds and the herbarium materials of A. grandiflora were defined. These patterns were detected using three methods of seed germination (in vitro culture, filter papers and potting soil) and morphometric approaches. A significant differentiation among populations' patterns of molecular, cytogenetic and phenotypic variation was detected within the A. grandiflora species complex. Presence of two closely related cytotypes (diploids 2n=2x=22 and tetraploids 2n=4x=44) was detected using both classical and more recent methods (chromosome number count and flow cytometry respectively). The species complex of A. grandiflora exhibits high variation in 2C-DNA value, the genome size ranges from 2.11 ± 0.74 pg to 2.70 ± 0.11 pg for the diploid populations and from 4.30 ± 1.51 pg to 5.27 ± 0.14 pg for the tetraploid populations. Moreover, the seeds of tetraploids germinate well and in high proportion than the seeds of the diploid ones. In addition, both acicular and linear leaves from the diploid populations differ significantly within the diploids and with the lanceolate leaves of the tetraploid ones. New protocol of seed germination for the tetraploids by in vitro culture after scarifying was described for th first time. The affected factors on seed germination percentages were determinated by an explanatory model of six predictors (altitude, longitude, latitude, ploidy levls, both period and condition of seed storage). Consequently, all these findings are fundamental for the determination of the evolutionarily significant units (ESUs) within A. grandiflora species complex and thus the definition of efficient restoration plans in the future. This study would consider as the preliminary signal for necessary revision for the intraspecific taxonomic keys problematic for this species complex.