Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide and in its advanced stage, has a poor clinical outcome. Resistance to chemotherapeutic agents, either intrinsic or acquired, is the primary cause of treatment failure in NSCLC. Tubulin binding agents (TBAs), such as paclitaxel and vinorelbine are important components in the treatment of NSCLC. Upregulation of the neuronal specific class III β-tubulin (β-III-tubulin) is frequently found in drug resistant cancer cell lines and human tumours, lending support that βIII-tubulin might play a role in the development of drug resistance in cancer cells. However, to date, compelling evidence supporting its direct role in drug resistance and response has been lacking. To address its role in NSCLC, RNA interference (RNAi) was employed to knock down βIII-tubulin expression in two drug naive NSCLC cell lines, Calu-6 and H460. Specific knockdown of βIII-tubulin resulted in increased sensitivity to TBAs and DNA damaging agents, two classes of agents that are commonly used in the treatment of NSCLC. Increased sensitivity to TBAs and DNA damaging agents in the βIII-tubulin knockdown cells was due to an increased propensity of the cells to undergo apoptosis, suggesting that this tubulin isotype may be a cellular survival factor. Interestingly, specific knockdown of βII- or βIVb-tubulin hypersensitised the cells to Vinca alkaloids but not taxanes, demonstrating that each isotype is unique in terms of drug-target interactions. Moreover, the β-tubulin isotype composition of a cell can influence response, and therefore resistance to TBAs. To determine whether βIII-tubulin differentially regulates microtubule behaviour and influences cell proliferation via an effect on microtubule dynamics, siRNAs were used to knockdown βIII-tubulin expression in H460 cells stably expressing GFP-βI-tubulin and the dynamic instability behaviour of individual microtubules was measured by time-lapse microscopy. In the absence of drug, silencing of βIII tubulin alone did not significantly affect the dynamic instability of interphase microtubules. However, at the IC50 for proliferation of either paclitaxel or vincristine, the overall dynamicity was suppressed significantly in the βIII-tubulin silenced cells as compared to the control, indicating that βIII-tubulin knockdown induces paclitaxel or vincristine sensitivity by enhancing the ability of these agents to suppress microtubule dynamics. At a concentration of drug that represented the IC50 for mitotic arrest, for either paclitaxel or vincristine, increased apoptosis induction was found to play a dominant role in βIII-tubulin knockdown, further supporting a role for βIII-tubulin as a cellular survival factor. Collectively, when βIII-tubulin is overexpressed in tumours cells, it is highly likely to be promoting cellular survival and resistance to TBAs. In addition to its proposed role in drug resistance, high expression of βIII-tubulin in tumours of non-neuronal origin such as NSCLC, has been positively correlated with the degree of tumour aggressiveness. H460 cells are known to display substrate- independent growth in soft agar and tumourigenicity in nude mice and provided an ideal model to investigate the role of βIII-tubulin in tumourigenesis. To address the role of βIII-tubulin, H460 cells stably expressing βIII-tubulin shRNA were generated, validated and examined using both in vitro and in vivo methods of tumourigenesis. Colony formation of H460 cells stably expressing βIII-tubulin shRNA was dramatically reduced in soft agar and significantly delayed tumour growth and reduced tumour incidence of subcutaneous xenografted tumours in nude mice when compared to respective controls. These results provide new insights into the function of βIII-tubulin and suggest that βIII-tubulin may play an important role in tumour development and progression in lung cancer. In conclusion, β-tubulin isotype status can serve as a valuable molecular marker capable of distinguishing patients with differential sensitivity to TBAs. These results not only shed new light on the role of specific β-tubulin isotypes in the response to TBAs, but also the role of βIII-tubulin in the biology of cancer that will lead to new treatment strategies for NSCLC.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide and in its advanced stage, has a poor clinical outcome. Resistance to chemotherapeutic agents, either intrinsic or acquired, is the primary cause of treatment failure in NSCLC. Tubulin binding agents (TBAs), such as paclitaxel and vinorelbine are important components in the treatment of NSCLC. Upregulation of the neuronal specific class III β-tubulin (β-III-tubulin) is frequently found in drug resistant cancer cell lines and human tumours, lending support that βIII-tubulin might play a role in the development of drug resistance in cancer cells. However, to date, compelling evidence supporting its direct role in drug resistance and response has been lacking. To address its role in NSCLC, RNA interference (RNAi) was employed to knock down βIII-tubulin expression in two drug naive NSCLC cell lines, Calu-6 and H460. Specific knockdown of βIII-tubulin resulted in increased sensitivity to TBAs and DNA damaging agents, two classes of agents that are commonly used in the treatment of NSCLC. Increased sensitivity to TBAs and DNA damaging agents in the βIII-tubulin knockdown cells was due to an increased propensity of the cells to undergo apoptosis, suggesting that this tubulin isotype may be a cellular survival factor. Interestingly, specific knockdown of βII- or βIVb-tubulin hypersensitised the cells to Vinca alkaloids but not taxanes, demonstrating that each isotype is unique in terms of drug-target interactions. Moreover, the β-tubulin isotype composition of a cell can influence response, and therefore resistance to TBAs. To determine whether βIII-tubulin differentially regulates microtubule behaviour and influences cell proliferation via an effect on microtubule dynamics, siRNAs were used to knockdown βIII-tubulin expression in H460 cells stably expressing GFP-βI-tubulin and the dynamic instability behaviour of individual microtubules was measured by time-lapse microscopy. In the absence of drug, silencing of βIII tubulin alone did not significantly affect the dynamic instability of interphase microtubules. However, at the IC50 for proliferation of either paclitaxel or vincristine, the overall dynamicity was suppressed significantly in the βIII-tubulin silenced cells as compared to the control, indicating that βIII-tubulin knockdown induces paclitaxel or vincristine sensitivity by enhancing the ability of these agents to suppress microtubule dynamics. At a concentration of drug that represented the IC50 for mitotic arrest, for either paclitaxel or vincristine, increased apoptosis induction was found to play a dominant role in βIII-tubulin knockdown, further supporting a role for βIII-tubulin as a cellular survival factor. Collectively, when βIII-tubulin is overexpressed in tumours cells, it is highly likely to be promoting cellular survival and resistance to TBAs. In addition to its proposed role in drug resistance, high expression of βIII-tubulin in tumours of non-neuronal origin such as NSCLC, has been positively correlated with the degree of tumour aggressiveness. H460 cells are known to display substrate- independent growth in soft agar and tumourigenicity in nude mice and provided an ideal model to investigate the role of βIII-tubulin in tumourigenesis. To address the role of βIII-tubulin, H460 cells stably expressing βIII-tubulin shRNA were generated, validated and examined using both in vitro and in vivo methods of tumourigenesis. Colony formation of H460 cells stably expressing βIII-tubulin shRNA was dramatically reduced in soft agar and significantly delayed tumour growth and reduced tumour incidence of subcutaneous xenografted tumours in nude mice when compared to respective controls. These results provide new insights into the function of βIII-tubulin and suggest that βIII-tubulin may play an important role in tumour development and progression in lung cancer. In conclusion, β-tubulin isotype status can serve as a valuable molecular marker capable of distinguishing patients with differential sensitivity to TBAs. These results not only shed new light on the role of specific β-tubulin isotypes in the response to TBAs, but also the role of βIII-tubulin in the biology of cancer that will lead to new treatment strategies for NSCLC.

Agmatine, an endogenous amine derived from decarboxylation of l-arginine catalyzed by arginine decarboxylase, has been proposed as a neurotransmitter or neuromodulator in the brain. In the present study, we examined whether agmatine has neuroprotective effects against repeated immobilization-induced morphological changes in brain tissues and possible effects of immobilization stress on endogenous agmatine levels and arginine decarboxylase expression in rat brains. Sprague-Dawley rats were subjected to 2 h immobilization stress daily for 7 days. This paradigm significantly increased plasma corticosterone levels, and the glutamate efflux in the hippocampus as measured by in vivo microdialysis. Immunohistochemical staining with β-tubulin III showed that repeated immobilization caused marked morphological alterations in the hippocampus and medial prefrontal cortex that were prevented by simultaneous treatment with agmatine (50 mg/kg/day), i.p.). Likewise, endogenous agmatine levels measured by high-performance liquid chromatography in the prefrontal cortex, hippocampus, striatum and hypothalamus were significantly increased by immobilization, as compared to controls. The increased endogenous agmatine levels, ranging from 92 to 265% of controls, were accompanied by a significant increase of arginine decarboxylase protein levels in the same regions. These results demonstrate that the administration of exogenous agmatine protects the hippocampus and medial prefrontal cortex against neuronal insults caused by repeated immobilization. The parallel increase in endogenous brain agmatine and arginine decarboxylase protein levels triggered by repeated immobilization indicates that the endogenous agmatine system may play an important role in adaptation to stress as a potential neuronal self-protection mechanism.

Agmatine is an endogenous amine derived from decarboxylation of arginine catalysed by arginine decarboxylase. Agmatine is considered a novel neuromodulator and possesses neuroprotective properties in the central nervous system. The present study examined whether agmatine has neuroprotective effects against repeated restraint stress-induced morphological changes in rat medial prefrontal cortex and hippocampus. Sprague-Dawley rats were subjected to 6 h of restraint stress daily for 21 days. Immunohistochemical staining with β-tubulin III showed that repeated restraint stress caused marked morphological alterations in the medial prefrontal cortex and hippocampus. Stress-induced alterations were prevented by simultaneous treatment with agmatine (50 mg/kg/day, i.p.). Interestingly, endogenous agmatine levels, as measured by high-performance liquid chromatography, in the prefrontal cortex and hippocampus as well as in the striatum and hypothalamus of repeated restraint rats were significantly reduced as compared with the controls. Reduced endogenous agmatine levels in repeated restraint animals were accompanied by a significant increase of arginine decarboxylase protein levels in the same regions. Moreover, administration of exogenous agmatine to restrained rats abolished increases of arginine decarboxylase protein levels. Taken together, these results demonstrate that exogenously administered agmatine has neuroprotective effects against repeated restraint-induced structural changes in the medial prefrontal cortex and hippocampus. These findings indicate that stress-induced reductions in endogenous agmatine levels in the rat brain may play a permissive role in neuronal pathology induced by repeated restraint stress.

In the present study, we examined the possible effect of chronic treatment with glucocorticoids on the morphology of the rat brain and levels of endogenous agmatine and arginine decarboxylase (ADC) protein, the enzyme essential for agmatine synthesis. Seven-day treatment with dexamethasone, at a dose (10 and 50 μg/kg/day) associated to stress effects contributed by glucocorticoids, did not result in obvious morphologic changes in the medial prefrontal cortex and hippocampus, as measured by immunocytochemical staining with β-tubulin III. However, 21-day treatment (50 μg/kg/day) produced noticeable structural changes such as the diminution and disarrangement of dendrites and neurons in these areas. Simultaneous treatment with agmatine (50 mg/kg/day) prevented these morphological changes. Further measurement with HPLC showed that endogenous agmatine levels in the prefrontal cortex and hippocampus were significantly increased after 7-day treatments with dexamethasone in a dose-dependent manner. On the contrary, 21-day treatment with glucocorticoids robustly reduced agmatine levels in these regions. The treatment-caused biphasic alterations of endogenous agmatine levels were also seen in the striatum and hypothalamus. Interestingly, treatment with glucocorticoids resulted in a similar change of ADC protein levels in most brain areas to endogenous agmatine levels: an increase after 7-day treatment versus a reduction after 21-day treatment. These results demonstrated that agmatine has neuroprotective effects against structural alterations caused by glucocorticoids in vivo. The parallel alterations in the endogenous agmatine levels and ADC expression in the brain after treatment with glucocorticoids indicate the possible regulatory effect of these stress hormones on the synthesis and metabolism of agmatine in vivo.

This thesis deals with the phylogeny of the lichen genus Usnea (Parmeliaceae, Ascomycetes). The relationships and the morphological variation among Usnea species has been studied, as well as the relationship of Usnea to allied genera. Two species, U. florida and U. subfloridana, which earlier were regarded to form two separate species have been synonymized. In an analysis based on sequence data these two taxa formed a monophyletic group of intermixed specimens. Usnea florida and U. subfloridana have earlier been regarded to form a species pair, but the species pairs concept cannot be applied in this case. The morphological characters traditionally used for species recognition of a number of European Usnea species have been analyzed regarding their reliability. The evolution and distribution of the morphological characters was studied in relation to a phylogeny based on sequence data. Most characters proved to be homoplastic in relation to the phylogeny. Few characters were consistent in a clade, and the same character could be inconsistent in another clade. Therefore a combination of several characters is recommended for species recognition. The relationship of Neuropogon to Usnea was investigated based on sequence data. Neuropogon showed to be closely related to Usnea subg. Usnea. The subgenera Eumitria and Dolichousnea formed the sister group to the clade comprising subg. Usnea and Neuropogon. Usnea is paraphyletic in this investigation. Eumitria is treated as a genus and the subgenus Dolichousnea is elevated to generic rank. The position of Usnea, Neuropogon, Eumitria, and Dolichousnea in the family Parmeliaceae was investigated based on a phylogeny obtained by sequence data. Protousnea probably forms the sister group to the clade of Usnea, Neuropogon, Eumitria, and Dolichousnea. Several monophyletic groups in the family Parmeliaceae were identified.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Cultivation of Walnut-pecan (Carya illinoinensis [Wangenh.] K. Koch) has intensified in recent years in the state of Rio Grande do Sul. However, the research related to plant pathology has not developed to the same degree, so producing pecan nuts have faced many unknown diseases in the state. The main objective of this study was: a) identify and characterize causal agents of new diseases on Walnut-pecan in Rio Grande do Sul, and as specific objectives: b) to collect and to identify pathogens in different locations in Rio Grande do Sul, c) evaluate the pathogenicity of isolates collected in plantations of Walnut-pecan from different locations in Rio Grande do Sul, d) perform the isolates morphological characterization collected from diseased plants of Walnut-pecan, and e) to identify the isolates species. For this purpose, samples were taken in seven cities in the state for isolating potentially pathogenic fungi which were also collected and analyzed soil samples to characterize the area and georeferencing. Pathogens, Fusarium spp. and Pestalotiopsis spp. which were not yet reported were tested for their pathogenicity. The first genus was tested by inoculation on substrate, and the second by leaf inoculation with spore suspension. The pathogenic isolates were characterized by mycelial growth, sporulation, conidial dimensions, colony pigmentation and formation of specific structures to each genus. The same isolates were also identified by molecular sequencing of the ITS and TEF-1α genes for Fusarium spp. and ITS and β-tubulin genes for Pestalotiopsis spp. Twelve pathogenic isolates of Fusarium spp. and eleven of Pestalotiopsis spp. were identified. The variables used on morphological characterization were able to differentiate the isolates, especially the width of conidia for Fusarium spp. and diameter of the colonies for Pestalotiopsis spp. The sequencing of the ITS regions and TEF-1α to Fusarium spp. confirmed the separation of isolates through morphological characteristics and identified five species: F. chlamydosporum, F. oxysporum, F. equiseti, Giberella fujikuroi species complex and F. graminearum species complex and for Pestalotiopsis spp. sequencing of the ITS regions and β-tubulin could identify some species, such as P. clavisora and P. cocculi, and other isolates remained without precise identification of the species, since the phylogeny of the genus is still poorly known. / O cultivo da nogueira-pecan (Carya illinoinensis [Wangenh.] K. Koch) tem se intensificado nos últimos anos no estado do Rio Grande do Sul. Entretanto, as pesquisas relacionadas aos problemas fitossanitários da espécie não se desenvolveram na mesma intensidade, de modo que muitos produtores do estado têm enfrentado enfermidades desconhecidas. Diante disto, o objetivo geral do presente trabalho foi identificar e caracterizar os agentes causais de novas doenças que atacam a nogueira-pecan no Rio Grande do Sul. Como objetivos específicos estabeleceram-se: a) coletar e identificar agentes patogênicos em diferentes localidades no Rio Grande do Sul; b) avaliar a patogenicidade de isolados coletados em plantios de nogueira-pecan de diferentes localidades no Rio Grande do Sul; c) caracterizar morfofisiologicamente os isolados coletados de plantas doentes de nogueira-pecan; e d) identificar, em nível de espécie, os isolados provenientes de plantas doentes de nogueira-pecan. Para tanto, foram realizadas coletas em sete municípios do estado, para isolamento de fungos potencialmente patogênicos. Também foram coletadas e analisadas amostras de solo para caracterização da área e georreferenciamento dos pontos. Os patógenos, ainda não relatados, Fusarium spp. e Pestalotiopsis spp., foram testados quanto a sua patogenicidade. O primeiro foi testado com inoculação em substrato, e o segundo com inoculação foliar por suspensão de esporos. Os isolados patogênicos foram caracterizados morfofisiologicamente através das variáveis crescimento micelial, esporulação, dimensões de conídios, pigmentação das colônias e formação de estruturas específicas de cada gênero. Os mesmos isolados também foram identificados molecularmente através de sequenciamento dos genes ITS e TEF-1α, para Fusarium spp., e ITS e β-tubulina, para Pestalotiopsis spp. Foram identificados doze isolados patogênicos de Fusarium spp. e onze de Pestalotiopsis spp. As variáveis utilizadas na caracterização morfofisiológica foram suficientes na diferenciação dos isolados, especialmente a largura dos conídios, para Fusarium spp. e o diâmetro das colônias, para Pestalotiopsis spp. O sequenciamento das regiões ITS e TEF-1α, para Fusarium spp., confirmou a separação dos isolados por meio das características morfofisiológicas e identificou cinco espécies, sendo elas F. chlamydosporum, F. oxysporum, F. equiseti, Giberella fujikuroi species complex e F. graminearum species complex. Para Pestalotiopsis spp., o sequenciamento das regiões ITS e β-tubulina permitiu que se identificassem algumas espécies, como é o caso de P. clavisora e P. cocculi. Outros isolados permaneceram sem identificação precisa da espécie, já que a filogenia do gênero ainda é pouco conhecida.

Les champignons mycorhiziens à arbuscules (CMA), classés dans le phylum Glomeromycota, ne peuvent pas être facilement identifiés par la morphologie de leurs spores et leurs mycélia à l'intérieur ou à l'extérieur des racines de leurs hôtes. Ce problème fondamental d'identification rend l'étude de leur diversité, en particulier dans leur habitat naturel (sol et racine) extrêmement difficile. Les gènes ribosomaux ont été largement utilisés pour développer des amorces spécifiques et en inférer des arbres phylogénétiques. Cependant, ces gènes sont très polymorphes et existent en plusieurs copies dans le génome des CMA, ce qui complique l’interprétation des résultats. Dans notre étude, nous avons étudié le polymorphisme intra- et inter-spécifique du gène β-tubuline, présent en faible nombre de copies dans le génome des CMA, afin d’obtenir de nouvelles séquences nucléotidiques pour développer des marqueurs moléculaires. Les gènes β-tubuline amplifiés à partir de l'ADN génomique de cinq espèces du genre Glomus ont été clonés et séquencés. L’analyse des séquences indique un polymorphisme intraspécifique chez trois espèces de CMA. Deux séquences paralogues très variables ont été nouvellement identifiées chez les G. aggregatum, G. fasciculatum et G. cerebriforme. Aucun polymorphisme n’a été détecté chez les G. clarum et G. etunicatum. Toutes les séquences montrent la présence de deux introns hautement variables. La majorité des substitutions ont été localisées dans les exons et sont synonymes à 90%. La conservation des acides aminés suggère un niveau élevé de sélection négative sur le gène β-tubuline et nous permet de confirmer que les CMA représentent un ancien groupe fongique (400 million d’années). L’analyse phylogénétique, réalisée avec vingt et une séquences nucléotidiques du gène β-tubuline, a révélé que les séquences des Glomaceae forment un groupe monophylétique bien supporté, avec les Acaulosporaceae et Gigasporaceae comme groupe frère. Les séquences paralogues nouvellement identifiées chez les G. aggregatum et G. fasciculatum n'ont pas été monophylétiques au sein de chaque espèce. Les oligonucléotides ont été choisis sur la base des régions variables et conservées du gène β-tubuline. Le test PCR des amorces β-Tub.cerb.F/ β-Tub.cerb.R a révélé des bandes spécifiques de 401 pb pour les séquences paralogues du G. cerebriforme. Deux paires d’amorces ont été développées afin d’identifier les séquences du groupe nommé Tub.1. Les tests PCR nous ont permis d’identifier certaines séquences du groupe Tub.1. Une paire d’amorce β-Tub.2.F/ β-Tub.2.R nous a permis d’identifier certaines séquences paralogues du groupe nommé Tub.2. L’analyse d’autres gènes combinée à celle du gène β-tubuline permettra le développement de marqueurs moléculaires plus spécifiques pour l’identification de CMA. / Arbuscular mycorrhizal fungi (AMF) are classified in the phylum Glomeromycota. This fungal group cannot be easily identified using morphology of their spores and hyphae inside or outside plant-roots. This fundamental problem renders the study of their diversity extremely difficult, particularly in their natural habitat (soil and roots). The ribosomal genes have been widely used to develop specific primers and to infer phylogenetic trees. However, these genes are highly polymorphic and exist in multiple copies in the genome of the AMF. This leads misinterpretation of the results. In our study, we analysed the intra- and inter specific polymorphism of the β-tubulin gene, which is present in low copy number in AMF genome, to obtain further nucleotide sequences to develop molecular markers. β-tubulin genes were sequenced from genomic DNA of five Glomus species. PCR amplified β-tubulin genes were cloned and sequenced. Intra- and inter-specific polymorphism analyses indicate the polymorphic nature of β-tubulin gene in three AMF species. Two highly variable β-tubulin paralogs were newly identified in G. aggregatum, G. fasciculatum and G. cerebriforme. No evidence for the presence of paralogs was found in G. clarum and G. etunicatum. All AMF sequences show the presence of two introns, which are highly variable. The majority of substitutions are located in the exons and 90% are synonymous. The conservation of amino-acids level suggests a high level of negative selection acting on the β-tubulin gene and allows us to consider that the AMF represent an ancient fungal group (400 million years). A phylogenetic analysis, carried out with twentyone β-tubulin nucleotidic sequences, revealed that the sequences of Glomeraceae form a highly supported monophyletic group with Acaulosporaceae and Gigasporaceae as sistergroup. The newly identified paralogs from G. aggregatum and G. fasciculatum were not found to be monophyletic within each species. Oligonucleotides were designed in the conserved and variable regions. PCR amplification test of β-Tub.cerb.F / β-Tub.cerb.R revealed specific bands of 401 bp for the G. cerebriforme paralogs. Two pairs of primers were developed to identify sequences of the group named Tub.1. PCR tests have allowed us to identify certain sequences of Tub.1 group. A primer pair of β-Tub.2.F / β-Tub.2.R we identified some sequences paralogous group named Tub.2. Analysis of other genes combined with that of β-tubulin gene enable the development of more specific molecular markers for AMF identification.

Les champignons mycorhiziens à arbuscules (CMA), classés dans le phylum Glomeromycota, ne peuvent pas être facilement identifiés par la morphologie de leurs spores et leurs mycélia à l'intérieur ou à l'extérieur des racines de leurs hôtes. Ce problème fondamental d'identification rend l'étude de leur diversité, en particulier dans leur habitat naturel (sol et racine) extrêmement difficile. Les gènes ribosomaux ont été largement utilisés pour développer des amorces spécifiques et en inférer des arbres phylogénétiques. Cependant, ces gènes sont très polymorphes et existent en plusieurs copies dans le génome des CMA, ce qui complique l’interprétation des résultats. Dans notre étude, nous avons étudié le polymorphisme intra- et inter-spécifique du gène β-tubuline, présent en faible nombre de copies dans le génome des CMA, afin d’obtenir de nouvelles séquences nucléotidiques pour développer des marqueurs moléculaires. Les gènes β-tubuline amplifiés à partir de l'ADN génomique de cinq espèces du genre Glomus ont été clonés et séquencés. L’analyse des séquences indique un polymorphisme intraspécifique chez trois espèces de CMA. Deux séquences paralogues très variables ont été nouvellement identifiées chez les G. aggregatum, G. fasciculatum et G. cerebriforme. Aucun polymorphisme n’a été détecté chez les G. clarum et G. etunicatum. Toutes les séquences montrent la présence de deux introns hautement variables. La majorité des substitutions ont été localisées dans les exons et sont synonymes à 90%. La conservation des acides aminés suggère un niveau élevé de sélection négative sur le gène β-tubuline et nous permet de confirmer que les CMA représentent un ancien groupe fongique (400 million d’années). L’analyse phylogénétique, réalisée avec vingt et une séquences nucléotidiques du gène β-tubuline, a révélé que les séquences des Glomaceae forment un groupe monophylétique bien supporté, avec les Acaulosporaceae et Gigasporaceae comme groupe frère. Les séquences paralogues nouvellement identifiées chez les G. aggregatum et G. fasciculatum n'ont pas été monophylétiques au sein de chaque espèce. Les oligonucléotides ont été choisis sur la base des régions variables et conservées du gène β-tubuline. Le test PCR des amorces β-Tub.cerb.F/ β-Tub.cerb.R a révélé des bandes spécifiques de 401 pb pour les séquences paralogues du G. cerebriforme. Deux paires d’amorces ont été développées afin d’identifier les séquences du groupe nommé Tub.1. Les tests PCR nous ont permis d’identifier certaines séquences du groupe Tub.1. Une paire d’amorce β-Tub.2.F/ β-Tub.2.R nous a permis d’identifier certaines séquences paralogues du groupe nommé Tub.2. L’analyse d’autres gènes combinée à celle du gène β-tubuline permettra le développement de marqueurs moléculaires plus spécifiques pour l’identification de CMA. / Arbuscular mycorrhizal fungi (AMF) are classified in the phylum Glomeromycota. This fungal group cannot be easily identified using morphology of their spores and hyphae inside or outside plant-roots. This fundamental problem renders the study of their diversity extremely difficult, particularly in their natural habitat (soil and roots). The ribosomal genes have been widely used to develop specific primers and to infer phylogenetic trees. However, these genes are highly polymorphic and exist in multiple copies in the genome of the AMF. This leads misinterpretation of the results. In our study, we analysed the intra- and inter specific polymorphism of the β-tubulin gene, which is present in low copy number in AMF genome, to obtain further nucleotide sequences to develop molecular markers. β-tubulin genes were sequenced from genomic DNA of five Glomus species. PCR amplified β-tubulin genes were cloned and sequenced. Intra- and inter-specific polymorphism analyses indicate the polymorphic nature of β-tubulin gene in three AMF species. Two highly variable β-tubulin paralogs were newly identified in G. aggregatum, G. fasciculatum and G. cerebriforme. No evidence for the presence of paralogs was found in G. clarum and G. etunicatum. All AMF sequences show the presence of two introns, which are highly variable. The majority of substitutions are located in the exons and 90% are synonymous. The conservation of amino-acids level suggests a high level of negative selection acting on the β-tubulin gene and allows us to consider that the AMF represent an ancient fungal group (400 million years). A phylogenetic analysis, carried out with twentyone β-tubulin nucleotidic sequences, revealed that the sequences of Glomeraceae form a highly supported monophyletic group with Acaulosporaceae and Gigasporaceae as sistergroup. The newly identified paralogs from G. aggregatum and G. fasciculatum were not found to be monophyletic within each species. Oligonucleotides were designed in the conserved and variable regions. PCR amplification test of β-Tub.cerb.F / β-Tub.cerb.R revealed specific bands of 401 bp for the G. cerebriforme paralogs. Two pairs of primers were developed to identify sequences of the group named Tub.1. PCR tests have allowed us to identify certain sequences of Tub.1 group. A primer pair of β-Tub.2.F / β-Tub.2.R we identified some sequences paralogous group named Tub.2. Analysis of other genes combined with that of β-tubulin gene enable the development of more specific molecular markers for AMF identification.