Contributions of Conversation Analysis to the study of traumatic brain injury: a single case report

A persistência de dificuldades no processo de comunicação em sujeitos acometidos por um traumatismo craneo-encefálico requer a utilização de instrumentos de avaliação, sensíveis às alterações lingüístico-cognitivas apresentadas, que possam facilitar a reintegração desses indivíduos na sociedade. O presente estudo de caso investiga as contribuições da Análise Conversacional, na avaliação das habilidades funcionais de um jovem de 27 anos, acometido por um traumatismo craneo-encefálico grave. Analisa uma interação conversacional espontânea por meio do estudo do mecanismo colaborativo da tomada de turno, do gerenciamento do tópico e das reformulações, comparando os resultados com testes formais de linguagem. Discute as contribuições dessa abordagem ao processo de reabilitação fonoaudiológica. Enquanto os testes formais não indicaram alterações de linguagem, a Análise Conversacional permitiu, ao analisar a seqüência de turnos e as reações mútuas dos interlocutores, identificar problemas comunicativos e verificar como os interlocutores lidavam com os mesmos. A Análise Conversacional mostrou-se sensível às alterações lingüístico-cognitivas apresentadas pelo traumatismo craneo-encefálico, fornecendo elementos concretos para, no processo de reabilitação, abordar, de forma organizada, o discurso mais utilizado no cotidiano.The persistence of communication impairments in individuals who sustained a traumatic brain injury requires the use of sensitive tools to evaluate the linguistic-cognitive problems they present, aiming at their reintegration in society. This single case study investigates the contributions of Conversation Analysis to the evaluation of the pragmatic abilities of a 27 year-old man who sustained a severe traumatic brain injury. It analyses a spontaneous conversational interaction through the study of the collaborative mechanism of turn-taking, topic management and repairs, comparing the results of this analysis with formal language tests. It discusses the contributions of this approach to develop intervention. While formal tests did not indicate the presence of language difficulties, the Conversation Analysis allowed, through the analysis of the sequence of turns and of the mutual reactions of both the interlocutors, the identification of communicative problems and the observation of how the speakers dealt with them. The results of this study revealed that Conversation Analysis is a sensitive approach to capture the linguistic-cognitive deficits caused by traumatic brain injury, providing concrete elements to be used in therapy in order to emphasize, in an organized way, the kind of discourse most used in daily life

CRISPR-barcoding pour l'étude fonctionnelle de mutations oncogéniques dans un contexte d'hétérogénéité intra-tumorale.

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.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

Functional analysis of oncogenic driver mutations through CRISPR-barcoding in a context of intratumoral heterogeneity

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

UTILISATION DE L'IMPLANTATION IONIQUE DANS LES TECHNOLOGIES MOS

L'implantation ionique, technique de dopage originale, s'est affirmée dans le domaine de la technologie MOS où elle est devenue un processus industriel. A partir des caractéristiques de cette technique, on dégagera les méthodes qui permettent de pallier différentes limitations de la technologie MOS classique (recouvrement grille drain, rapport tension de seuil oxyde épais – tension de seuil oxyde lin) et on montrera les applications originales qu'il est possible d'envisager avec l'implantation ionique. Des exemples de technologies seront présentés (technologie déplétion-enrichissement à canal P, technologie MOS complémentaire sur substrat isolant) précisant les possibilités de l'implantation ionique tant au niveau de la structure des circuits intégrés réalisés que des performances atteintes

Range chart for Tertiary ostracodes from ODP Hole 101-628A (Table 1)

The Oligocene to Pliocene section from Hole 628A supplied about 100 species of Tertiary ostracodes. Deep-sea psychrospheric? species (Bradleya cf. dictyon, Agrenocythere cf. gosnoldia, Cardobairdia spp., Henryhowella sp., Cytheropteron spp., etc.) are present throughout the section. Starting in the Miocene, neritic species (Hulingsina sp., Puriana spp., Caudites spp., Loxoconcha fischeri, Cytherelloidea sp., etc.) dominate. Redeposition of these species from the continental shelf seems to be penecontemporaneous with sedimentation. Variations in the assemblages indicate biostratigraphic position. Species having an ecologic or stratigraphic importance are discussed and illustrated

Semantic, phonemic and verb fluency in individuals with mild cognitive impairment

INTRODUÇÃO: A identificação precoce de risco de desenvolver processos demenciais é importante para que os indivíduos possam beneficiar-se de ações dirigidas a aspectos cognitivos e funcionais. Provas de fluência verbal são geralmente incluídas nas avaliações para o diagnóstico precoce da doença de Alzheimer, pois contribuem para detectar alterações de linguagem e de funções executivas. As provas de fluência verbal mais frequentemente utilizadas são as de fluência semântica (FS) e fonêmica (FF) que solicitam respectivamente a emissão em um minuto do maior número de palavras que pertencem a uma determinada categoria semântica ou que começam com uma letra específica. Estudos recentes têm mostrado que, com relação à FS e à FF, as provas de fluência de verbos (FVe) \"coisas que as pessoas podem fazer\" são tarefas mais sensíveis para detectar precocemente alterações cognitivas em indivíduos com comprometimento cognitivo leve (CCL). No entanto, estes dados precisam ser replicados. Em contraste com a maioria das pesquisas que analisa o número total de palavras produzidas em um minuto, o estudo do desempenho segundo o modelo de distribuição temporal do comportamento verifica a variação do número de palavras produzidas nos quartis sucessivos de 15 segundos. Este método pode auxiliar a compreender o papel destas tarefas como instrumento diagnóstico. Como este modelo preconiza que, em tarefas complexas, mais recursos executivos são necessários para completá-las no decorrer do tempo, a análise comparativa dos quartis pode auxiliar a detectar declínios cognitivos associados ao processamento executivo. Cada modalidade de fluência verbal apresenta restrições linguístico-cognitivas específicas. Isto significa que o estudo concomitante das diferentes modalidades pode auxiliar a compreender os mecanismos cognitivos subjacentes a estas provas. OBJETIVOS: O presente estudo teve como objetivo comparar o desempenho entre indivíduos idosos saudáveis e com CCL nas provas de FS, FF e FVe, por meio do método tradicional de contar o número total de itens gerados em um minuto. Além de mostrar a utilidade diagnóstica destas provas, visa compreender seu papel no diagnóstico e analisar os mecanismos cognitivos subjacentes às provas, isolando a contribuição dos recursos executivos por meio do modelo de distribuição temporal do comportamento. Para isto, compara o desempenho entre os grupos nos diferentes quartis de 15 segundos que compõem cada tarefa, e busca detectar diferenças entre os grupos, quando o número de palavras disponíveis no estoque lexical diminui e os indivíduos precisam implementar mecanismos executivos de busca adicionais. MÉTODO: Foram examinados 30 indivíduos com CCL, pareados por idade e nível de escolaridade com indivíduos saudáveis. O desempenho de cada grupo foi examinado por meio da análise do número total de palavras corretas produzidas em um minuto nas provas de FS (animais, itens do supermercado), FF (FAS) e FVe (ações). Adicionalmente, analisou-se a distribuição temporal do desempenho em cada modalidade, subdividindo o tempo de produção verbal em quatro quartis de 15 segundos. RESULTADOS: Os resultados apontam para diferenças significativas no número total de itens gerados entre uma amostra variada de indivíduos com CCL e controles apenas para a prova de FVe, confirmando pesquisas anteriores que mostram que esta prova permite capturar precocemente os déficits cognitivos associados ao CCL. O estudo da distribuição temporal permitiu detectar diferenças significativas entre os grupos apenas na prova de FVe. Nesta modalidade, é provável que no decorrer do tempo menos palavras fiquem disponíveis na rede semântica, e que os recursos executivos disponíveis de indivíduos com CCL não sejam suficientes para completar a tarefa. Diferenças associadas aos recursos executivos necessários para o processamento dos verbos em relação aos substantivos podem explicar as diferenças de desempenho entre os grupos. CONCLUSÕES: A FVe permite detectar precocemente a presença de alterações cognitivas no CCL e diferencia-se das provas de FS e FF. O estudo da distribuição temporal do itens gerados contribui para o entendimento das alterações linguístico-cognitivas subjacentes a estas provas, e mostra que o que torna a FVe mais sensível às alterações cognitivas presentes no CCL é, provavelmente, sua alta demanda de recursos executivos. Estudos a respeito da complexa inter-relação entre linguagem e funções cognitivas podem auxiliar a compreender o limite entre o envelhecimento saudável e aquele associado a processos neuro-degenerativosINTRODUCTION: Early Identification of the risk of developing dementia processes is important so that individuals may benefit from actions directed towards cognitive and functional aspects. Verbal fluency tasks are generally included in the assessments for the early diagnosis of Alzheimer\'s disease since they contribute to detect language and executive dysfunction. The most frequently used verbal fluency tasks are the semantic (SeF) and phonemic (PhoF) fluencies that respectively involves the generation of as many words as possible belonging to a certain semantic category or that begin with a specific letter in one minute. Recent studies have shown that, regarding SeF and PhoF, verb fluency (VeF) tasks \"things people may do\" are more sensitive to detect cognitive dysfunctions in individuals with mild cognitive impairment (MCI). Nevertheless, these data must be replicated. Contrary to most researches that analyze the total number of words produced in one minute, the performance study according to the temporal distribution model checks the variation of the number of words produced in the successive 15-second quartiles. This method can help understand the role of these tasks as a diagnostic instrument. Since in complex tasks this model states that more executive processing resources are necessary to complete them throughout time, the comparative analysis of the quartiles may help detect cognitive declines associated to executive processing. Each verbal fluency modality presents specific linguistic-cognitive restrictions. This means that the concomitant study of the different modalities may help understand the cognitive mechanisms underlying the tasks. OBJECTIVES: The present study aims at comparing the performance between healthy elderly individuals and others with MCI in SeF, PhoF and VeF through the traditional method of counting the total number of items generated in one minute. Besides showing the diagnostic utilities of these tasks, it aims at understanding its role in the diagnosis and analyzing the cognitive mechanisms underlying these tasks, isolating the contribution of executive resources through the temporal distribution model. For such, it compares the performance between the groups in the different 15-second quartiles that compose each task and tries detecting differences between the groups when the number of words available in the lexical stock is reduced and the individuals must implement additional executive-search mechanisms. METHOD: Thirty individuals with MCI were examined and paired with healthy individuals by age and schooling level. The performance of each group was analyzed by evaluating the total number of correct words produced in one minute in SeF (animals, supermarket items), PhoF (FAS) and VeF (actions). Additionally, it analyzed the temporal distribution of the performance in each modality, subdividing the verbal-production time into four 15-second quartiles. RESULTS: The results indicate significant differences in the total number of items generated between a varied sample of individuals with MCI and controls only for the VeF task, confirming previous researches showing that this task allows for the early detection of cognitive deficits associated with MCI. The temporal distribution study allowed for the detection of significant differences between the groups only in the VeF task. In this modality, it is probable that throughout time less words are available in the semantic network and that executive resources of individuals with MCI are not enough to complete the task. Differences associated with executive resources necessary to process the verbs in comparison to the nouns may explain the performance differences between the groups. CONCLUSIONS: VeF allows for the early detection of cognitive dysfunction in MCI and differentiates itself from SeF and PhoF tasks. The temporal distribution study of the items generated contributes to understanding the linguistic-cognitive dysfunction underlying the tasks and shows that what makes VeF more sensitive to cognitive dysfunction in MCI is probably its high demand for executive processing resources. Studies on the complex inter-relation between language and cognitive functions may help understand the limit between healthy aging and aging associated with neuro-degenerative processe

CRISPR/Cas9 editing of the genome for cancer modeling

International audienceThe CRISPR/Cas9 revolution has democratized access to genome editing in many biological fields, including cancer research. Cancer results from the multistep accumulation of mutations that confer to the transformed cells certain biological hallmarks typical of the malignant phenotype. One of the major goals in cancer research is to characterize such mutations and assess their implication in the oncogenic process. Through CRISPR/Cas9 technology, genetic aberrations identified in a patient's tumor can now be easily recreated in experimental models, which can then be used for basic research or for more translational applications. Here we review the different CRISPR/Cas9 strategies that have been implemented to reca-pitulate oncogenic mutations in both in vitro and in vivo systems, including novel strategies to model tumor evolution and genetic heterogeneity