Procedimentos clínicos complementares realizados após o preparo químico-cirúrgico de canais radiculares visam potencializar a limpeza e desinfecção após a fase de preparo. O objetivo deste trabalho foi avaliar, por métodos moleculares baseados em rDNA e rRNA, a eficácia antimicrobiana de procedimentos complementares de primeira e de segunda sessão, realizados após o PQC em dentes com periodontite apical. Baseado em estudo piloto prévio, amostras microbiológicas dos canais radiculares de 20 dentes unirradiculares com periodontite apical foram coletadas na primeira sessão clínica: após a cirurgia de acesso (S1), após o PQC realizado com Sistema Reciproc e NaOCl 2,5% (S2), após a utilização do instrumento XP-endo Finisher (S3a), e após a ativação ultrassônica (S3b). Na segunda sessão clínica as amostras foram coletadas após medicação intracanal entre sessões por 14 dias (S4), e após o repreparo dos canais na segunda sessão de tratamento (S5). As amostras foram submetidas à extração de DNA e RNA. O RNA foi submetido à reação de transcrição reversa (RT-PCR) para confecção da fita dupla de DNA complementar (cDNA). DNA e cDNA foram submetidos a reações de qPCR, com iniciadores universais para a região 16S rRNA do domínio Bacteria. A atividade metabólica das bactérias foi verificada através da relação entre os níveis de rRNA e rDNA das amostras baseados nos dados dos ensaios de qPCR. Os dados foram analisados pelo teste de Wilcoxon para amostras pareadas (p < 0,05). Todas amostras S1 foram positivas para bactérias (mediana: 1,79 x 105 cópias de rDNA). Doze canais (60%) permaneceram infectados em S2, com uma redução significativa de rDNA (mediana: 7,58 x 103; P = 0,0001). Em S3a e S3b, o número de canais infectados reduziu para 11 (55%) e 10 (50%), respectivamente, e aumentou para 14 (70%) em S4; porém não houve diferenças significativas entre os níveis de rDNA bacteriano quando essas amostras foram comparadas às amostras S2 ou comparadas entre si (P > 0,05). A prevalência de canais infectados voltou a cair em S5 para 6 (30%) e o número cópias de rDNA bacteriano detectado reduziu de maneira significativa quando comparado às amostras S4 (p = 0,0061). Nas amostras positivas para os 2 métodos, os níveis de rRNA foram significativamente maiores do que os níveis de rDNA nas amostras S1 (p = 0,0007) e S4 (p = 0,0499), indicando um alto metabolismo bacteriano. A relação dos níveis de rRNA e de rDNA revelou uma redução do metabolismo de bactérias totais em S2, S3a e S3b e um aumento significativo do metabolismo bacteriano em S4 (p = 0,0173) quando comparado a S3b. Concluiu-se que: o PQC promoveu redução dos níveis e da atividade metabólica de bactérias nos canais radiculares; o uso do instrumento XP-endo Finisher e ativação ultrassônica não contribuiu para uma desinfecção adicional na primeira sessão; após o uso de Ca(OH)2 como medicação intracanal, houve um aumento no metabolismo de bactérias persistentes; o repreparo dos canais radiculares após medicação intracanal, na segunda sessão, promoveu maior desinfecção do que os procedimentos realizados na primeira sessão do tratamento de dentes com periodontite apical. / Supplementary procedures performed after the chemical-surgical preparation of root canals aim to enhance cleaning and disinfection in endodontic treatment. The objective of this clinical study was to evaluate using molecular microbiological techniques based on rDNA and rRNA, the antimicrobial efficacy of supplementary disinfection procedures performed in multiple sessions after the chemical-surgical preparation in teeth with apical periodontitis. Based on a previous pilot study, microbiological samples of the root canals of 20 unirradicular teeth with apical periodontitis were taken after the access surgery (S1), after the chemical-surgical preparation using Reciproc and 2,5% NaOCl (S2) after the XP-endo Finisher (S3a), after ultrasonic activation (S3b), after intracanal medication for 14 days (S4), and after re-preparation of the root canals in the second treatment appointment (S5). The samples were submitted to DNA and RNA extraction. The RNA was subjected to the reverse transcription reaction (RT-PCR) to make the complementary DNA double strand (cDNA). The effect of endodontic procedures on bacterial reduction was determined by rDNA-based qPCR using universal primers for the 16S rRNA region of the Bacteria domain. The metabolic activity of the bacteria was assessed by the rRNA/rDNA ratio based on qPCR assay data. Data were analyzed by the Wilcoxon signed-rank test (P < 0.05). All S1 samples were positive for bacteria (median 1.79 x 105 rDNA copies). Twelve root canals (60%) remained infected in S2, with a significant reduction of rDNA (median: 7.58 x 103; P = 0.0001). In S3a and S3b, the number of infected teeth decreased to 11 (55%) and 10 (50%), respectively, and increased to 14 (70%) in S4; however there were no significant differences between bacterial rDNA levels when these samples were compared to the S2 samples or compared to each other (P> 0.05). The prevalence of infected canals dropped back to 6 (30%) in S5 and the number of bacterial rDNA detected significantly reduced when compared to S4 samples (p = 0.0061). In samples with positive reactions for both methods, rRNA levels were significantly higher than the rDNA levels in S1 (p = 0.0007) and S4 (p = 0.0499), indicating a high metabolic activity of bacteria. The results of the rRNA/rDNA ratio revealed a reduction in the metabolism of total bacteria in S2, S3a and S3b and a significant increase in metabolic activity of bacteria in S4 (p = 0.0173) when compared to S3b. It was concluded that: PQC promoted reduction of the levels and metabolic activity of bacteria in the root canals; the use of XP-endo Finisher plus ultrasonic activation did not contribute to an additional disinfection in the first session; after the use of Ca(OH)2 as intracanal medication, there was an increase in the metabolismo of persistente bacteria; the repreparation of the root canals in the second session after the use of intracanal medication, promoted greater disinfection than the procedures performed in the first session of the treatment of teeth with apical periodontitis.

Wie sich in den letzten Jahren gezeigt hat, ist Fettgewebe nicht nur ein inerter Fettspeicher, sondern produziert auch eine Vielzahl endokrin wirksamer Substanzen, die unter anderem auch an der Blutdruckregulation beteiligt sind. Da Adipositas ein wichtiger Risikofaktor für die Entwicklung der Hypertonie ist, sollte im Rahmen dieser Dissertation ein System zur quantitativen Untersuchung der Genexpression in Primärkulturen humaner Adipocyten entwickelt werden und dessen Funktionalität am Beispiel der hormonellen Regulation der Gene des Renin-Angiotensin-Systems demonstriert werden. Dies beinhaltete die Etablierung der Adipocytenisolierung und -kultivierung, eines Stimulationsassays, die Entwicklung einer der besonderen Größe und dem hohen Fettgehalt der Zellen angepaßten Zellzahl- und Vitalitätsbestimmungsmethode, die Untersuchung vier verschiedener RNA-Extraktionsmethoden auf ihre Eignung für Adipocyten und die Etablierung eines besonders sensitiven RT-PCR Systems zur Untersuchung der Genexpression mittels einer fluoreszenzmarkierten Sonde. Exemplarisch konnte anhand der Renin-Angiotensin-System-Gene die Funktionalität der Methoden demonstriert werden, indem nicht nur die Genexpression aller Komponenten des Renin-Angiotensin-Systems in humanen Adipocyten nachgewiesen wurden, sondern auch gezeigt werden konnte, dass Hydrocortison sowohl die Genexpression als auch die Dichte des Angiotensin II Typ 1-Rezeptors in der Adipocytenmembran stimuliert. Dieser Aspekt könnte möglicherweise nicht nur bei der besonderen Adipositasform des Cushing-Syndroms, sondern auch für die Entstehung der zentralen Adipositas von Bedeutung sein. / Adipose tissue has functions above-and-beyond storing fat. It also produces a variety of different endocrine substances, some of which influence blood pressure regulation. Obesity is a well known risk factor for the development of hypertension Thus, the genes regulating expression of vasoactive molecules in adipose tissue, possibly contributing to an increase in blood pressure are of great interest. The aim of this work was to develope a system for quantitative gene expression analysis in primary cultured human adipocytes and to demonstrate its utility for studying the hormonal regulation of genes encoding the renin-angiotensin-system. We established procedures for the isolation and culture of human adipocytes, as well as a stimulatory assay. We also developed methods for the determination of cell number and vitality. Above this, four RNA extraction protocols were evaluated regarding their suitability for adipocytes, and a very sensitive RT-PCR system for gene expression analysis using fluorescent labeled probes was established. As an example for the functionality of these methods we showed that all genes of the renin-angiotensin-system are expressed in human adipocytes. We also demonstrated that hydrocortisone stimulates the gene expression as well as the density of the angiotensin II receptor type 1 on cultured human adipocytes. This finding may be of interest for the development of the obesity phenotype found in cushing syndrome, but could also contribute to the development of central obesity.

Les mycotoxines sont des molécules toxiques produites par de nombreuses espèces fongiques. Les seules mycotoxines avérées aujourd’hui cancérigènes pour l’homme sont les aflatoxines. Elles sont produites par le genre Aspergillus principalement et sont retrouvées tout au long de la chaine alimentaire (champs, stockage, transformation, etc.). A cause du réchauffement climatique, la France devient de plus en plus exposée à la présence de ces mycotoxines. Afin de limiter l’exposition des consommateurs, de nombreuses stratégies de prévention ou de décontamination sont développées. Dans ce contexte, nous avons recherché à mettre au point un système de lutte biologique permettant de prévenir la production d’aflatoxines sur le maïs au champ. Pour cela, nous avons choisi des bactéries issues du sol et déjà connues pour être commercialisées pour la lutte biologique, les actinomycètes. Nous avons étudié l’interaction in vitro sur boites de Pétri entre Aspergillus flavus, principal producteur d’aflatoxines, et certains actinomycètes. Nous avons démontré que l’interaction peut réduire la concentration en aflatoxines mesurée par HPLC. De plus, certains isolats bactériens sont aussi capables de réduire, en culture pure, la concentration d’aflatoxine B1 dans le milieu. Des premiers tests d’adsorption ont été réalisés pour comprendre la nature de ce mécanisme. Par ailleurs, une étude approfondie via RT-qPCR sur 6 souches bactériennes du genre Streptomyces sp. A montré que celles-ci étaient capables d’impacter l’expression de différents gènes impliqués dans la voie de biosynthèse chez A. flavus et A. parasiticus. Enfin, nous avons complété les données déjà existantes sur l’impact de facteurs environnementaux (température, disponibilité en eau et du temps d’incubation) sur la production d’aflatoxines. / Mycotoxins are toxic contaminants of foodstuffs produced by a wide range of fungal species. Aflatoxins are the only mycotoxins carcinogenic for humans. They are mainly produced by the Aspergillus genus and can be found at each step of the agrofood chain (e.g. field, storage, process). Due to climate changes, France is starting to be exposed to aflatoxins. In order to limit the consumer exposure, many prevention or decontamination techniques have been developed. To this aim, we started the development of a biocontrol against aflatoxins accumulation for maize field application. Actinomycetes, are soil-borne bacteria that has already been commercialized as biocontrol. In Petri dishes, we studied the in vitro interaction between some actinomycetes and Aspergillus flavus, the main aflatoxins producer. We revealed that the interaction reduced the aflatoxins content (monitored by HPLC). Moreover, some bacterial isolates were able to reduce pure-aflatoxin B1 added in the medium. To understand this mechanism, adsorption tests has been conducted. Otherwise, RT-qPCR methodology was used to study the impact of Streptomyces-Aspergillus sp. on aflatoxin gene expression. Finally, the current knowledge of the impact of environmental factors (temperature, water activity and incubation time) on aflatoxins production was supplemented.