The incubation period is crucial for development and overall quality of a chick. The selection for fast growing broilers has allowed the birds to reach market weight at a faster rate making the incubation period a larger portion of a broiler's life. A faster growth rate can lead to the release of more metabolic heat inside of the egg toward the second half of incubation because the embryo shifts to a homeothermic state. More heat being released into the incubator can cause the incubation temperature to rise if the incubator is not electronically regulated or cannot be ventilated properly due to malfunction. A high incubation temperature can impact the hatchability, growth, and development of the chick. This thesis provides a more in-depth analysis of the effects of high incubation temperature (37.5°C versus 39.5°C) on the developing small intestine and yolk sac, which provide the chick with nutrients posthatch and during embryogenesis. Studying these organs and mechanisms occurring during this time could potentially indicate why chicks from eggs subjected to a higher incubation temperature are not developing and growing properly. Chicks from eggs incubated at a higher temperature had lower body weights, lower hatchability and lower villus height in the duodenum, jejunum, and ileum. There were also differences seen in the depth of the crypt, which is the site for stem cells. Chicks from eggs incubated at a higher temperature had a lower crypt depth in the duodenum and jejunum. There was no difference in the expression of the intestinal stem cell marker olfactomedin 4 (Olfm4) and mucin 2, which is secreted by goblet cells and forms mucus. In the yolk sac, heat shock proteins (HSP) 70 and 90 were elevated at embryonic day 15, and HSP90 still remained elevated at embryonic day 17. Chicks from eggs incubated at a higher temperature showed greater expression of peptide transporter 1 and avian beta-defensin 10 mRNA at embryonic day 13. Even though small intestinal morphology was impacted early posthatch and expression of genes in the yolk sac were elevated at embryonic day 13, there does not seem to be a long-lasting effect on the development of the small intestine or the yolk sac. It is still important to study the impact of the incubation environment to understand the development and growth of the chicks and how different incubation factors can impact the overall hatchability and health of the chick. / Master of Science / The incubation period is crucial for development and overall quality of a chick. The selection for fast growing broilers has allowed the birds to reach market weight at a faster rate making the incubation period a larger portion of a broiler’s life. A faster growth rate can lead to the release of more metabolic heat inside of the egg toward the second half of incubation because the embryo shifts to a homeothermic state. More heat being released into the incubator can cause the incubation temperature to rise if the incubator is not electronically regulated or cannot be ventilated properly due to malfunction. A high incubation temperature can impact the hatchability, growth, and development of the chick. This thesis provides a more in-depth analysis of the effects of high incubation temperature (37.5°C versus 39.5°C) on the developing small intestine and yolk sac, which provide the chick with nutrients posthatch and during embryogenesis. Studying these organs and mechanisms occurring during this time could potentially indicate why chicks from eggs subjected to a higher incubation temperature are not developing and growing properly. Chicks from eggs incubated at a higher temperature had lower body weights, lower hatchability and lower villus height in the duodenum, jejunum, and ileum. There were also differences seen in the depth of the crypt, which is the site for stem cells. Chicks from eggs incubated at a higher temperature had a lower crypt depth in the duodenum and jejunum. There was no difference in the expression of the intestinal stem cell marker olfactomedin 4 (Olfm4) and mucin 2, which is secreted by goblet cells and forms mucus. In the yolk sac, heat shock proteins (HSP) 70 and 90 were elevated at embryonic day 15, and HSP90 still remained elevated at embryonic day 17. Chicks from eggs incubated at a higher temperature showed greater expression of peptide transporter 1 and avian beta-defensin 10 mRNA at embryonic day 13. Even though small intestinal morphology was impacted early posthatch and expression of genes in the yolk sac were elevated at embryonic day 13, there does not seem to be a long-lasting effect on the development of the small intestine or the yolk sac. It is still important to study the impact of the incubation environment to understand the development and growth of the chicks and how different incubation factors can impact the overall hatchability and health of the chick.

Nitrification is the biological process whereby ammonia is converted first to nitrite by ammonia-oxidizing bacteria, and then the nitrite is subsequently converted to nitrate by nitrite-oxidizing bacteria. Ammonia and nitrite levels are closely monitored during treatment of wastewater due to their toxicity to other biological processes. Sybron Chemicals, Inc., is a company that manufactures a nitrifying bioaugmentation culture (1010N) that is used to enhance the naturally occurring levels of biological nitrification. The microbial population of the 1010N product has been examined using a combination of conventional bacteriological methods and modern molecular techniques, with the goal of developing nucleic acid probes that can be used to detect the product in an environmental sample. Small regions of the 16S rRNA genes of the bacteria in 1010N (and two new nitrifying enrichment cultures) were amplified via the polymerase chain reaction (PCR) and analyzed via temperature gradient gel electrophoresis (TGGE). TGGE is a procedure that allows for separation and visualization of individual PCR products that are the same size, based on differences in their sequence. Two of the predominant PCR products in 1010N were purified from the TGGE gel matrix, reamplified via PCR, and sequenced to allow for phylogenetic analysis and nucleic acid probe design. Coincidentally, two strains (NS500-9 and MPN2) that had been isolated from the 1010N mixed consortium and grown in pure culture were found, via TGGE, to have identical 16S rRNA sequences to the PCR products under investigation. Nearly the full-length 16S rRNA genes from these two organisms were PCR amplified, cloned, and sequenced in order to provide a basis for more accurate phylogenetic analysis. The two dominant organisms in the 1010N product, NS500-9 and MPN2, were thereby found to be most closely related to Nitrosomonas and Nitrobacter, respectively, in the existing database. Using the nucleic acid sequences of the cloned DNA, organism-specific DNA probes were designed for both NS500-9 and MPN2. Unfortunately, difficulties were encountered in using the probes to monitor 1010N activity levels via quantitative dot blot hybridizations (rRNA-DNA). Therefore, efforts were redirected to using the TGGE semi-quantitatively with an internal PCR standard (Brüggeman, et al., 2000) to estimate original cell numbers of 1010N within a mixed consortium. This method was not applicable to our system due to substantial preferential binding of the primers to template other than the standard. Samples from a laboratory-scale bioreactor, bioaugmented with 1010N, were used in an attempt to correlate an increase in activity with a detectable shift in population via TGGE. No detectable shift in population was detected in these samples even though the system exhibited increased levels of nitrification. Therefore, the sensitivity of the TGGE system was also examined by determining the limits of detection when 1010N was present in activated sludge. In both whole cell spiking experiments and genomic DNA spiking experiments, it was found that 1010N must be present at a level of at least 5% of the total population in order to be detected. While this provides some information about microbial populations, in order to evaluate the biological activity of a system, nucleic acid probes should be used in a rRNA based study. / Master of Science

Yes / Curiouser and curiouser! A biarylpyrimidine ligand (see picture: N blue, H cyan, S yellow) shows a marked structure and sequence selectivity for the poly(dA)⋅poly(rU) hybrid duplex. An intercalative binding site was discovered where the ligand occupies a surprising ten base pairs. A strong correlation between hybrid duplex and DNA triplex binding indicates new directions for ligand design.

Yes / Dual submicromolar trypanocidal–antiplasmodial compounds have been identified by screening and chemical synthesis of 4-aminoquinoline-based heterodimeric compounds of three different structural classes. In Trypanosoma brucei, inhibition of the enzyme trypanothione reductase seems to be involved in the potent trypanocidal activity of these heterodimers, although it is probably not the main biological target. Regarding antiplasmodial activity, the heterodimers seem to share the mode of action of the antimalarial drug chloroquine, which involves inhibition of the haem detoxification process. Interestingly, all of these heterodimers display good brain permeabilities, thereby being potentially useful for late stage human African trypanosomiasis. Future optimization of these compounds should focus mainly on decreasing cytotoxicity and acetylcholinesterase inhibitory activity.

9 ABSTRACT Tatiana Labudová: Genetic changes in neuroectodermal tumours detected by molecular biological methods. Charles University in Prague, Faculty of Science, Department of Anthropology and Human Genetics Thesis, 75 pages,8 supplements, 2012 This thesis is concerned to neuroectodermal tumours that make a major group of infant tumour diseases. Genetic material gained from patients with neuroectodermal tumours was examined using comparative genomic hybridization (CGH) and interphasic fluorescence in situ hybridization (I-FISH). The aim of this Thesis is to prove chromosomal changes and to create the whole genetic profile. According to these profiles can be determined tumourgenetic cascade or specific genetic changes that lead to malignant tumours. In some cases (f.e. neuroblastoms) the genetic profile helps us to determine a subtype of disease and it's biological behaviour. Keywords: tumour diseases, neuroblastoma, CNS tumours, pheochromocytoma, Ewing's sarcoma, neuroectodermal tumour, comparative genomic hybridization, interphase fluorescence in situ hybridization

O objetivo do presente estudo clínico randomizado foi avaliar in vivo, por meio da técnica de biologia molecular Checkerboard DNA-DNA Hybridization: 1) A contaminação de bráquetes metálicos por 4 espécies bacterianas de microrganismos cariogênicos (Streptococcus mutans, Streptococcus sobrinus, Lactobacillus casei e Lactobacillus acidophilus); e 2) A eficácia da utilização do gluconato de clorexidina a 0,12% (Periogard&reg;) sob a forma de bochechos, sobre esses microrganismos. Participaram do estudo 39 pacientes de 11 a 33 anos de idade, em tratamento com aparelho ortodôntico fixo, nos quais foram colados 2 bráquetes metálicos novos, nos pré-molares. Os pacientes do Grupo Controle (n=20) foram orientados a fazer 2 bochechos semanais com solução placebo, durante 30 dias. Os pacientes do Grupo Experimental (n=19) foram orientados a fazer bochechos com solução à base de gluconato de clorexidina a 0,12% (Periogard&reg;), da mesma forma que o grupo Controle. Decorridos 30 dias, os 2 bráquetes foram removidos de cada paciente e processados para detecção das 4 cepas bacterianas, pela técnica Checkerboard DNADNA Hybridization. Os resultados obtidos foram analisados por meio do teste nãoparamétrico de Kruskal-Wallis, utilizando o software SAS (Statistical Analysis System). O nível de significância adotado foi de 5%. De acordo com os resultados obtidos, observou-se que S. mutans, S. sobrinus, L. casei e L. acidophilus foram detectados em 100% das amostras dos bráquetes de ambos os grupos. No entanto, os bráquetes do grupo Controle encontravam-se mais densamente contaminados por S. mutans e S. sobrinus (p<0,01). No grupo Experimental, embora as quantidades de S. mutans, S. sobrinus, L. casei e L. acidophilus tenham sofrido redução numérica após o uso dos bochechos com solução de gluconato de clorexidina a 0,12%, a comparação com os valores observados no grupo Controle evidenciou diferença estatisticamente significante apenas para S. mutans (p=0,03). Concluiu-se que os bochechos com solução de gluconato de clorexidina a 0,12% podem ser úteis, na prática clínica, com a finalidade de reduzir os níveis de microrganismos cariogênicos, em pacientes portadores de aparelhos ortodônticos fixos. / The purpose of this randomized clinical study was to evaluate in vivo, by the checkerboard DNA-DNA hybridization biomolecular technique: 1) The contamination of metallic brackets by four cariogenic bacterial strains (Streptococcus mutans, Streptococcus sobrinus, Lactobacillus casei and Lactobacillus acidophilus); and 2) The efficacy of 0.12% chlorhexidine gluconate mouthrinses (Periogard&reg;) against these microorganisms. Thirty-nine 11-33-year-old patients under treatment with fixed orthodontic appliances were enrolled in the study and had 2 new metallic brackets bonded to the premolars. For the patients of the control group (n=20), mouthrinses with a placebo solution were prescribed twice a week during 30 days. The patients randomized to the experimental group (n=19) were instructed to use a 0.12% chlorhexidine gluconate solution (Periogard&reg;) as an oral rinse twice a week during 30 days, in the same way as in control group. After this period, the 2 brackets were removed from each patient and processed for detection of the 4 bacterial strains by checkerboard DNA-DNA hybridization. The obtained data were analyzed statistically by the non-parametric Kruskal-Wallis test using the SAS (Statistical Analysis System) software. A level of significance of 5% was set for all analysis. S. mutans, S. sobrinus, L. casei and L. acidophilus were detected in 100% of the samples from brackets of both groups. However, the brackets of the control group were more heavily contaminated by S. mutans and S. sobrinus (p<0.01). In the experimental group, although S. mutans, S. sobrinus, L. casei and L. acidophilus counts decreased after rinsing with the 0.12% chlorhexidine gluconate solution, the comparison with the values obtained in the control group showed statistically significant difference only for S. mutans (p=0.03). In conclusion, the use of 0.12% chlorhexidine gluconate mouthrinses can be useful in clinical practice to reduce the levels of cariogenic microorganisms in patients under treatment with fixed orthodontic appliances.

Empregando a técnica de biologia molecular Checkerboard DNA-DNA Hybridization e o teste Limulus Amebocyte Lysate, os objetivos do presente estudo clínico randomizado in vivo foram avaliar, em bráquetes ortodônticos metálicos: 1) A presença de 16 espécies de microrganismos periodontopatogênicos Gram-negativos pertencentes aos complexos laranja e vermelho, por meio de sondas de DNA; 2) A quantidade de endotoxina bacteriana presente; e 3) A eficácia da utilização do gluconato de clorexidina a 0,12%, sob a forma de bochechos, na redução da contaminação pelas 16 espécies de microrganismos periodontopatogênicos Gram-negativos e na redução da quantidade de endotoxina bacteriana. Participaram do estudo 33 pacientes de 11 a 33 anos de idade, em tratamento com aparelho ortodôntico fixo, nos quais foram colocados randomicamente 3 bráquetes metálicos novos nos pré-molares. Os pacientes do Grupo Controle (n=17) fizeram 2 bochechos semanais com solução placebo, durante 30 dias. Os pacientes do Grupo Experimental (n=16) fizeram bochechos com solução à base de gluconato de clorexidina a 0,12% (Periogard®), da mesma forma que o grupo Controle. Decorridos 30 dias, os 3 bráquetes foram removidos de cada paciente e processados para a detecção dos microrganismos, pela técnica Checkerboard DNA-DNA Hybridization, e para a quantificação da endotoxina bacteriana por meio do teste Limulus Amebocyte Lysate. Os resultados obtidos foram analisados por meio dos testes não-paramétricos de Kruskal-Wallis, Mann-Whitney e pós-teste de Dunn, utilizando os softwares SAS e Graphpad Prism. O nível de significância adotado foi de 5%. De acordo com os resultados obtidos, observou-se que todos os bráquetes dos pacientes do Grupo Controle encontravam-se densamente contaminados pelos microrganismos avaliados. Nesse grupo, as espécies bacterianas do complexo laranja apresentaram-se em maiores quantidades, em relação às espécies do complexo vermelho (p<0,01). A mediana da quantidade de endotoxina para este grupo foi de 0,6673 EU/ml. Quando comparado ao grupo Controle, observou-se que o número total de microrganismos no grupo Experimental foi estatisticamente menor, com mediana de 29.150.000 no grupo Controle e de 13.130.000 no grupo Experimental (p=0,01). Quando os microrganismos foram avaliados por complexos, foi observada diferença estatisticamente significante entre os grupos Controle e Experimental para o complexo laranja (p=0,04), com contagens menores de bactérias após os bochechos com clorexidina. Por outro lado, observou-se que a quantidade de endotoxina no grupo Experimental foi maior, com mediana de 1,2199 EU/ml (p=0,02). Concluiu-se que os bochechos com solução de gluconato de clorexidina a 0,12% podem ser úteis, na prática clínica, com a finalidade de reduzir os níveis de microrganismos periodontopatogênicos Gram-negativos, em pacientes portadores de aparelhos ortodônticos fixos. No entanto, em função do aumento da quantidade de endotoxina bacteriana após o uso dos bochechos com clorexidina, estudos adicionais são necessários com a finalidade de desenvolver procedimentos clínicos ou agentes antimicrobianos que tenham ação sobre a endotoxina presente nos bráquetes metálicos. / Using the biomolecular technique Checkerboard DNA-DNA Hybridization and the Limulus Amebocyte Lysate (LAL) assay, the purposes of the present randomized clinical study were to evaluate in orthodontic metallic brackets: 1) The presence of 16 Gram-negative periodontopathogenic microbial species of the orange and red complexes by using DNA probes; 2) The amount of bacterial endotoxin; and 3) The efficacy of 0.12% chlorhexidine gluconate mouthwashes in reducing the contamination by the evaluated microbial species and the amount of bacterial endotoxin. Thirty-three 11-33-year-old patients undergoing orthodontic treatment with fixed appliances were enrolled in the study and all subjects had 3 new metallic brackets bonded to different premolars in a randomized manner. The patients in the Control group (n=17) were instructed to use a placebo mouthwash twice a week, while those in the Experimental group (n=16) were instructed to use a 0.12% chlorhexidine gluconate mouthwash (Periogard®) in the same way. After 30 days, the 3 brackets were removed from each patient and processed for detection of the microorganisms by the Checkerboard DNADNA hybridization technique, and for quantification of bacterial endotoxin by the LAL assay. The data were analyzed statistically by the non-parametric Mann-Whitney, Kruskal-Wallis and Dunn\'s post tests using SAS and GraphPad Prism softwares. A significance level of 5% was set for all analyses. The brackets of the patients in the Control group were densely contaminated by the evaluated microbial species. In this group, the number of bacterial species of the orange complex was larger compared to the number of bacterial species of the red complex (p<0.01). The median of the amount of bacterial endotoxin for this group was 0.6673 EU/ml. The Experimental group had a significantly smaller number of microorganisms than the Control group (median 13,130,000 versus 29,150,000; p=0.01). When the microorganisms were analyzed by complex, there was statistically significant difference between the Control and Experimental groups for the orange complex (p=0.04) with smaller counts of bacteria after use of chlorhexidine oral rinses. On the other hand, there was a greater amount of bacterial endotoxin in the Experimental (median of 1,2199 EU/ml; p=0.02). In conclusion, 0.12% chlorhexidine oral rinse can be useful in the clinical practice to reduce the levels of Gram-negative periodontopathogenic microorganisms in patients with fixed orthodontic appliances. Considering the increase in the amount of bacterial endotoxin after use of chlorhexidine oral rinses, further research is necessary to develop clinical procedures or antimicrobial agents with action against the endotoxin in the metallic brackets

O objetivo do presente estudo clínico randomizado foi avaliar in vivo, por meio da técnica de biologia molecular Checkerboard DNA-DNA Hybridization: 1) A contaminação de bráquetes metálicos por 4 espécies bacterianas de microrganismos cariogênicos (Streptococcus mutans, Streptococcus sobrinus, Lactobacillus casei e Lactobacillus acidophilus); e 2) A eficácia da utilização do gluconato de clorexidina a 0,12% (Periogard&reg;) sob a forma de bochechos, sobre esses microrganismos. Participaram do estudo 39 pacientes de 11 a 33 anos de idade, em tratamento com aparelho ortodôntico fixo, nos quais foram colados 2 bráquetes metálicos novos, nos pré-molares. Os pacientes do Grupo Controle (n=20) foram orientados a fazer 2 bochechos semanais com solução placebo, durante 30 dias. Os pacientes do Grupo Experimental (n=19) foram orientados a fazer bochechos com solução à base de gluconato de clorexidina a 0,12% (Periogard&reg;), da mesma forma que o grupo Controle. Decorridos 30 dias, os 2 bráquetes foram removidos de cada paciente e processados para detecção das 4 cepas bacterianas, pela técnica Checkerboard DNADNA Hybridization. Os resultados obtidos foram analisados por meio do teste nãoparamétrico de Kruskal-Wallis, utilizando o software SAS (Statistical Analysis System). O nível de significância adotado foi de 5%. De acordo com os resultados obtidos, observou-se que S. mutans, S. sobrinus, L. casei e L. acidophilus foram detectados em 100% das amostras dos bráquetes de ambos os grupos. No entanto, os bráquetes do grupo Controle encontravam-se mais densamente contaminados por S. mutans e S. sobrinus (p<0,01). No grupo Experimental, embora as quantidades de S. mutans, S. sobrinus, L. casei e L. acidophilus tenham sofrido redução numérica após o uso dos bochechos com solução de gluconato de clorexidina a 0,12%, a comparação com os valores observados no grupo Controle evidenciou diferença estatisticamente significante apenas para S. mutans (p=0,03). Concluiu-se que os bochechos com solução de gluconato de clorexidina a 0,12% podem ser úteis, na prática clínica, com a finalidade de reduzir os níveis de microrganismos cariogênicos, em pacientes portadores de aparelhos ortodônticos fixos. / The purpose of this randomized clinical study was to evaluate in vivo, by the checkerboard DNA-DNA hybridization biomolecular technique: 1) The contamination of metallic brackets by four cariogenic bacterial strains (Streptococcus mutans, Streptococcus sobrinus, Lactobacillus casei and Lactobacillus acidophilus); and 2) The efficacy of 0.12% chlorhexidine gluconate mouthrinses (Periogard&reg;) against these microorganisms. Thirty-nine 11-33-year-old patients under treatment with fixed orthodontic appliances were enrolled in the study and had 2 new metallic brackets bonded to the premolars. For the patients of the control group (n=20), mouthrinses with a placebo solution were prescribed twice a week during 30 days. The patients randomized to the experimental group (n=19) were instructed to use a 0.12% chlorhexidine gluconate solution (Periogard&reg;) as an oral rinse twice a week during 30 days, in the same way as in control group. After this period, the 2 brackets were removed from each patient and processed for detection of the 4 bacterial strains by checkerboard DNA-DNA hybridization. The obtained data were analyzed statistically by the non-parametric Kruskal-Wallis test using the SAS (Statistical Analysis System) software. A level of significance of 5% was set for all analysis. S. mutans, S. sobrinus, L. casei and L. acidophilus were detected in 100% of the samples from brackets of both groups. However, the brackets of the control group were more heavily contaminated by S. mutans and S. sobrinus (p<0.01). In the experimental group, although S. mutans, S. sobrinus, L. casei and L. acidophilus counts decreased after rinsing with the 0.12% chlorhexidine gluconate solution, the comparison with the values obtained in the control group showed statistically significant difference only for S. mutans (p=0.03). In conclusion, the use of 0.12% chlorhexidine gluconate mouthrinses can be useful in clinical practice to reduce the levels of cariogenic microorganisms in patients under treatment with fixed orthodontic appliances.

Empregando a técnica de biologia molecular Checkerboard DNA-DNA Hybridization e o teste Limulus Amebocyte Lysate, os objetivos do presente estudo clínico randomizado in vivo foram avaliar, em bráquetes ortodônticos metálicos: 1) A presença de 16 espécies de microrganismos periodontopatogênicos Gram-negativos pertencentes aos complexos laranja e vermelho, por meio de sondas de DNA; 2) A quantidade de endotoxina bacteriana presente; e 3) A eficácia da utilização do gluconato de clorexidina a 0,12%, sob a forma de bochechos, na redução da contaminação pelas 16 espécies de microrganismos periodontopatogênicos Gram-negativos e na redução da quantidade de endotoxina bacteriana. Participaram do estudo 33 pacientes de 11 a 33 anos de idade, em tratamento com aparelho ortodôntico fixo, nos quais foram colocados randomicamente 3 bráquetes metálicos novos nos pré-molares. Os pacientes do Grupo Controle (n=17) fizeram 2 bochechos semanais com solução placebo, durante 30 dias. Os pacientes do Grupo Experimental (n=16) fizeram bochechos com solução à base de gluconato de clorexidina a 0,12% (Periogard®), da mesma forma que o grupo Controle. Decorridos 30 dias, os 3 bráquetes foram removidos de cada paciente e processados para a detecção dos microrganismos, pela técnica Checkerboard DNA-DNA Hybridization, e para a quantificação da endotoxina bacteriana por meio do teste Limulus Amebocyte Lysate. Os resultados obtidos foram analisados por meio dos testes não-paramétricos de Kruskal-Wallis, Mann-Whitney e pós-teste de Dunn, utilizando os softwares SAS e Graphpad Prism. O nível de significância adotado foi de 5%. De acordo com os resultados obtidos, observou-se que todos os bráquetes dos pacientes do Grupo Controle encontravam-se densamente contaminados pelos microrganismos avaliados. Nesse grupo, as espécies bacterianas do complexo laranja apresentaram-se em maiores quantidades, em relação às espécies do complexo vermelho (p<0,01). A mediana da quantidade de endotoxina para este grupo foi de 0,6673 EU/ml. Quando comparado ao grupo Controle, observou-se que o número total de microrganismos no grupo Experimental foi estatisticamente menor, com mediana de 29.150.000 no grupo Controle e de 13.130.000 no grupo Experimental (p=0,01). Quando os microrganismos foram avaliados por complexos, foi observada diferença estatisticamente significante entre os grupos Controle e Experimental para o complexo laranja (p=0,04), com contagens menores de bactérias após os bochechos com clorexidina. Por outro lado, observou-se que a quantidade de endotoxina no grupo Experimental foi maior, com mediana de 1,2199 EU/ml (p=0,02). Concluiu-se que os bochechos com solução de gluconato de clorexidina a 0,12% podem ser úteis, na prática clínica, com a finalidade de reduzir os níveis de microrganismos periodontopatogênicos Gram-negativos, em pacientes portadores de aparelhos ortodônticos fixos. No entanto, em função do aumento da quantidade de endotoxina bacteriana após o uso dos bochechos com clorexidina, estudos adicionais são necessários com a finalidade de desenvolver procedimentos clínicos ou agentes antimicrobianos que tenham ação sobre a endotoxina presente nos bráquetes metálicos. / Using the biomolecular technique Checkerboard DNA-DNA Hybridization and the Limulus Amebocyte Lysate (LAL) assay, the purposes of the present randomized clinical study were to evaluate in orthodontic metallic brackets: 1) The presence of 16 Gram-negative periodontopathogenic microbial species of the orange and red complexes by using DNA probes; 2) The amount of bacterial endotoxin; and 3) The efficacy of 0.12% chlorhexidine gluconate mouthwashes in reducing the contamination by the evaluated microbial species and the amount of bacterial endotoxin. Thirty-three 11-33-year-old patients undergoing orthodontic treatment with fixed appliances were enrolled in the study and all subjects had 3 new metallic brackets bonded to different premolars in a randomized manner. The patients in the Control group (n=17) were instructed to use a placebo mouthwash twice a week, while those in the Experimental group (n=16) were instructed to use a 0.12% chlorhexidine gluconate mouthwash (Periogard®) in the same way. After 30 days, the 3 brackets were removed from each patient and processed for detection of the microorganisms by the Checkerboard DNADNA hybridization technique, and for quantification of bacterial endotoxin by the LAL assay. The data were analyzed statistically by the non-parametric Mann-Whitney, Kruskal-Wallis and Dunn\'s post tests using SAS and GraphPad Prism softwares. A significance level of 5% was set for all analyses. The brackets of the patients in the Control group were densely contaminated by the evaluated microbial species. In this group, the number of bacterial species of the orange complex was larger compared to the number of bacterial species of the red complex (p<0.01). The median of the amount of bacterial endotoxin for this group was 0.6673 EU/ml. The Experimental group had a significantly smaller number of microorganisms than the Control group (median 13,130,000 versus 29,150,000; p=0.01). When the microorganisms were analyzed by complex, there was statistically significant difference between the Control and Experimental groups for the orange complex (p=0.04) with smaller counts of bacteria after use of chlorhexidine oral rinses. On the other hand, there was a greater amount of bacterial endotoxin in the Experimental (median of 1,2199 EU/ml; p=0.02). In conclusion, 0.12% chlorhexidine oral rinse can be useful in the clinical practice to reduce the levels of Gram-negative periodontopathogenic microorganisms in patients with fixed orthodontic appliances. Considering the increase in the amount of bacterial endotoxin after use of chlorhexidine oral rinses, further research is necessary to develop clinical procedures or antimicrobial agents with action against the endotoxin in the metallic brackets

A substituição clínica de dentes naturais perdidos por implantes osteointegrados tem representado uma das primeiras opções terapêuticas para a reabilitação de pacientes total ou parcialmente edêntulos. Apesar dos excelentes índices de sucesso demonstrados pelas restaurações implanto-suportadas, alguns fatores permanecem não esclarecidos, principalmente no que diz respeito à remodelação óssea ao redor dos implantes osteointegrados. Desta forma, o objetivo deste estudo foi avaliar a relação do nível de instalação dos implantes dentários com os parâmetros clínicos, com a remodelação óssea peri-implantar e com a colonização bacteriana, em implantes de plataforma regular, submetidos à carga imediata. Implantes de plataforma regular foram instalados em dois diferentes níveis em relação à crista óssea ao nível ósseo e supra ósseo (1 mm). No total, trinta e cinco implantes em 9 pacientes (idade média de 62,4 11,2 anos) foram avaliados radiograficamente no momento da instalação dos implantes (T1) e 6 meses após (T2), momento no qual também foram feitas análises clínicas e coleta de amostras para o teste microbiológico. Nos exames radiográficos foram analisadas a perda óssea, a partir de mensurações lineares da distância entre um ponto fixo do componente protético e o ponto mais coronário do contato osso-implante, e a densidade óptica alveolar obtida a partir de regiões ósseas de interesse (ROIs). As análises clínicas consistiram na avaliação da profundidade de sondagem e na mensuração do volume do fluido gengival peri-implantar. O perfil bacteriano dos sítios avaliados foi caracterizado por meio do método de análise de checkerboard DNA-DNA hybridization. Os testes estatísticos realizados mostraram não haver relação entre o nível de instalação dos implantes em relação à crista óssea e a remodelação óssea alveolar, tanto com relação à perda óssea (p = 0,725), como com relação à densidade óptica alveolar (p = 0,975). Também não foi possível estabelecer uma correlação entre a remodelação óssea e parâmetros clínicos como profundidade de sondagem e volume do fluido gengival peri-implantar. Com relação ao perfil bacteriano, não foram encontradas diferenças estatisticamente significativas entre os grupos avaliados para nenhuma das 40 bactérias analisadas. / The replacement of missing teeth with osteointegrated dental implants represents one of the first therapeutic options for the rehabilitation of totally or partially edentulous patients. In spite of clinical success rates for implant-supported restorations, some factors remain nuclear, especially those related to alveolar bone remodeling around osteointegrated implants. Thus, the aim of this study was to evaluate the relation between the installation level of dental implants with bone remodeling and bacterial colonization, in regular platform implants, under an immediate loading protocol. Dental implants with regular platform were inserted in different levels related to the bone crest on the level of bone crest and above the bone crest. In total, thirty-five implants in 9 patients (mean age 62,4 11,2 years) were radiographically evaluated in the moment of implant installation (T1) and 6 months after (T2), when clinical analysis and sample collecting for microbiologic test were performed. In radiographic exams were analyzed the marginal bone loss, from linear measurements of the distance between a fixed point of prosthetic components and the most coronal point of bone-implant contact, and optical alveolar density, from bone regions of interest (ROIs). Clinical analysis consisted of probing depth evaluation and peri-implant crevicular fluid volume mensuration. The bacterial profile of selected sites was characterized by checkerboard DNA-DNA hybridization method. Statistic tests showed no relation between implant insertion levels and alveolar bone remodeling, with relation to marginal bone loss (p = 0,725) and optical alveolar density (p = 0,975). It was not also possible to establish a correlation among alveolar bone remodeling and clinical parameters as probing depth and peri-implant crevicular fluid volume. With respect to the bacterial profile, no statistically significant differences were found between the study groups, for none of the 40 bacterial specimens analyzed.