Background: Trauma is a major cause of death and disability. Adverse events, such as prolonged prehospital time, hypoxia, hypotension and/or hyperventilation have been reported to correlate to poor outcome. Adequate cortisol levels are essential for survival after major trauma. In hypotensive critically ill patients, lack of sufficient amount of cortisol can be suspected, and a concept of critical illness related corticosteroid insufficiency has been proposed. Corticosteroid therapy has many adverse effects in critically ill patients and should only be given if life-saving. Correct measurement of serum cortisol levels is important but difficult in critically ill patients with capillary leakage. Estimation of the free and biologically active cortisol is preferable. In serum less than 10% of cortisol is free and biologically active and not possible to measure with routine laboratory methods. Salivary cortisol can be used as a surrogate for free cortisol, but salivary production is reduced in critically ill patients. Liver resection could reduce cortisol levels due to substrate deficiency. Aims: 1. Evaluate the occurrence of early adverse events in patients with traumatic brain injury and relate them to outcome. 2. Assess cortisol levels over time after trauma and correlate to severity of trauma, sedative/analgesic drugs and cardiovascular function. 3. Evaluate if saliva stimulation could be performed without interfering with salivary cortisol levels. 4. Assess cortisol levels over time after liver resection in comparison to other major surgery. Results: There was no significant correlation between prehospital time ³60 minutes, hypoxia (saturation <95%), hypotension (systolic blood pressure <90 mmHg), or hyperventilation (ETCO2 <4.5 kPa) and a poor outcome (Glasgow Outcome Scale 1-3) in patients with traumatic brain injury. Cortisol levels decreased significantly over time after trauma, but there was no correlation between low (<200 nmol/L) serum cortisol levels and severity of trauma. Infusion of sedative/analgesic drugs was the strongest predictor for a low (<200 nmol/L) serum cortisol. The odds ratio for low serum cortisol levels (<200 nmol/L) was 8.0 for patients receiving continuous infusion of sedative/analgesic drugs. There was no significant difference between unstimulated and stimulated salivary cortisol levels (p=0.06) in healthy volunteers. Liver resection was not associated with significantly lower cortisol levels compared to other major surgery. Conclusion: There was no significant correlation between early adverse events and outcome in patients with traumatic brain injury. Cortisol levels decreased significantly over time in trauma patients. Low cortisol levels (<200 nmol/L) were significantly correlated to continuous infusion of sedative/analgesic drugs. Saliva stimulation could be performed without interfering with salivary cortisol levels. Liver resection was not associated with low cortisol levels compared to other major surgery.

Prolonged strenuous exercise affects the circulating numbers and functions of immune cells. These effects are thought to be largely mediated by the actions of elevated circulating stress hormones and alterations in regulatory cytokines. Although the effects of a single acute bout of exercise on immune system function are quite well established, it is still not clear how time of day and repeated bouts of prolonged exercise on the same day influence immune function. It is of particular interest to understand the effects of nutritional supplementation on immunoendocrine responses. Therefore, the aims of the studies described in this thesis were to determine the effects of two bouts of prolonged cycling and carbohydrate supplementation on immunoendocrine responses. The saliva collection study showed that the use of a swab for collecting saliva is not an ideal method because it affects the results of saliva composition (Chapter 4). The comparison of the effects of exercise at different times of day on immunoendocrine responses showed that a single bout of prolonged exercise performed in the afternoon induces a larger perturbation in the redistribution of leukocytes into the circulation than an identical bout of morning exercise, which maybe due to higher hypothalamic-pituitaryadrenal (HP A) activation and. circadian rhythms. However, in terms of oral mucosal immunity, performing prolonged cycling at different times of day does not differently affect the salivary responses. The second compared with the first of two bouts of prolonged exercise on the same day induces a greater HP A activation, a larger leukocyte trafficking into the circulation, a decreased neutrophil degranulation response to lipopolysaccharide (LPS) on per cell basis and a lower saliva flow rate, but does not increase plasma interleukin-6 (IL-6), or change saliva immunoglobulin A (slgA) secretion rate (Chapter 5). Furthermore, carbohydrate (CHO) ingestion during any period of two bouts of prolonged exercise shows limited beneficial effect in blunting these higher responses in the second exercise bout compared with the first identical exercise bout on the same day (Chapter 6, 7 and 8). The determination of the effects of CHO ingestion on exercise-induced immunoendocrine responses showed that when two bouts of exercise are performed on the same day, the greater benefit in terms of circulating immunoendocrine responses is obtained by feeding CHO at the earliest opportunity (Chapter 6, 7 and 8). A 3-h interval is insufficient for recovery of leukocyte mobilisation and neutrophil function from the impact of previous exercise whether subjects consumed placebo or CHO during exercise or recovery (Chapter 5, 6, 7 and 8). However, an 18-h interval is sufficient for full recovery of all immunoendocrine variables that were measured in this thesis from the impact of two bouts of prolonged exercise (Chapter 8).

Post-exercise carbohydrate-protein consumption and tapering periods during training periodisation have been proposed as effective recovery strategies in several sports; however, limited attention has been given to judo. Apart from training and recovery, effective competition performance can also be influenced by several stimuli on the competition day, which may be manifested as distinct endocrine responses. The main objective of this thesis was to influence effective competition performance in judo, through examining strategies that can aid recovery from intense exercise/training and examining endocrine responses to competition. Three experimental studies on recovery were completed (chapters 3-5) followed by an observational study on a judo competition day (chapter 6) in elite, national level, male judo athletes. Studies 1 and 2 examined the effects 1000 ml of post-exercise chocolate milk (CM) consumption compared with water (W) following an intense judo training session (chapter 3) and five days of intense judo training with concomitant weight loss (chapter 4) on the recovery of salivary cortisol (sC), salivary testosterone (sT), salivary testosterone:cortisol (sT/C) ratio, salivary secretory IgA (SIgA) absolute concentrations and secretion rate, muscle soreness, mood state and judo-related performance. Study 1 (n=10) did not show any beneficial effects of acute CM consumption on aspects of recovery of any of the measured variables, except for a lower perception of soreness (p<0.05) and a tendency for better push-up performance (p=0.09). Study 2 (n=12) showed that post-exercise CM consumption resulted in significantly lower sC levels, a tendency for higher sT/C ratio (p=0.07), better judo-related performance, lower muscle soreness and reduced mood disturbance (p<0.05) with W. In addition, post-exercise consumption of CM resulted in a 1.1% decrease in body weight, indicating that CM is an effective recovery beverage during periods of intense judo training without affecting intentional weight loss. Study 3 (n=11) examined the effects of a 2-week exponential taper following 2 weeks of intense judo training on recovery of the aforementioned variables. Within 12 days of tapering there were evidence of enhanced performance, lower sC, higher sT and higher sT/C ratio, higher SIgA secretion rate, lower muscle soreness and reduced mood disturbance, indicating that a tapering period of ~10 days is an effective recovery strategy for optimising judo performance. Study 4 observed the responses of sC, sT, SIgA absolute concentrations and SIgA secretion rate and self-measured anxiety state in the winners (n=12) and losers (n=11) of a judo competition. Winners presented significantly higher morning sC levels and higher cognitive anxiety in anticipation of the competition, as well as a tendency for higher SIgA secretion rate (p=0.07) and significantly higher saliva flow rate mid-competition. These findings indicate that winners experienced higher arousal levels and that anticipatory sC might have some predictive value for winning performance in judo. This thesis concludes that nutrition and tapering are both important aspects of effective recovery; CM can be an effective nutritional recovery aid during periods of intense judo training and tapering for 7-12 days can optimise judo performance and can be implemented prior to competitions. In addition, elevated sC levels in anticipation of a judo competition and higher levels of arousal could have some predictive value for winning performance in judo. Further research could focus on strategies to increase levels of arousal in anticipation of competition.

Neste trabalho desenvolvemos um procedimento simples para a separação de nanoesferas ocas de carbono (do inglês Carbon Spherical Shells ou CSS) em diâmetros entre 400 e 500 nm utilizando centrifugação corroborado pelas análises realizadas na microscopia eletrônica de varredura e de transmissão. A análise de sua composição química, realizada através da técnica de fotoelétrons excitados por raios X, indicou que as CSS são constituídas de 79% de carbono e 21% de oxigênio em sua superfície, apresentando grupos funcionais carbonila e hidroxila. Plataformas sensoriais distintas foram obtidas formando filmes homogêneos das CSS sobre o eletrodo de carbonno vítreo GCE (do inglês glassy carbon electrode ou GCE). Como resultado dos experimentos eletroanalíticos, observou-se o aumento da sensitividade do eletrodo GCE/CSS de acordo com a diminuição do diâmetro (500 até 400 nm) das CSS. As plataformas sensoriais GCE/CSS com 400 nm de diâmetro apresentaram maior sensitividade (0.02 &mu;A µmol L-1) com um limite detecção de 0.2 &mu;mol L-1. Os eletrodos GCE/CSS foram estáveis, apresentando pequena interferência de espécies concomitantes presentes na amostra e seu desempenho na quantificação de paracetamol em suor mostrou-se estatisticamente equivalente ao método padrão baseado em cromatografia líquida. / We applied a simple strategy, based upon centrifugation, to separate carbon spherical shells (CSS), in sizes varying from 400 to 500 nm, which is shown by the micrographs obtained in the Scanning and Transmission Electron microscopy analysis. In their surface, carbonyl and hydroxyl groups were present, constituting a composition of 21% of oxygen and 79% of carbon. The CSS were casted on a glassy carbon electrode\'s (GCE) surface, forming a thin film, and the resulting platform was used as a sensor. A trend was observed in the results obtained by the electroanalytical experiments: as the size of the CSS were reduced, the sensibility of the GCE/CSS platform towards paracetamol detection increased. The best attained result, namely the platform with the GCE and the 400 nm diameter CSS, have shown promising results, achieving sensitivity\'s value of 0.02 &mu;A &mu;mol-1 L. The proposed sensors were stable, displaying little interference from another species coexisting in the samples, and its performance towards paracetamol detection were statistically identical to the standard method for paracetamol detection based upon liquid chromatography.

Submitted by Rosana Moreira (rosanapsm@outlook.com) on 2018-11-20T18:27:45Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AtividadeImunomoduladoraAntioxidante.pdf: 20324950 bytes, checksum: 23513f8e4b2fff677439e7129e334b3f (MD5) / Approved for entry into archive by Edisangela Bastos (edisangela@ufpa.br) on 2019-01-31T17:21:46Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AtividadeImunomoduladoraAntioxidante.pdf: 20324950 bytes, checksum: 23513f8e4b2fff677439e7129e334b3f (MD5) / Made available in DSpace on 2019-01-31T17:21:46Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AtividadeImunomoduladoraAntioxidante.pdf: 20324950 bytes, checksum: 23513f8e4b2fff677439e7129e334b3f (MD5) Previous issue date: 2017-07-04 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A sepse e uma disfuncao organica causada por uma resposta imune desregulada a uma infeccao. A mesma tem em sua abordagem terapeutica inicial a utilizacao de antimicrobianos de amplo espectro, os quais nao sao suficientes para o controle da infeccao, sendo necessario a busca por novas terapias focadas na interrupcao da resposta inflamatoria em cascata produzida pela invasao e disseminacao de agentes patogenicos. Dessa forma, a saliva do Aedes aegypti apresenta caracteristicas imunomoduladoras, com potencial de inibicao de citocinas pro-inflamatorias, bem como a presenca de peptideos ativadores de oxido nitrico. Com isso, seria de grande interesse investigar o efeito imunomodulador da saliva em modelo animal de sepse. Neste sentido, camundongos foram pre-tratados com a saliva do Aedes aegypti sendo a sepse induzida pelo modelo de transfixacao do coto-cecal. Apos 12 e 24 horas as amostras foram coletadas, e avaliado a sobrevida, carga bacteriana, migracao leucocitaria e parametros oxidativo (NO, EROs, MDA, TEAC, DPPH e GSH). A saliva melhorou o prognostico dos animais, aumentando a sobrevida e o peso. Alem disso, reduziu a carga bacteriana e aumentou o influxo de monocitos no sangue. A saliva, tambem, mostrou efeito antioxidante por reduzir a producao de especies reativas e aumentar a capacidade antioxidante, alem de diminuir a peroxidacao lipidica. Assim, a saliva foi capaz de inibir os danos causados pela sepse em animais in vivo melhorando seu prognostico. / Sepsis is an organ dysfunction caused by a dysregulated immune response to an infection, the initial therapeutic approach to sepsis are broad spectrum antimicrobials, which is not sufficient for control of infection, requiring association with other therapies focused in the interruption of the inflammatory response chain, provided by the pathogens. In this way, the Aedes Aegypti’s saliva presents immunomodulary features, with a potential pro inflammatory cytokine inhibition, as well as the presence of nitric oxide peptides activators. Therefore, it would be a great interest to search the saliva’s immunomodulator effect in animal model sepses. In this regards, mices were pre treated with Aedes aegypti saliva, and sepsis was induced by the cecal ligation and puncture. After 12 and 24 hours, the samples were collected, and evaluated the survival rate, bacteria level, leukocyte migration, and oxidative parameters (NO, EROs, MDA e TEAC). The saliva improved the animal prognostic, increasing the survival rate and weight. Furthermore, decreased the bacterial levels and increased the influx of monocytes. The saliva, in addition, presented antioxidant effects by reducing production of the reatives species, and increasing the antioxidant capacity, other than decrease the lipid peroxidation. Thus, the saliva was capable to inhibit damages caused by sepsis in animals in vivo, improving its prognostic.

O objetivo do presente estudo clínico foi compreender o efeito do uso prévio de agentes antimicrobianos na diversidade e a estrutura do microbioma procariótico de saliva, biofilme supragengival e canal radicular de dentes com infecção endodôntica aguda. Realizaram-se coletas microbiológicas de saliva (S), biofilme supragengival (BS) e canal radicular (CR) de pacientes que não utilizaram antibióticos (G1: n=6) e de pacientes que utilizaram antibióticos (G2: n=6). Para a caracterização das comunidades de procariotos por meio de sequenciamento de alto rendimento, foram produzidos pools de seis amostras para cada um dos sítios. A região hipervariável V3-V4 do gene 16S rRNA foi amplificada e a plataforma Illumina MiSeq foi empregada para análise das sequências geradas. Foram determinadas a presença e abundância relativa das unidades taxonômicas operacionais (OTUs) em cada amostra. Procedeu-se a análise de alfa-diversidade para cada amostra, considerando-se as métricas de Simpson, dominância, estimativa de riqueza de Chao-I e o Índice de Shannon. Os valores obtidos foram comparados por meio de testes estatísticos. Para a análise dos índices de beta-diversidade, empregou-se o método de agrupamento UPGMA, com jackknifing e método de comparação UniFrac com peso. A representação gráfica tridimensional da beta-diversidade foi realizada por meio de análise de coordenadas principais. A técnica de PCR gene específico foi empregada para determinar a presença de genes relacionados à resistência bacteriana para agentes beta-lactâmicos (blaTEM, blaZ, ampC, mecA), macrolídeos (ermB, ermC, mefA), tetraciclinas (tetM, tetQ, tetW), lincosamidas (lnuB, lsaB) e vancomicina (vanA, vanD, vanE). A similaridade para a presença/ausência de genes de resistência nas amostras de S, BS e CR em G1 e G2 foi determinada por meio de coeficiente de agrupamento, utilizando-se o método UPGMA com distância Euclidiana. Todos os pacientes apresentavam infecção endodôntica aguda, caracterizada pela presença de dor espontânea, necrose pulpar e dor à percussão vertical. Aumento de volume foi observado em 8/12 pacientes. Os pacientes do Grupo 2 utilizaram beta-lactâmicos previamente à consulta (amoxicilina = 5; cefalexina = 1). Há predomínio de integrantes do domínio Bactéria em todas S, BS e CR. Archaea pertencentes ao gênero Methanobrevibacter foram encontradas apenas em amostras de CR, constituindo menos de 1% do total de OTUs (G1-CR = 0,319%; G2-CR = 0,014%). Há predomínio de bactérias dos filos Firmicutes e Bacteroidetes em amostras de S, BS e CR. Há redução intensa no percentual de OTUs pertencentes ao Filo Firmicutes em amostras de saliva, quando antibiótico foi utilizado (G1-S = 75,371; G2-S = 35,242). Comportamento oposto ocorreu neste ecossistema para integrantes do Filo Bacteroidetes (G1-S = 12,657; G2-S = 33,947). Tanto em G1 quanto em G2, bactérias do gênero Streptococcus predominam em amostras de S e BS. Em canais radiculares, maiores percentuais de OTUs foram observados para os gêneros Porphyromonas e Prevotella. As métricas de alfadiversidade indicam que o uso prévio de antimicrobiano parece oportunizar o estabelecimento de espécies antes menos abundantes, especialmente em saliva (dominância: G1-S>G2-S; índice de Shanon: G1-S<G2-S; índice de Simpson: G1- S<G2-S; índice de Chao-1: G1-S<G2-S). A análise de beta-diversidade mostra proximidade entre G1-BS e G2-BS; há distanciamento entre G1-S e G2-S. As amostras G1-CR e G2-CR estão mais distantes de S e BS, sugerindo maior seleção imposta pelo ecossistema do CR aos procariotos. Os genes de resistência mais frequentemente detectados foram tetM, tetQ, tetM, ermB e mefA. Não foram detectados genes vanA, vanD, vanE, blaZ, mecA, lnuB e ermC. O gene ermB foi frequentemente detectado em amostras de S, BS e CR em ambos os grupos. Em pacientes que não utilizaram antibiótico, o gene tetM e o gene tetQ foram detectados simultaneamente em S, BS e CR (tetM = 4/6; tetQ = 3/6). A análise multivariada não demonstrou nível de agrupamento alto entre amostras de um mesmo paciente, de um mesmo ecossistema, ou de um mesmo grupo. A partir dos resultados do presente estudo, observou-se que a utilização de agente antimicrobiano betalactâmico parece alterar parâmetros composicionais de comunidades de procariotos na cavidade bucal. Entretanto, particularidades relativas a cada ecossistema podem modular a extensão deste efeito, parecendo ser mais intensos em amostras de S do que em BS e CR. / The present clinical study aimed to assess the effect of antibiotics over the diversity and structure in prokaryotic communities of saliva, supragengival biofilm and root canal of teeth with acute primary infections. Samples of saliva (S), supragengival biofilm (BS) and root canal of teeth with acute primary infections (CR) were collected from patients, and were grouped according with the previous use of antibiotic (G1 = no antibiotics; G2 = antibiotics). Pooled samples for each community were evaluated. DNA sequencing was performed with MiSeq (Illumina). The V3-V4 hypervariable region from the 16S rRNA gene was amplified. The presence and relative abundance of the operational taxonomic units (OTUs) was determined for each sample. Alpha-diversity analysis was performed with the Simpson’s index, dominance, Chao-1 richness index and Shannon’s index. Statistical analysis was carried out to compare their values for each community.Beta-diversity was computed through UPGMA clustering and jackknifing. The principal coordinate analysis employed weighted UniFrac. Gene-specific PCR was employed to detect resistance genes to lactamics (blaTEM, blaZ, ampC, mecA), macrolides (ermB, ermC, mefA), tetracyclines (tetM, tetQ, tetW), lincosamides (lnuB, lsaB) e vancomycin (vanA, vanD, vanE). The UPGA clustering analysis with Euclidean distance was applied to investigate the existence of similar groups of samples. A dendrogram was constructed to show the arrangement of the sample groups produced by clustering. All the patients had primary acute endodontic infections, with spontaneous pain, pulp necrosis and tenderness on percussion. Swelling was observed for 8 out of 12 patients. Patients from G2 had lactamics before the urgency appointment (amoxicillin = 5; cephalexin = 1). Bacteria were predominant in S, BS and CR samples. Archaea belonging to the genus Methanobrevibacter were only detected in RC samples and comprised less than 1% of all the OTUs G1-CR = 0.319%; G2-CR = 0.014%). The great majority of the detected OTUs in S, BS, and CR belonged to the phyla Firmicutes and Bacteroidetes. Reduction in the percentage of Firmicutes was observed in G2-S (G1-S = 75.371%; G2-S = 35.242%). A distinct behavior was demonstrated by the Bacteroidetes (G1- S = 12.657%; G2-S = 33.947%). Components belonging to the genus Streptococcus were predominant in S and BS, for both G1 and G2. CR harbored high percentage of species belonging to the genus Porphyromonas and Prevotella. The alpha-diversity indexes demonstrated that the G2-S had an increase in low abundant species (dominance: G1- S>G2-S; Shanon index: G1-S<G2-S; Simpson index: G1-S<G2-S; Chao-1 index: G1-S<G2- S). Beta-diversity showed that G1-BS and G2-BS had close spatial distribution. G1-CR and G2-CR were more distant from S and BS samples. The RC may promote a most intense species selection, due to environmental conditions. The most frequently detected genes associated with resistance to antibiotics were tetM, tetQ, tetM, ermB, and mefA. The genes vanA, vanD, vanE, blaZ, mecA, lnuB, and ermC were not detected in the samples. The gene ermB was frequently detected in S, BS and CR samples. In patients from G1, tetM and tetQ were detected simultaneously in all the three environments (tetM = 4/6; tetQ = 3/6). There was no clustering behavior for samples belonging to different environments in the same patients and between the same environment samples from different groups. An overall analysis from the data allows for suggesting that the use of lactamic agents may alter compositional parameters from prokaryotic communities in the oral cavity to different extents. Specific environmental characteristics from each site may modulate the effect that seemed to be more intense for S than for BS and CR.

Lors de la prise d'un repas sanguin par le moustique Aedes (Ae) aegypti, le virus de la dengue (DENV) est transmis à l'homme avec la salive du moustique. Ce mélange complexe est en partie déposé dans le compartiment cutané extravasculaire lors de la piqûre de moustique. Il est donc important de prendre en compte la triade virus-vecteur-hôte vertébré dans les mécanismes de transmission de ce virus à l'hôte vertébré. Des analyses de génomique et protéomique des glandes salivaires d'Ae. Aegypti infectées ou non par le DENV nous ont permis de mettre en évidence dans les glandes salivaires de moustiques infectés, la surexpression d'un gène codant pour un peptide antimicrobien (PAM) cationique. Nous avons démontré, que ce PAM possède une activité antibactérienne et antivirale contre les virus de la dengue et du chikungunya. Nos travaux soulignent l'importance chez les invertébrés, du compartiment « glandes salivaires » dans la réponse immunitaire innée du moustique. Nous avons également démontré que les kératinocytes humains sont des cellules permissives pour le DENV et que l'infection de ces cellules stimule la réponse immunitaire innée antivirale. Nos travaux démontrent que des extraits de glandes salivaires d'Ae. Aegypti augmentent l'infection du virus de la dengue dans les kératinocytes humains. Nous avons par la suite identifié une protéine salivaire d'Ae. Aegypti (34kDa), qui augmente l'infection du DENV en supprimant la production d'interféron. L'ensemble de ces travaux ont permis de contribuer aux connaissances sur la transmission du DENV, mais aussi d'identifier de nouvelles cibles potentielles pour le contrôle de la réplication virale. / Dengue virus (DENV) transmission is initiated when a blood-feeding Aedes (Ae) aegypti mosquito injects saliva, together with the virus, into the epidermis of its mammalian host. Studies of DENV should, therefore, take into account the triad virus-vector-vertebrate host. We have used functional genomic and proteomic analyses, of the salivary glands of female Ae. Aegypti, to demonstrate that this compartment harbors a potent immune response against DENV, represented by the production of an antimicrobial peptide (AMP). This AMP was found to exert, in addition to its anti-bacterial activity, an anti-viral activity against DENV and Chikungunya. Our data demonstrate, for the first time, the permissiveness of human epidermal keratinocytes to DENV infection. Remarkably, DENV replication in keratinocytes contributes to the establishment of anti-viral innate immunity that might occur shortly after the mosquito bite. To investigate the role of Ae. aegypti saliva in DENV transmission to man, primary human keratinocytes were infected with DENV in the presence of Ae. aegypti salivary gland extract. We show that Ae. aegypti saliva enhances dengue virus infection of human keratinocytes by suppressing innate immune responses. Furthermore, we have found a 34-kDa protein, in the saliva of Ae.aegypti, that strongly enhances DENV replication by suppressing type-I IFN production. This study provides new insights into the role of Ae. aegypti salivary glands and saliva in DENV transmission. The data presented here provide novel targets for the control of DENV replication in mammalian hosts.

Neste trabalho desenvolvemos um procedimento simples para a separação de nanoesferas ocas de carbono (do inglês Carbon Spherical Shells ou CSS) em diâmetros entre 400 e 500 nm utilizando centrifugação corroborado pelas análises realizadas na microscopia eletrônica de varredura e de transmissão. A análise de sua composição química, realizada através da técnica de fotoelétrons excitados por raios X, indicou que as CSS são constituídas de 79% de carbono e 21% de oxigênio em sua superfície, apresentando grupos funcionais carbonila e hidroxila. Plataformas sensoriais distintas foram obtidas formando filmes homogêneos das CSS sobre o eletrodo de carbonno vítreo GCE (do inglês glassy carbon electrode ou GCE). Como resultado dos experimentos eletroanalíticos, observou-se o aumento da sensitividade do eletrodo GCE/CSS de acordo com a diminuição do diâmetro (500 até 400 nm) das CSS. As plataformas sensoriais GCE/CSS com 400 nm de diâmetro apresentaram maior sensitividade (0.02 &mu;A µmol L-1) com um limite detecção de 0.2 &mu;mol L-1. Os eletrodos GCE/CSS foram estáveis, apresentando pequena interferência de espécies concomitantes presentes na amostra e seu desempenho na quantificação de paracetamol em suor mostrou-se estatisticamente equivalente ao método padrão baseado em cromatografia líquida. / We applied a simple strategy, based upon centrifugation, to separate carbon spherical shells (CSS), in sizes varying from 400 to 500 nm, which is shown by the micrographs obtained in the Scanning and Transmission Electron microscopy analysis. In their surface, carbonyl and hydroxyl groups were present, constituting a composition of 21% of oxygen and 79% of carbon. The CSS were casted on a glassy carbon electrode\'s (GCE) surface, forming a thin film, and the resulting platform was used as a sensor. A trend was observed in the results obtained by the electroanalytical experiments: as the size of the CSS were reduced, the sensibility of the GCE/CSS platform towards paracetamol detection increased. The best attained result, namely the platform with the GCE and the 400 nm diameter CSS, have shown promising results, achieving sensitivity\'s value of 0.02 &mu;A &mu;mol-1 L. The proposed sensors were stable, displaying little interference from another species coexisting in the samples, and its performance towards paracetamol detection were statistically identical to the standard method for paracetamol detection based upon liquid chromatography.

Orientador: Ana Cláudia de Melo Stevanato Nakamune / Banca: José Mauro Granjeiro / Banca: Célio Percinoto / Resumo: A saliva é coletada através de métodos simples e não invasivos, além de ser facilmente armazenada. A coleta não-traumática é atrativa especialmente para crianças e quando repetidas coletas são requeridas. A desvantagem da saliva como uma ferramenta de diagnóstico é a variabilidade, que ocorre em decorrência dos fatores fisiológicos e do modo de coleta. Numa primeira etapa o trabalho teve por objetivo estudar as atividades das enzimas fosfatase alcalina (FAlc), fosfatase ácida total (FAT) e fosfatase ácida resistente ao tartarato (TRAP) na saliva total não estimulada de crianças, investigando as influências de fatores como sexo, faixa etária e horário de coleta, bem como a correlação das enzimas com a taxa de fluxo salivar. Nesta etapa, 120 crianças saudáveis de ambos os sexos, nas faixas etárias de 1-5 e 6-12 anos de idade tiveram as amostras de saliva coletada entre 8:00-10:00 horas ou entre 14:00-16:00 horas. A segunda parte do trabalho teve por objetivo avaliar a correlação das atividades enzimáticas da FAT, TRAP, proteína tirosina fosfatase de baixa massa molecular relativa (PTP-BMr) e FAlc na saliva e no soro de crianças, além de analisar a influência do sexo e da faixa etária na atividade das enzimas, no soro e na saliva total. Para tanto 32 crianças de ambos os sexos, nas faixas etárias de 1-5 anos e 6-12 anos de idade tiveram as amostras de saliva e sangue coletadas entre 8:00-10:00 horas. Os resultados da primeira etapa sugerem que as atividades da FAT, TRAP e FAlc são influenciadas, de formas distintas, pelos fatores sexo, faixa etária e horário de coleta e que não existe correlação entre as atividades das enzimas e a taxa de fluxo salivar. / Abstract: Saliva can be collected by simple, non-invasive methods and is easily stored. The non-traumatic collection is specially appealing for children and when repeated collections are required. The main disadvantage of the saliva as a diagnosis tool is the variability that happens due to the physiologic factors and dependence on mode of the collection. In a first stage the work had for objective to study the activities of the enzymes alkaline phosphatase (ALP), total acid phosphatase (TAP) and tartrate-resistant acid phosphatase (TRAP) in the whole unstimulated saliva of children, investigating the influences of factors as sex, age group and time of collection, as well as the correlation of the enzymes with the salivary flow rate. In this stage, 120 healthy children of both sexes, in the age groups of 1-5 and 6-12 years of age had the saliva samples collected between 8:00-10:00 hours or between 14:00-16:00 hours. In a second stage, the work had for objective to evaluate the correlation of the enzymatic activities of TAP, TRAP, low molecular weight protein tyrosine phosphatase (LMW-PTP) and ALP in the saliva and in the children's serum, besides analyzing the influence of the sex and of the age group in the activity of the enzymes, in the serum and in the whole unstimulated saliva. For so much 32 children of both sexes, in the 1-5 year-old age groups and 6-12 years of age had the saliva samples and blood collected between 8:00-10:00 hours. The results of the first stage suggest that the activities of TAP, TRAP and ALP are influenced, in different ways, for the factors sex, age group and time of collection and that correlation doesn't exist between the activities of the enzymes and the salivary flow rate. / Mestre

Die Speicheldrüsen der Schmeißfliege Calliphora vicina produzieren bei Stimulierung mit dem Neurohormon Serotonin (5-Hydroxytryptamine, 5-HT) einen KCl-reichen Primärspeichel. Der transepitheliale K+-Transport wird durch eine apikal lokalisierte vakuoläre H+-ATPase (V-ATPase) energetisiert. Stimulierung der Speicheldrüsen mit 5-HT aktiviert die apikale V-ATPase, die Protonen aus der Zelle in das Drüsenlumen transportiert. Trotz des auswärts gerichteten Protonentransportes führt die 5-HT-Stimulierung kurioserweise zu einer intrazellulären Ansäuerung. Die Ursachen dieser 5-HT-induzierten Ansäuerung waren unzureichend untersucht. Deshalb war das Ziel dieser Arbeit die Identifikation aller Transporter, die an der intrazellulären pH-(pHi)-Regulation in unstimulierten Speicheldrüsen von Calliphora vicina beteiligt sind und an der Entstehung und Regulation der 5-HT-induzierten pHi-Änderungen mitwirken. Von besonderem Interesse war hierbei die funktionelle Mitwirkung der V-ATPase, deren Beteiligung an der pHi-Regulation in tierischen Zellen bisher wenig untersucht war. Wesentliche Ergebnisse dieser Arbeit waren: • Messungen des pHi-Wertes in der unstimulierten Drüse zeigten, dass vor allem die V-ATPase und mindestens ein Na+-abhängiger HCO3--Transporter an der Aufrechterhaltung des Ruhe-pHi beteiligt sind. • Zur Wiederherstellung des Ruhe-pHi nach einer intrazellulären Ansäuerung (NH4Cl-Vorpuls) tragen ebenfalls im Wesentlichen die V-ATPase und mindestens ein Na+-abhängiger HCO3--Transporter bei. Der Na+/H+-Antiporter hat in der unstimulierten Drüse keinen messbaren Einfluss auf den Ruhe-pHi. • Die Wiederherstellung des Ruhe-pHi nach einer intrazellulären Alkalisierung (Na-acetat-Vorpuls) ist Cl--abhängig, aber auch unter extremen Bedingungen waren die Zellen noch in der Lage sich vollständig von einer intrazellullären Alkalisierung zu erholen. Einen entscheidenden Anteil daran hat offenbar die hohe intrazelluläre Puerkapazität. • Ein Na+-abhängiger Glutamat-Transporter ist per se kein pHi-regulierender Transporter, seine Aktivität hat jedoch Einfluss auf den Ruhe-pHi in der unstimulierten Speicheldrüse von Calliphora vicina. • 10 nM 5-HT induzieren in den Calliphora Speicheldrüsen eine intrazelluläre Ansäuerung. An dieser Ansäuerung ist der Na+/H+-Antiporter entscheidend beteiligt. Auch eine klare Cl--Abhängigkeit der 5-HT-induzierten Ansäuerung konnte beobachtet werden. Wahrscheinlich ist eine gekoppelte Aktivität von Na+/H+-Antiporter und Cl-/HCO3--Antiporter. • Messungen mit einem O2-empndlichen Fluoreszenzfarbstoff zeigten, dass Stimulierung der Speicheldrüsen mit 5-HT die Zellatmung aktivierte. Der cAMP- und der IP3/Ca2+-Weg tragen auf komplexe Weise zu der 5-HT-induzierten Aktivierung der Zellatmung und damit auch zu den 5-HT-induzierten pHi-Änderungen bei. • Mit molekularbiologischen Untersuchungen ist es gelungen den Na+-abhängigen Glutamat-Transporter, den Na+/H+-Antiporter, die Carboanhydrase und die Untereinheit C der V-ATPase in den Calliphora Speicheldrüsen direkt nachzuweisen. Zudem konnte erstmals der direkte Nachweis für die Expression eines nH+/K+-Antiporters in den Speicheldrüsen von Calliphora vicina erbracht werden. Diese Arbeit trug ganz wesentlich zum Verständnis der pHi-Regulation in der unstimulierten und stimulierten Speicheldrüse von Calliphora vicina bei. Mechanismen die zur Aufrechterhaltung und Wiederherstellung des Ruhe-pHi nach einer intrazellulären Ansäuerung bzw. Alkalisierung beitragen, konnten mit pHi-Messungen und auch molekularbiologisch nachgewiesen werden. Die Mechanismen, welche die 5-HT-induzierte intrazelluläre Ansäuerung verursachen, konnten ebenfalls aufgeklärt werden. Zudem wurde an den Calliphora Speicheldrüsen eine neue optische Methode zur Messung des O2-Verbrauchs in tierischen Geweben etabliert. / The tubular salivary glands of the blowfly Calliphora vicina consist of a single layer of epithelial cells. Stimulation with the neurohormone serotonin (5-hydroxytryptamine,5-HT) induces the secretion of a KCl-rich primary saliva. Transepithelial K+-transport is energized by a vacuolar-type H+-ATPase (V-ATPase) which is located in the apical membrane. 5-HT stimulates the apical V-ATPase which transports protons out of the cells into the lumen of the glands. Despite this outward directed proton transport, 5-HT stimulation leads to an intracellular acidication. The causes of this intracellular acidication were poorly understood. Therefore the aim of this thesis was the identication of all pHi regulating transporters which are involved in pHi regulation in the unstimulated salivary glands of Calliphora vicina and which contribute to the 5-HT-induced pHi changes. Of special interest was the functional role of the V-ATPase,whose contribution to pHi regulation in animal cells is, as yet, not well studied. Key results were: • pHi measurements in unstimulated glands showed that mainly the V-ATPase and at least one Na+-dependent HCO3--transporter are involved in maintenance of resting pHi. • V-ATPase and at least one Na+-dependent HCO3--transporter are also necessary for the recovery from an intracellular acidication (NH4Cl prepulse). • Recovery from an intracellular alkali load (Na-acetate prepulse) is partially Cl--dependent. • A Na+ dependent gluatamate-transporter is present in Calliphora salivary glands and its activity aects the resting pHi. • 10 nM 5-HT induce an intracellular acidication. This acidication is Na+-dependent, EIPA-sensitive and also Cl--dependent. No DIDS-sensitivity was observed. A coupled activity of a Na+/H+-antiporter and a Cl-/HCO3- -antiporter was suggested. • Using O2-sensitive fluorescent microbeads I could show that 5-HT stimulation of the Calliphora salivary glands activates cellular respiration. The cAMP and Ca2+-signalling pathways contribute in a complex manner to the 5-HT-induced activation of cellular respiration and consequently, also to the 5-HT-induced intracellular acidication. • The expression of a Na+ dependent glutamate-transporter, a Na+/H+-antiporter, a carbonic anhydrase, subunit C of the V-ATPase and a nH+/K+-antiporter were determined on mRNA level by RT-PCR. This thesis contributes signicantly to the understanding of pHi regulation in unstimulated and stimulated salivary glands of Calliphora vicina. Mechanisms which contribute to the maintenance and recovery of resting pHi were identied by using pHi measurements and molecular biological techniques. Mechanisms which are responsible for the 5-HT-induced intracellular acidication were also clarified. Furthermore a new optical method for measuring O2 consumption in animals cells was established by using the Calliphora salivary glands as a model.