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Thesis ( MPH.) -- University of Limpopo, 2022 / Background Multidrug-resistant tuberculosis (MDR-TB) has recently resulted to be in an emergence state globally and this of constitute a big challenge for TB control and the goals of the World Health Organization’s End TB Strategy. Aminoglycosides (AG) were often used as part of treatment of life-threatening illnesses such as MDR-TB for decades, however their adverse effects are widely described and hearing loss is one of the major side effects. The risk factors for hearing loss in patients treated with AG include the dose and duration of AG, infection with human immunodeficiency virus (HIV), older age and persons exposed to a high level of noise while the damage can be total and permanent. Severe hearing impairment has been reported to occur among patients treated for MDR-TB with injectable drugs, especially among the elderly and patients infected with human immunodeficiency virus, however, Bedaquiline containing regimens have demonstrated improved outcomes over injectable containing regimens in the long-term treatment of MDR-TB. Methods The objective of the current study was to investigate the burden of hearing loss amongst MDR-TB patients on bedaquiline at Zithulele Hospital in Eastern Cape Province. Therefore, the current study followed a quantitative retrospective approach using simple random sampling to select MDR-TB patients treated with bedaquiline and having a baseline audiogram be the initiation of treatment. The data was captured in a Microsoft Excel spreadsheet and then transferred to Statistical Package for Social Sciences (SPSS) Version 20 for data analysis in which categorical variables were presented as percentages and frequencies, while continuous variables was presented as mean, median and standard deviation lastly, comparison of categorical variables was done using a Chi-Squared test, whereas continuous variables were compared using a t-test. P-value of <0.05 will be considered significant. Results The mean age for the participants was 39.2 years with standard deviation of 11.8 and there was no statistical significance difference between the age groups (p value = 0.178). There no was a statistical significance difference between the employment status (p value = 0.794), previous use of injectables (p value = 0.360) and type of hearing of loss (p value = 0.536). Majority of the MDR-TB patients on bedaquiline did not have hearing loss at 67% while those who had gradual hearing loss and sudden hearing loss were 26.8% and 6.2% respectively. There was no statistical significance difference between males and females in both the right and left ears, however, the right ear results appeared to be slightly worse than the left ear results. It was found that both males and females had a high frequency hearing loss in the left ears of 26.8% and 22.2% respectively as compared to the right ears with of 25.9% and 1.6% respectively. The was a statistical significance difference between the age groups in both ears for hearing loss at p-value <0.001. The overall prevalence of hearing loss was found to be 32.9% and hearing loss at 20dB or more loss at any frequency was low at 11.9% while hearing loss at 10B or more loss at any frequency was the highest at 32.9% followed by loss response at 3 consecutive frequencies at 26.2%. Hearing loss was increasing with increasing age from 8.3% in age group and age was significantly associated with hearing loss as older patients were 2.2 times more likely to have a hearing loss at a degree of 20dB and 4.4 times more likely to have a hearing loss at a degree of 10dB. Previous use of injectables was also significantly associated with hearing loss as patients who used injectables previously were 11.5 times more likely to have a hearing loss at degree of 10dB, 5.6 and 11.3 times more likely to have a hearing loss at loss response at 3 consecutive frequencies and overall hearing loss respectively. Conclusion South Africa has a high burden of drug-resistant tuberculosis (DRTB) and until recently, ototoxic aminoglycosides were predominant in treatment regimens. Drug resistant TB treatment with bedaquilines caused clinically and statistically significant deterioration of hearing loss in patients, most prominently at high frequencies. Although public health interventions to prevent hearing loss have been deemed cost effective and have meaningful individual and economic implications, hearing loss and its prevention consistently receive inadequate attention as a global public health priority. Despite the serious impacts of hearing loss, little is known regarding prevalence of ototoxic hearing loss after treatment for DR-TB. Therefore, when the use of injectable ototoxic medications is unavoidable, audiological ototoxicity monitoring is essential to optimise hearing-related outcomes.

Les aminosides sont majoritairement utilisés pour le traitement d’infections causées par des bactéries Gram-négatif. En raison de leur index thérapeutique étroit, les aminosides doivent être administrés avec des doses adéquates afin d’optimiser la guérison clinique tout en minimisant les risques de toxicité. De plus, le suivi thérapeutique posologique est d’autant plus important pour les populations spéciales. En effet, ces dernières, telles que les patients aux soins intensifs, peuvent présenter des conditions physiopathologiques pouvant influencer la pharmacocinétique des aminosides. Ce projet, séparé en trois volets, a permis la description des habitudes de posologies et de suivi thérapeutique des aminosides auprès des patients aux soins intensifs du Québec à l’aide d’un questionnaire. De plus, ce projet inclut également une revue des modèles pharmacocinétiques par approche populationnelle (PopPK) des aminosides pour des patients aux soins intensifs. Finalement, ce projet consiste en l’évaluation de la performance prédictive des modèles de gentamicine avec une base de données-patients provenant de deux établissements de santé du Québec. Le volet 1, sous forme d’un questionnaire, a obtenu un taux de réponse de 64.7%, représentant 42% des lits aux soins intensifs de la province. Les régimes posologiques administrés de façon uniquotidienne, sont plus utilisés que l’administration multiquotidienne avec des doses allant de 5 à 7 mg/kg pour la gentamicine et la tobramycine. L’amikacine est très peu utilisé dans les établissements du Québec. Les cibles thérapeutiques respectaient généralement les cibles recommandées dans la littérature. Le volet 2 a permis la description de six, onze et cinq modèles PopPK d’amikacine, de gentamicine et de tobramycine respectivement. Les modèles à deux compartiments décriraient mieux la pharmacocinétique de l’amikacine et de la tobramcyine, tandis que les modèles à un compartiment décriraient mieux la pharmacocinétique de la gentamicine. Les covariables les plus souvent considérées comme significatives étaient la clairance rénale et le poids corporel. Dans le volet 3, malgré qu’une performance prédictive adéquate a été déterminée auprès des 4 modèles évaluées avec la base de données-patients du Québec, de la variabilité demeure présente concernant la prédiction des concentrations et l’application de ces modèles dans un contexte doit ainsi se faire avec prudence. À partir du meilleur modèle, des régimes posologiques a priori ont pu être simulés. / Aminoglycosides are mostly used for treatment of severe Gram-negative infections. Due to their narrow therapeutic index, aminoglycosides should be administered following adequate dosing regimens in order to optimize clinical efficacy while minimizing the risks of toxicity. Moreover, therapeutic drug monitoring is even more important for frail populations such as the critically ill patients. In fact, the latter often present pathophysiological changes that may influence aminoglycosides’ pharmacokinetics. This project was divided in three parts. Firstly, a survey was developed to describe the usual dosing and monitoring practices of aminoglycosides in critically ill patients in the province of Quebec. This project also includes a literature review of aminoglycosides population pharmacokinetic (PopPK) models in critically ill patients. Finally, this project also consists of evaluating the predictive performance of gentamicin PopPK models with a validation dataset composed of patients from two Quebec institutions. The survey had a response rate of 64.7%, therefore representing 42% of all intensive care unit beds in the province. Once-daily-dose regimens are more used than multiple-daily-dose regimens. Most common gentamicin and tobramycin administered dose regimens ranged from 5 to 7 mg/kg. Amikacin is rarely used in Quebec’s institutions. Therapeutic targets were generally in-line with findings from the literature. The literature review described six, eleven and five amikacin, gentamicin and tobramycin PopPK models, respectively. Amikacin and tobramycin pharmacokinetics were mostly described by bi-compartment models whereas gentamicin pharmacokinetics were mostly described by single-compartment model. Most common covariates used were renal clearance and bodyweight. In the third part of this project, although an adequate predictive performance was determined in all four evaluated models, variability in the predicted concentrations by the model still remains. Therefore, usage of these models in clinical settings should be done cautiously. Based on the best performing model, a priori dosing regimens were simulated.

Thesis (MScMedSc (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: South Africa is currently facing a major multidrug-resistant tuberculosis (MDR-TB) epidemic and has one of the highest incidences in the world. Aminoglycoside antibiotics are commonly used in this country as a treatment against MDR-TB. A well known side-effect of aminoglycosides is permanent hearing loss and this is thought to have a significant genetic component. To date, at least six mutations in the mitochondrial genome are known to confer susceptibility to aminoglycosideinduced hearing loss. It is imperative that we investigate the frequency of these mutations in our populations and determine whether certain sub-groups are at increased risk. The aim of the present study was therefore to investigate the genetic aetiology of aminoglycoside-induced hearing loss in the South African population. A multiplex method using the ABI Prism® SNaPshotTM Multiplex system was optimised to screen for six mutations in the MT-RNR1: A1555G, C1494T, T1095C, 961delT+C(n), A827G and T1291C. A total of 115 MDR-TB patients from the Brooklyn Chest Hospital in Cape Town who were receiving high doses of either streptomycin, kanamycin or capreomycin were recruited for this study. Furthermore, 439 control samples, comprising of 93 Afrikaner, 104 Caucasian, 112 Black and 130 Mixed Ancestry individuals were recruited and screened for the presence of the six mutations. Identification of novel variants in the MT-RNR1 and the entire mitochondrial genome was performed using High Resolution Melt analysis (HRM) and whole mitochondrial DNA sequencing, respectively. A total of 97 family members from a South African family known to harbour the A1555G mutation were recruited and genotyped using SNaPshot analysis. In addition, mitochondrial functioning in the presence of different streptomycin drug concentrations, in transformed lymphoblasts of an individual harbouring the A1555G, was assessed by means of the MTT colorimetric assay. Detection of heteroplasmic mutations was performed using PCRRestriction Fragment Length Polymorphism (RFLP) analysis and UN-SCAN-IT software. We successfully developed a robust and cost-effective method that detects the presence of all six mutations simultaneously. The method worked equally well on both blood (from adults) and buccal swabs (from children). The C1494T, T1095C and T1291C mutations were not detected in any of the MDR-TB or control groups. Alarmingly, the A1555G mutation was detected in 0.9% of the Black control samples and in 1.1% of the Afrikaner controls (in one sample in the heteroplasmic state 25%). The A827G mutation was present at a frequency of 0.9% in the MDR-TB patients and in 1.1% of the Afrikaner controls. The 961delT + insC(n) mutation was found in relatively high frequencies in both the MDR-TB patients (3.5%) and control groups (1.1% of the Afrikaner, 1.5% of the Mixed Ancestry and 7.1% of the Black samples). Similarly, the T961G mutation was III detected at high frequencies in the Caucasian (2.9%) and Afrikaner (3.2%) controls. Screening for novel variants in MT-RNR1 in MDR-TB patients experiencing ototoxicity revealed two novel variants (G719A and T1040C). However, G719A and T1040C are not likely to be pathogenic since they were detected in ethnic-matched controls: Mixed Ancestry (20.7%) and Black (1.8%) controls. Furthermore, a total of 50 novel variants were identified within the mitochondrial genome of eight MDR-TB patients with ototoxicity. Only five of the 50 variants (one in the MT-TH, ND3, COX3 and two in the CYTB gene) were shown to reside at positions that are evolutionarily conserved across five species from human to frog, and the four variants in the protein coding genes resulted in missense changes. A total of 76 of the 97 family members recruited were found to be A1555Gpositive (on mitochondrial haplogroup L0d) and are therefore at risk of developing irreversible hearing loss. Genes and variants known to act as genetic modifiers: tRNASer(UCN), homozygous A10S in TRMU and 35delG in GJB2 were not present in this family. For the MTT assay, decreased mitochondrial functioning of cells harbouring the A1555G mutation in the presence of streptomycin were (compared to wild type) observed but this was not statistically significant (p-value: 0.615- 0.999). The high frequency of the A1555G mutation (0.9%) in the Black population in South Africa is of concern given the high incidence of MDR-TB in this particular ethnic group. However, future studies with larger numbers of samples are warranted to determine the true frequencies of the aminoglycoside deafness mutations in the general South African population. Our data suggests that the 961delT + insC(n) and T961G variants are common non-pathogenic polymorphisms due to the high frequencies observed in controls (>1%). The identification of the first novel variants within protein coding genes that could possibly be associated with aminoglycoside-induced hearing loss holds great possibilities with regards to the identification of a second gene involved in drug induced hearing loss. Future studies where the possible effect of these variants on the normal functioning of these genes could be assessed would contribute greatly to this field of research. All 76 A1555Gpositive members of the family were given genetic reports and counseled about their risk and that of their children for developing hearing loss due to aminoglycoside use. The development of a rapid and cost-effective genetic method facilitates the identification of individuals at high risk of developing hearing loss prior to the start of aminoglycoside therapy. This is of critical important in a low-resource country like South Africa where, despite their adverse sideeffects, aminoglycosides will be continue to be used routinely and are accompanied with very limited or no audiological monitoring. Future studies and greater public awareness is therefore needed to address this serious problem. / AFRIKAANSE OPSOMMING: Suid Afrika beleef tans „n grootskaalse tuberculose epidemie (veral weerstandige vorme van tuberculose) (MDR-TB), met een van die hoogste voorkomssyfers in die wêreld. Aminoglikosied antibiotikums word baie algemeen gebruik in Suid Afrika vir die behandeling van MDR-TB. ‟n Bekende newe effek van die middels is permanente gehoor verlies en dit is van mening dat dit gekoppel is aan „n genetiese component. Daar is tans ses mutasies in die mitochondriale genoom wat vatbaarheid tot aminoglikosied-geinduseerde gehoor verlies veroorsaak. Daarom is dit van uiterse belang dat die frekwensie van die mutasies in ons populasies bepaal word sodat daar vasgestel kan word watter groepe „n hoë risiko het om gehoor verlies te kan ontwikkel. Die ABI Prism® SNaPshotTM Multipleks sisteem is gebruik en geoptimiseer om te toets vir die ses mutasies in die MT-RNR1: C1494T, T1095C, 961delT+C(n), A827G and T1291C. „n Totaal van 115 MDR-TB pasiente van die Brooklyn Chest Hospital in Kaap Stad is gewerf vir die studie. Hierdie pasiente ontvang daaglikse hoë dosese van een van die volgende aminoglikosiede: streptomycin, kanamycin of capreomycin. Verder is „n totaal van 439 kontrole DNA monsters gewerf vanuit die volgende etniese groepe: 93 Afrikaner, 104 Blank, 112 Swart and 130 Kleurling. Hierdie monsters is ook getoets vir die ses mutatsies. Hoë Resolusie Smelt analise (HRS) is gebruik om nuwe DNS volgorde veranderinge in die MT-RNR geen te identifiseer. Die hele mitochondriale genoom is blootgestel aan DNA volgorde bepaling in „n poging om nuwe DNS volgorde verandering in die genoom te identifiseer wat moontlik betrokke kan wees by aminoglikosied-geinduseerde gehoor verlies. „n Total van 97 lede van „n Suid Afrikaanse familie waar die A1555G mutasie teenwoordig is, is deur middle van die SNaPshot metode gegenotipeer. Verder is die normale funcitoneering van die mitochondrion in getransformeerde witbloed selle, getoets in die teenwoordigheid van verskillende konsentrasies streptomycin met behulp van die MTT kleurmetrie toets. Deteksie van heteroplasmiese mutasies is gedoen deur middle van die PCR-RFLP tegniek en alle analises is gedoen op die UN-SCAN-IT program. Ons was suksesvol in die ontwikkeling van „n vinnige, koste effektiewe en kragtige tegniek wat al ses die mutasies in MT-RNR1 in een reaksie kan optel. Hierdie tegniek het goed gewerk met DNA monsters van bloed en van selle verkry vanuit die wangholte (geneem van kinders jonger as 12 jaar). Die C1494T, T1095C en T1291C mutasies is glad nie waargeneem in enige van ons MDR-TB patiente of kontroles nie. Skrikwekkend is die hoë frekwensie (0.9%) waarby die A1555G mutasie in die Swart kontrole groep waargeneem is. Hierdie mutasie is ook in 1.1% van die Afrikaner kontrole groep opgemerk in heteroplasmie van 25%. Die A827G mutasie was teenwoordig in 0.9% en 1.1% van die MDR-TB patiente en Afrikaner kontrole monsters, onerskeidelik. Die 961delT + insC(n) mutasie is opgemerk in baie hoë frekwensies in beide die MDR-TB (3.5%) en kontrole groepe (1.1% van die Afrikaner, 1.5% van die Kleurling en 7.1% van die Swart monsters). Die T961G mutasie is ook in hoë frekwensies in slegs die Blanke (2.9%) en die Afrikaner (3.2%) kontrole groepe waargeneem. Nuwe DNS volgorde veranderinge in MT-RNR1 is gesoek in „n groep MDR-TB patiente wat gehoor verlies ondervind. Slegs twee nuwe verandering is ontdek (G719A en T1040C). Dit is onwaarskynlik dat hierdie veranderinge patogenies is siende dat hulle teen frekwensies van 20.7% en 1.8% waargeneem is in die Kleurling en Swart kontrole groepe onderskeidelik. Tydens die soeke na nuwe DNS volgorde veranderinge wat moontlik geassosieer is met aminoglikosied-geinduseerde gehoor verlies in die mitochondriale genoom is 50 onbekende veranderinge ontdek (een in die MT-TH, ND3, COX3 en twee in die CYTB gene). Die veranderinge is verder ondersoek vir evolusionêre konservasie op beide die nukliotied en amino suur vlak van mens to padda. Dit is bevind dat 76 uit die 97 familie lede positief is vir die A1555G mutasie en het dus „n hoë risiko om aminoglikosied-geinduseerde gehoor verlies te ontwikkel as hul bloot gestel word aan hierdie antibiotikums. Verder is gevind dat hierdie familie op die L0d mitochondriale haplogroep lê. Geen van die sogenaamde genetiese modifiseerde gene of DNS volgorde veranderinge in hierdie gene (tRNASer(UCN), A10S in TRMU in homosigotiese vorm en die 35delG in GJB2) is gevind in die familie nie. Die MTT toets het „n afname in die mitochondriale funksioneering van selle waar die A1555G mutasie teenwoordig was getoon, alhoewel die verskil tussen selle wat nie die A1555G mutasie het nie, nie statisties betekenisvol was nie (p-waarde: 0.615-0.999). Die hoë frekwensie van die A1555G mutasie (0.9%) in die Swart populasie van Suid Afrika is skrikwekkend siende dat die voorkomssyfer van MDR-TB in hierdie groep baie hoog is. Toekomstige studies met grooter getalle is nodig om die ware frekwensie van die mutasies geassosieer met aminoglikosied-geinduseerde gehoor verlies in die algemende Suid Afrikaanse populasie te bepaal. Ons data dui aan dat die 961delT + insC(n) en die T961G mutasies slegs algemene nie-patogeniese polimorphismis is siende dat dit in sulke hoë frekwensies (>1%) in kontroles opgemerk is. Die identifiseering van die eerste DNS volgorde veranderinge in proteïen kodeerende gene wat moontlik geassosieer is met aminoglikosied-geinduseerde gehoor verlies hou groot en belowende moontlikehede in, interme van die identifiseering van „n tweede geen. Toekomstige studies waarin die effek van hierdie veranderinge op die normale funktioneering van hierdie gene ondersoek word sal „n besondere groot bydrae lewer tot hierdie veld van navorsing. Al 76 van die A1555G positiewe familie lede is voorsien van genetiese verslae en het berading ontvang in verband met hul risiko en die risiko van hul kinders om aminoglikosied-geinduseerde gehoor verlies te ontwikkel. Die ontwikkeling van „n kragtige, vinnige en koste-effektiewe genetiese metode vergemaklik die vinnige identifiseering van hoë risiko individue vir die ontwikkeling van gehoor verlies voordat hulle met hul aminoglikosiede behandeling begin. Dit is veral noodsaaklik in „n derde wêreld land soos Suid Afrika waar, ten spyte van hul gevaarlike newe effekte, aminoglikosied antibiotikums steeds gebruik sal word. Daarom is grooter publieke bewusmaking nodig om hierdie problem te probeer oplos en te verhoed.

[pt] Neste trabalho, métodos baseados em diferentes abordagens em eletroforese capilar (CE) foram propostos. No caso da determinação de compostos organoestanhos ou OTs (difenilestanho e monofenilestanho) em fluidos biológicos, foi usada abordagem de eletroforese capilar por zona (CZE) hifenada com a espectrometria de massas (do tipo quadrupolo) com fonte de plasma indutivamente acoplado (CE-ICP-MS). As condições de análise foram estudadas no modo univariado visando otimizar a composição da solução eletrolítica (tampão acetato 5,0 mmol L(-1), pH 2,8) e obter os parâmetros instrumentais (45 graus Celsius, mais 30 kV e 30 s de tempo de introdução hidrodinâmica de amostra). A solução de complementação foi uma solução aquosa 5,0 mmol L(-1) de NH4NO3 contendo 10 por cento metanol em volume e 1,0 Mg L(-1) de Cspositivo, com pH ajustado para 2,8 com tampão acetato. A vazão dessa solução foi mantida em 40 ML min(-1). Os OTs foram diluídos em solução de metanol:tampão acetato de sódio 50:50 por cento v/v ou apenas em tampão acetato de sódio pH 2,8. As condições de detecção do ICP-MS foram ajustadas em 1200 W, 15 L min(-1) de vazão de argônio para formação do plasma, 1 L min(-1) de vazão de argônio auxiliar. A vazão de argônio do nebulizador foi ajustada diariamente. Os isótopos de estanho 120Sn e 118Sn foram monitorados, assim como o 133Cspositivo para controlar a eficiência e estabilidade do processo de nebulização. A resposta linear do método ficou entre 0,050 a 2,0 mg L(-1) de Sn (0,42 a 17 Mmol L-(1)). Os limites de detecção (LOD) e de quantificação (LOQ) em termos de Sn foram de 15 Mg L(-1) (0,13 Mmol L(-1)) e 50 Mg L-1 (0,42 Mmol L(-1)), calculados utilizando a menor concentração dos picos dos analitos que podem ser diferenciados do sinal de fundo. A repetibilidade para o tempo de migração e área dos picos ficou próximo a 5 por cento. O método foi aplicado na análise de urina, sangue total e plasma fortificados com os OTs. Recuperações entre 75 e 95 por cento foram obtidas. No caso da determinação de sete pesticidas da classe das estrobilurinas (azoxistrobina, 9 dimoxistrobina, fluoxastrobina, picoxistrobina, piraclostrobina, trifloxistrobina e kresoxim-metil) em sopas infantis, foi usada a cromatografia eletrocinética capilar micelar (MEKC) com detecção fotométrica (no UV) com capilar de caminho óptico estendido. Um estudo multivariado, usando um planejamento 33 Box Behnken, indicou que a melhor separação para os pesticidas foi com solução aquosa de eletrólito composto por tampão tetraborato de sódio (5,1 mmol L(-1), pH 9,0) contendo 51 mmol L(-1) de dodecil sulfato de sódio (SDS) e 24 por cento acetonitrila (ACN) em volume. As condições instrumentais foram 25 C e mais 30 kV de diferença de potencial aplicada, 45 s de tempo de introdução hidrodinâmica de amostra e detecção em 210 nm. Para aumentar o poder de detecção, foi usada a concentração dos analitos no capilar. Para tal, as soluções de padrões e amostras foram dissolvidas em solução tampão tetraborato de sódio 45 mmol L(-1): acetonitrila 80:20 por cento v/v. As curvas analíticas apresentaram comportamento linear e os valores de LOD ficaram entre 7,0 Mg L(-1) ou 18 nmol L(-1) (piraclostrobina) a 15 Mg L-1 ou 33 nmol L(-1) (fluoxastrobina). Os valores de LOQ ficaram entre 21 Mg L(-1) ou 54 nmol L(-1) (piraclostrobina) a 45 Mg L(-1) ou 98 nmol L(-1) (fluoxastrobina). A repetibilidade ficou entre 1,7 a 7,9 por cento para a área de pico e entre 0,25 a 0,71 por cento para o tempo de migração. A precisão intermediária, avaliada com análises realizadas em diferentes dias, apresentou valores entre 1,3 a 5,3 por cento para a área de pico e entre 0,06 a 0,90 por cento para o tempo de migração. O método foi aplicado na análise de sopas prontas infantis fortificadas com as estrobilurinas. Os pesticidas foram extraídos aplicando o método QuEChERS com ajuste de pH com tampão acetato e limpeza com extração em fase sólida dispersiva. Os resultados das análises obtidos com um método cromatográfico adaptado da literatura foram estatisticamente iguais aos alcançados com o método proposto. A CZE foi o modo de separação escolhido para mostrar o potencial da determinação indireta de aminoglicosídeos com medição de fluorescência de pontos quânticos (excitação com laser de diodo em 410 nm) amplificada na presença dos analitos. A fotoluminescência dos pontos quânticos (nanopartículas de CdTe modificados com ácido tioglicólico monodispersas em solução) foi mais intensa em solução tampão (pH 8,0) contendo entre 5 e 10 mmol L(-1) de tetraborato de sódio. A interação no capilar entre aminoglicosídeos (neomicina e canamicina) e os pontos quânticos provocou aumento de fotoluminescência dependente do pH do meio (indício de interação de natureza 10 eletrostática). Alguns parâmetros de mérito foram avaliados com uma faixa linear curta (0,1 a 1,0 mol L(-1) para canamicina e 0,03 a 0,5 mol L(-1) para neomicina). Os valores mínimos detectados de 0,1 Mmol L(-1) ou 58 Mg L(-1) (canamicina) e 0,03 Mmol L(-1) ou 27 Mg L(-1) (neomicina) mostram que essa é uma abordagem interessante para a determinação sensível de aminoglicosídeos. / [en] In this work, analytical methods based on different approaches using capillary electrophoresis (CE) have been proposed. For the determination of organotins or OTs (diphenyltin and monophenyltin) in biological fluids, the separation using capillary zone electrophoresis (CZE) was applied using tandem with inductively coupled plasma mass spectrometry (CE-ICP-MS). The conditions for the analysis were optimized in an unvaried way aiming to find the conditions for the electrolyte solution (acetate buffer, 5.0 mmol L (-1), pH 2.8) and the employed instrumental parameters (45C, 30 kV and 30 s of the time for hydrodynamic introduction of the sample). A complementary solution was composed by NH4NO3 5.0 mmol L(-1), 10 por cento v/v of methanol and 1.0 g L(-1) of Cspositive in acetate buffer with pH adjusted to 2.8. The flow of this solution was set to 40 ML min(-1). The OTs were diluted either in a methanol: acetate buffer 50:50 por cento v/v solution or only in sodium acetate buffer pH 2.8. The conditions for detection by ICP-MS were set to 1200 W, 15 L min(-1) for the Ar plasma flow and 1,0 L min(-1) for the auxiliary Ar. The nebulizer Ar flow was adjusted daily. The monitored tin isotopes were 120Sn 118Sn. The isotope 133Cs was also monitored in order to control the efficiency and stability of the nebulization. The method presented a linear response between 0.05 and 2.0 mg L(-1) (0.42 a 17 Mmol L(-1)) for Sn. The value for the limits of detection (LOD) and for the limits of quantification (LOQ) for Sn were 15 Mg L(-1) (0.13 Mmol L-1) e 50 Mg L(-1) (0.42 Mmol L(-1)), calculated based on the lowest concentration of the analyte peaks that can be differentiated from the background signal. The repeatability for migration time and peak area was approximately 5 per cent. The method was applied in the analysis of organotin fortified blood and urine samples with recoveries between 75 and 95 per cent. In the case of the determination of seven strobilurin class pesticides (azoxystrobin, dimoxystrobin, fluoxastrobin, picoxystrobin, pyraclostrobin, trifloxystrobin and kresoxim-methyl) in baby food (vegetable and fruit soups), 12 the micellar electrokinetic capillary chromatography (MEKC) was used using photometric detection (UV) in a capillary with extended optical path. A multivariate study, with 33 Box Behnken design, indicated the best composition for the electrolytic solution to separate the seven pesticides: a sodium tetraborate buffer (5.1 mmol L(-1), pH 9.0) solution containing 51 mmol L(-1) sodium dodecyl sulfate and acetonitrile (24 por cento in volume). The instrumental conditions were 25C, 30 kV of applied voltage, 45 s for hydrodynamic introduction of the sample and detection at 210 nm. To increase the detection power, the concentration of the analytes into the capillary was used by using the Normal Stacking Mode. For this purpose, the solutions of standards and samples were prepared in 45 mmol L(-1) sodium tetraborate buffer solution: acetonitrile 80:20 por cento v/v. The analytical curves presented a linear behavior and the LOD values were between 7.0 Mg L (-1) or 18 nmol L(-1) (pyraclostrobin) to 15 Mg L(-1) or 33 nmol L(-1) (fluoxastrobin). The LOQ values were between 21 Mg L(-1) or 54 nmol L(-1) (pyraclostrobin) a 45 Mg L(-1) ou 98 nmol L(-1) (fluoxastrobin). The repeatability was between 1.7 to 7.9 por cento for the peak area and between 0.25 to 0.71 por cento for the migration time. The intermediate precision, evaluated by the analysis performed in different days were between 1.3 to 5.3 por cento for the peak area and between 0.06 and 0.90 per cent for the migration time. The method was applied in the analysis of baby food spiked with strobilurin. Pesticides were extracted using the QuEChERS method with pH adjustment with acetate buffer and clean-up using the dispersive solid phase extraction. The analysis results were statistically identical to those obtained with a chromatographic method adapted from the literature. The CZE separation mode was chosen to evaluate the potential of the indirect determination of aminoglycosides through the amplified photoluminescence from quantum dots (excitation laser diode 410 nm) in the presence of the analytes. The photoluminescence from quantum dots (monodispersed CdTe nanoparticles modified with thioglycolic acid) was more intense in buffer solution (pH 8.0) containing between 5 and 10 mmol L(-1) sodium tetraborate. The interaction between aminoglycosides (kanamycin and neomycin) and quantum dots inside the capillary caused the increasing of fluorescence in a pH-dependent way (indicating the electrostatic nature for the interaction). A few figures of merit were evaluated with a short linear range (0.1) to 1.0 Mmol L(-1) for kanamycin and 0.03 to 0.5 Mmol L(-1) for neomycin). The 13 minimum values detected were 0.1 nmol L(-1) or 58 Mg L(-1) (kanamycin) and 0.03 nmol L(-1) or 27 Mg L(-1) (neomycin) showing that the proposed approach can be used to detect aminoglycosides in a relatively sensitive way.

De acordo com a Organização Mundial de Saúde (World Health Organization - WHO), aproximadamente 40 milhões de pessoas ao redor do mundo estão infectadas com HIV/AIDS. Atualmente, a epidemia tem sido controlada em grande parte do mundo ocidental, porém, projeções sugerem que, até o fim desta década, o número de incidência da doença poderá duplicar. Apesar das significantes melhoras na morbidade e mortalidade de pacientes infectados pelo HIV, o rápido surgimento de cepas resistentes aos agentes anti-HIV, além dos efeitos adversos e o alto custo de fármacos de última geração, torna-se necessário o continuo desenvolvimento de novas classes de agentes anti-HIV. A transcrição e multiplicação do RNA viral são dependentes das interações seqüência-específica entre duas proteínas reguladoras virais essenciais, Tat e Rev, com seus respectivos sítios no RNA, TAR e RRE. Durante a última década, os aminoglicosídeos foram introduzidos como ligantes universais do RNA, sendo capazes de se ligar ao TAR e ao RRE. A literatura apresenta diversos aminoglicosídeos que são capazes de se ligar ao TAR e inibir a interação Tat-TAR bem como, inibir competitivamente a ligação da proteína Rev ao RRE, como, por exemplo, a neomicina e tobramicina. Considerando a importância dos aminoglicosídeos e análogos nucleosídicos, conhecidamente eficazes na terapia antirretroviral, o trabalho foi direcionado para a síntese de conjugados de aminociclitol, 2- desoxi-estreptramina, e adenosina, bem como, dímeros de adenosina via estratégia de click chemistry por reação de cicloadição azido-alcino catalisada por Cu(I) (CuAAC). Para a síntese destes produtos, o precursor adenosina foi convertido no derivado 5\'-azido-5\'- desoxi-adenosina, o qual foi condensado com diversos diinos terminais comerciais, contendo diferentes grupos espaçantes, com a finalidade de explorar suas influências nas propriedades eletrônicas e estéricas nos conjugados de interesse frente à atividade anti-HIV. Os derivados alcinos presentes na posição C-5\' de adenosina, via grupo triazol, foram empregados para a síntese dos monômeros nucleosídeo-aminociclitóis, assim como, na síntese de dímeros nucleosíde0-aminociclitóis, via reação de cicloadição 1,3-dipolar, na presença de CuSO4, quantidade catalítica, e ascorbato de sódio, para geração in situ de Cu(I). Adicionalmente, alguns dos compostos, na concentração de 1mM, foram testados empregando o ensaio de ELISA para detecção da presença de proteína viral p24 em linhagem células H9 e avaliação de sua atividade antirretroviral. De acordo com o ensaio biológico, um dos compostos preparados apresentou, proporcionalmente ao crescimento da linhagem H9 (HIV) controle, atividade de inibição de formação da proteína viral p24 similar ao composto padrão zidovudina (AZT). Além disso, outros dois compostos também apresentaram um resultado relevante uma vez que suas atividades foram similares ao composto padrão lamivudina (3TC). Estes resultados sugerem que a presença de uma cadeia metilênica mais extensa, como cadeia lateral ou grupo espaçante, pode influenciar positivamente a atividade biológica por efeito hidrofóbico ou estérico. Por fim, os ensaios de viabilidade celular demonstraram que os compostos testados não foram citotóxicos nas condições testadas. / According to World Health Organization - WHO about 40 million of people are infected with HIV/AIDS. Currently, the epidemic has been controlled largely in the western world, since, projections suggest that, until this decade end, the disease incidence could increase. Despite significant improvements in morbidity and mortality of HIV-patients, quick emergence of resistant strains to anti-HIV agents, in addition to adverse effects and high cost of recent drugs, becomes necessary the ongoing development of new classes of HIV agents. Transcription and translation of the viral RNA are dependent of sequence-specific interactions among two essential viral regulatory proteins, Tat and Rev, and their corresponding TAR and RRE sites in HIV-1 RNA. Over the past decade, aminoglycosides were established as universal RNA linkers, being able to link to TAR and RRE. The literature reports several aminoglycosides that bind to TAR and inhibit Tat-TAR interaction, as well as, competitively inhibit the bind between Rev protein and RRE, such as: neomycin and tobramycin. Considering the importance of nucleosides analogs, effective in antiretroviral therapy, and aminoglycosides, the work was driven to the synthesis of aminocyclitol, 2- deoxy-streptamine, conjugated to the adenosine, as well as, conjugated dimers of adenosine by molecular duplication via click chemistry strategy involving copper-catalysed azide-alkyne cycloaddition reaction (CuAAC). For the synthesis of these products, the starting material adenosine was converted to 5\'-azide-5\'-deoxy-adenosine, which was conjugated with several commercials terminal dialkynes containing different intercalating groups in order to explore the influences of the steric and electronic properties of conjugates towards anti-HIV activity. Alkynes derivated at C-5\' position of adenosine, via triazole group, were used in the synthesis of nucleoside-linked aminocyclitols, as well as, nucleoside conjugated dimers by 1,3-dipolar cycloaddition reaction under microwave-assisted conditions (MW), using the catalytic system CuSO4/sodium ascorbate for the in situ generation of Cu(I). Additionally, several compounds, at concentration of 1mM, were tested in vitro by ELISA for detection of p24 protein in H9 cells to antiretroviral evaluation. According with the biologic assay, one of the compounds showed inhibition of p24 protein production similar to zidovudine (AZT), when compared to H9 cells line growth control, Furthermore, two compounds also showed important activities similar to lamivudine (3TC). These results suggest that the presence of a longer methylenic chain, as side chain or intercalating groups, could influence positively in the biologic activity due to hydrophobic or steric effects. Ultimately, the cell viability assays showed that compounds were not cytotoxic in the tested conditions.

Das Ziel dieser Arbeit war die Synthese niedermolekularer Verbindungen, die an die prä-miRNA binden und dadurch die Reifung zur miRNA inhibieren. Daher sollten die RNA-Binder 2-Desoxystreptamin sowie Neamin mit Alkinen funktionalisiert und durch Kupfer-katalysierte Azid-Alkin 1,3-dipolare Cycloaddition (CuAAC) mit verschiedenen bivalenten Aziden verknüpft werden. Im Rahmen dieses Projekts wurde der synthetische Zugang zu den benötigten Alkin- sowie Azid-funktionalisierten Grundbausteinen optimiert. Ferner wurde ein effektives und zuverlässiges Protokoll für die CuAAC erarbeitet, welches es ermöglichte, 88 Testsubstanzen in guter Ausbeute und hoher Reinheit zu isolieren. Anschließend wurde die Substanzbibliothek in einem BRCA-Reifungsassay unter kompetitiven Bedingungen auf die Inhibition der miRNA-Reifung getestet. Dabei wurden mehrere potente Inhibitoren der miRNA-Reifung mit IC50-Werten von bis zu 0.5 µM identifiziert. Der zweite Schwerpunkt dieser Arbeit lag auf der Etablierung einer chemo-enzymatische RNA-Funktionalisierungsstrategie um prä-miRNA-Sonden herzustellen: In-vitro-Transkriptionen mit der T7-RNA-Polymerase sowie Ligationen mit der T4 RNA Ligase 1 waren das Fundament der enzymatischen RNA-Synthese, während die Funktionalisierung der RNA durch CuAAC erzielt wurde. Dafür wurden die neuen Verbindungen O-(5‘-Guanosin)-O-propargylmonophosphat sowie 3‘,5‘-O,O-Bisphosphat-5-ethinyluridin synthetisiert, durch in-vitro-Tran¬skription an das 5‘-Ende von prä-miRNAs eingeführt und anschließend durch CuAAC mit einem Fluoreszenzlöscher modifiziert. Das Uridinbisphosphat wurde durch CuAAC mit einem Fluorophor markiert und anschließend effizient mit der T4 RNA Ligase 1 an das 3‘-Ende verschiedener prä-miRNAs ligiert. Darüber hinaus war es auch möglich, das Alkin-modifizierte Uridinbisphosphat an das 3‘-Ende von prä-miRNAs zu ligieren, dieses durch eine zweite Ligation an eine definierte interne Position zu verschieben und abschließend durch CuAAC zu funktionalisieren. / The objective of this work was the synthesis of small molecules, which bind to pre-miRNAs to prevent their maturation to fully active miRNAs. To create a substance library of bivalent inhibitors, the RNA binding motifs 2-deoxystreptamine as well as neamine were alkyne modified and linked with several bisazides via a copper catalyzed alkyne-azide cycloaddition (CuAAC). Hence, optimized syntheses of the basic building blocks along with an effective and reliable CuAAC-protocol were established. 88 test substances were isolated in good yield and high purity. Finally they were analyzed with regard to their potential to selectively inhibit the miRNA maturation. For this purpose, the assay was performed under competitive conditions with a set of two pre-miRNA-pairs. The initial screening revealed several inhibitors with IC50-values in the lower µM range. The second focus of this work was on the development of a synthetic access to alkyne modified ribonucleotides to establish a chemo-enzymatic functionalization strategy for RNAs using in-vitro-transcription, ligation and CuAAC. In this context, the syntheses of 3‘,5‘-O,O-bisphosphate-5-ethinyl uridine and O-(5‘-guanosine)-O-propargyl monophos-phate are described for the first time. The guanosine monophosphate was used as transcription starter to address the 5’-end of RNA and was consecutively labeled with an azido-tagged quencher. The uridine bisphosphate was conjugated with a fluorophor and introduced to the 3’-end of RNAs by T4 RNA ligase 1. Moreover, the uridine bisphosphate can be ligated to the 3’-end without a fluorophor attached, to serve as a connecting point for a further ligation with an oligonucleotide of any length. Thereby, the former terminal alkynylated uridine was shifted to a defined internal position by successive enzymatic reactions and was successfully derivatized with a fluorophor by CuAAC.

No description available.

Selenoproteine spielen eine wichtige Rolle in der antioxidativen Abwehr und bei Immunreaktionen. Der Selen(Se)metabolismus wird von Hepatozyten gesteuert, die das Se-Transportprotein Selenoprotein P (SEPP) synthetisieren und sezernieren. SEPP nimmt bei kritischen Erkrankungen, z. B. Sepsis ab und führt zu niedrigen Se-Spiegeln. Sepsis triggert die übermäßige Produktion von proinflammatorischen Zytokinen. Aminoglykosid-Antibiotika (AG), die oft bei schwerer Sepsis eingesetzt werden, induzieren Fehlinterpretationen der mRNA inklusive des Stoppcodons UGA welches für die Selenoprotein-Biosynthese notwendig ist. Es wurden daher die molekularen Wechselwirkungen zwischen den Zytokinen IL-6, IL-1b und TNFa, AG und dem Se-Status mit der Biosynthese in Leberzelllinien untersucht. IL-6 führte zu einer starken Reduktion der SEPP-mRNA und einer dosisabhängigen Reduktion von SEPP. Parallel dazu reduzierte IL-6 das Transkriptlevel, die Proteinexpression und die Enzymaktivität der Typ-I-Dejodase (DIO1). Auf die Expression der antioxidativ-wirkenden Glutathionperoxidasen (GPX) wirkte IL-6 isozymspezifisch; während die Transkriptkonzentrationen von GPX2 anstiegen und die von GPX4 abnahmen, blieb GPX1 unbeeinflusst. Die IL-6-abhängigen Effekte bestätigten sich auch in Reportergenassays von SEPP-, DIO1-, GPX2- und GPX4-Promotorkonstrukten. Um die Wirkungen von AG auf die Selenoprotein-Translation besser zu verstehen, wurden die SECIS-Elemente von GPX1-, GPX4- und SEPP-Transkripten in ein Reportersystem kloniert und auf eine Regulation durch AG und Se analysiert. Die Ergebnisse zeigen, dass der korrekte Se-Einbau vom Se-Status, von der AG-Konzentration und dem spezifischen SECIS-Element abhängig ist. Auf transkriptionaler und translationaler Ebene führten AG zu stark erhöhten SEPP-Spiegeln, während die Expression und Enzymaktivität von GPX und DIO1 nur in geringerem Ausmaß beeinflusst wurden. Eine Analyse der Se-Beladung zeigte, dass der Se-Gehalt von SEPP stark durch AG reduziert und vom Se-Status abhängig war. / Selenoproteins play important roles in antioxidant defence and immunoregulation. Selenium (Se) metabolism is controlled by hepatocytes synthesizing and secreting the Se-transporter selenoprotein P (SEPP) declining in critical illness, e.g., sepsis. Sepsis triggers excessive production of pro-inflammatory cytokines. Aminoglycoside (AG) antibiotics applied in sepsis in induce mRNA misinterpretation including the stop codon UGA required during selenoproteins biosynthesis. The molecular interplay between the cytokines IL-6, IL-1b and TNFa, AG and Se-status on selenoprotein expression was investigated in hepatic-derived cell lines. IL-6 strongly reduced the level of SEPP mRNA and secreted SEPP in a dose-dependent manner. Likewise, expression of selenoenzyme type 1 deiodinase (DIO1) declined at the transcript, protein and enzyme activity level. The effects of IL-6 on the expression of antioxidative-acting glutathione peroxidases (GPX) were isozyme-specific; while transcript level of GPX2 increased and those of GPX4 decreased, GPX1 remained unaffected. IL-6-dependent effects were reflected in reporter gene experiments of selenoprotein promoter constructs. Characterising the effects of AG on selenoprotein translation, the SECIS-elements of GPX1, GPX4 and SEPP transcripts were cloned into a reporter system and analysed for their response to AG and Se. The results indicate that the correct co-translational Se-insertion depends on the Se-status, AG concentration and the specific SECIS-element. At both transcriptional and translational levels, SEPP levels were strongly increased in response to AG, whereas the expression and enzyme activity of GPX and DIO1 were affected to a lower degree. Analysis Se-status indicate that the Se-content of SEPP was strongly reduced by AG and depends on Se-status.

Made available in DSpace on 2016-01-26T12:51:25Z (GMT). No. of bitstreams: 1 joaofernandopicollodeoliveira_dissert.pdf: 2216002 bytes, checksum: 172981ec9e4daa13d8e1e6872337867d (MD5) Previous issue date: 2008-12-10 / Nephrotoxicity is the main adverse effect of aminoglycoside use. There are few information about its prevalence and risk factor in intensive care unit patients. Objectives: To assess the prevalence, mortality and risk factors for aminoglycoside nephrotoxicity in ICU patients. Casuistic and Method: In order to assess the prevalence of, and risk factors for aminoglycoside nephrotoxicity in the ICU, 360 consecutive patients starting aminoglycoside therapy in the ICU with a baseline calculated GFR (cGFR) &#8805;30 ml/min/1.73 m2 were evaluated. Results: Of them, 209 (58%) developed aminoglycoside-induced nephrotoxicity (AKI, decrease in cGFR >20% from baseline), while 151 did not (non-AKI). Both groups had similar baseline cGFR. The AKI group developed a lower cGFR nadir (45 ± 27 vs. 79 ± 39 ml/min/1.73 m2, p<0.001), was older (56 ± 18 y vs. 52 ± 19 y, p=0.033), had a higher prevalence of diabetes (19.6% vs. 9.3%, p=0.007), used other nephrotoxic drugs (51% vs. 38%, p=0.024) and iodinated contrast more frequently (18% vs. 8%, p=0.0054), showed higher prevalence of hypotension (63% vs. 44%, p=0.0003), shock (56% vs. 31%, p<0.0001), and jaundice (19% vs. 8%, p=0.0036). Mortality was 44.5% in the AKI and 29.1% in the non-AKI groups (p=0.0031). A logistic regression model identified as significant (p<0.05) independent factors affecting aminoglycoside-induced nephrotoxicity baseline cGFR<60 ml/min/1.73 m2 (OR 0.42), diabetes (OR 2.13), simultaneous use of other nephrotoxins (OR 1.61) or iodinated contrast (OR 2.13), and hypotension (OR 1.83). Conclusion: The AKI was frequent among ICU patients using aminoglycoside, and it was associated with high mortality. The presence of diabetes, hypotension, simultaneous use of other nephrotoxic drugs, and iodinated contrast were independent risk factors for the development of aminoglycoside-induced nephrotoxicity. / Nefrotoxicidade é a principal complicação do uso de aminoglicosídeos. Existem poucas informações sobre a prevalência e os fatores de risco para nefrotoxicidade por aminoglicosídeos em paciente internados em unidades de terapia intensiva. Objetivos: Avaliar a prevalência, a mortalidade e os fatores de risco para nefrotoxicidade por aminoglicosídeos em pacientes internados em Unidade de Terapia Intensiva geral. Casuística e Métodos: Foram avaliados a prevalência, os fatores de risco e a mortalidade da nefrotoxicidade por aminoglicosídeo de 360 pacientes internados em terapia intensiva com filtração glomerular calculada por fórmula (MDRD, RFG) basal &#8805;30ml/min/ 1,73m2, que iniciaram o uso do antibiótico na terapia intensiva; nefrotoxicidade foi definida como queda >20% na RFG em relação ao RFG basal. Resultados: Entre os pacientes estudados 209 (58%) desenvolveram nefrotoxicidade (IRA) e 151 não alteraram a função renal (não IRA). Ambos os grupos (média±desvio padrão) tinham RFG basal similar (89&#61617;42 ml/min/ 1,73m2 no grupo IRA versus 84±42 ml/min/ 1,73m2 no grupo não IRA). O grupo IRA teve menor nadir de RFG (45±27 ml/min/ 1,73m2 versus 79±39 ml/min/ 1,73m2, p<0,001), idade maior (56&#61617;18 anos versus 52&#61617;19 anos, p=0,033), maior prevalência de diabetes (19,6% versus 9,3%, p=0,007), uso simultâneo mais freqüente de outras drogas nefrotóxicas (51% versus 38%, p=0,024) e contraste (18% versus 8%, p=0,0054), maior prevalência de hipovolemia (44% versus 27%, p=0,001), hipotensão (63% versus 44%, p=0,0003), choque (56% versus 31%, p<0,0001) e icterícia (19% versus 8%, p=0,0036). A mortalidade foi 44,5% no grupo IRA e 29,1% no grupo não IRA (p=0,0031). A análise por regressão logística identificou como fatores de risco independente para a nefrotoxicidade por aminoglicosídeo, RFG basal <60ml/min/1,73m2 [OR 0,42 (IC 95% 0,24-0,72, p=0,02)], diabetes [OR 2,13 (IC 95% 1,01-4,49, p=0,046)], uso simultâneo de outras drogas nefrotóxicas [OR 1,61 (IC 95% 1,00-2,59, p=0,048)], uso de contraste iodado [OR 2,13 (IC 95% 1,02-4,43, p=0,043)] e hipotensão [OR 1,83 (IC 95% 1,14-2,94, p=0,012)]. Conclusões: Nefrotoxicidade por aminoglicosídeo foi freqüente e associada a alta mortalidade em pacientes de UTI. A presença de diabetes, hipotensão, uso simultâneo de outras drogas nefrotóxicas e contraste iodado foram fatores de risco independentes para o desenvolvimento de nefrotoxicidade.