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Le procédé de granulation en voie humide nécessite l'ajout d'un agent d’enrobage ou liant, typiquement composé d'agents tensioactifs, d'eau, de plastifiant et de charge hydrophobe. Cependant, dans les procédés de granulation en voie sèche, l'agent d’enrobage est ajouté sous la forme de fines particules solides. L’objectif de ce travail est double : d’une part, examiner le comportement des particules dans les systèmes secs et aqueux aux échelles microscopique et mésoscopique, et d’autre part, développer des méthodologies prédictives permettant de choisir le liant adéquat et formuler la bonne solution d’enrobage. Dans le cadre de cette étude, nous avons utilisées l'hydroxypropyl-méthylcellulose (HPMC) et la cellulose d'éthyle (EC) comme agents d’enrobage, polyvinylpyrrolidone (PVP) et la cellulose microcristalline (MCC) généralement utilisés comme liants, l'acide stéarique (SA) qui est une charge hydrophobe, et le polyéthylène glycol (PEG) comme plastifiant. Tous ces matériaux sont largement utilisés dans les industries alimentaires et pharmaceutiques. La réussite d’une granulation dépend de l’affinité entre les particules primaires et le liant. Afin de prédire l'affinité liant-substrat en milieu sec et en milieu aqueux, nous avons comparé deux approches; la première est basée sur le travail de l'adhésion alors que la seconde s’appuie sur le concept de résistance à la traction idéale. L’équation de résistance à la traction idéale a été étendue aux systèmes ternaires dans le but de l’appliquer pour la granulation en milieu aqueux. Les approches développées ont été ensuite confrontées aux données expérimentales sur différent systèmes (composées de PVP, MCC, HPMC, SA, EC, PEG et l'eau). Nous avons ainsi trouvé que l’approche basée sur le travail d'adhésion semble donner de meilleures prédictions des affinités. Les deux approches prédisent que le HPMC est un bon liant pour le MCC. Les résultats indiquent également que le PEG a une bonne affinité avec le HPMC et le SA. Nous avons ensuite étudié la structure des agglomérats formés dans les formulations colloïdales utilisées dans les procédés d’enrobage. Pour ce faire, nous nous sommes appuyés sur des analyses expérimentales et des simulations mésoscopiques. Ces dernières reposent sur l’utilisation de la méthode de dynamique des particules dissipatives (DPD) dans laquelle les composés sont décrits comme un ensemble de billes souples (approche « coarse-grain ») interagissant selon le modèle de Flory-Huggins. Les interactions répulsives entre les billes ont été évaluées en utilisant le paramètre de solubilité (δ) calculé par simulation moléculaire tout-atome. Les résultats de simulation DPD ont été comparés aux résultats expérimentaux obtenus par plusieurs voies : cryogénique-MEB, analyse de distribution de taille de particule et par la technique DSC. Les résultats de la simulation DPD montrent que le polymère HPMC est un meilleur agent stabilisant pour le SA que le PVP et le MCC. En outre, HPMC est capable de recouvrir la particule de SA d'une couche épaisse et d’y pénétrer en profondeur, empêchant ainsi l’agglomération et la croissance des cristaux de SA. Néanmoins, HPMC est incapable de stabiliser les particules de SA lorsque celles-ci sont en quantités élevées (supérieurs à 10% (w/w)). Nous constatons également que le PEG se diffuse à l'intérieur des chaînes de HPMC entrainant l’extension de ce dernier, formant ainsi un polymère composite lisse. Les résultats expérimentaux montrent des tendances similaires; l’analyse de la distribution de taille de particule indique qu’en présence de HPMC, pour de faible pourcentages de SA (au-dessous de 10% (w/w)), la majorité des particules de SA sont inférieures à 1 μm de diamètre. Les images MEB révèlent que HPMC entoure les cristaux de SA avec un film texturé et ancre sur leur surface. / Wet granulation process requires the addition of a coating agent or binder, typically composed of surfactants, water, plasticizers and fillers. In dry granulation however, the coating agent is added to the system in the form of fine solid particles. Our goals are to investigate the particles behaviour and agglomeration mechanism in dry and aqueous systems at the micro and meso scales, and also, to develop predictive methodologies and theoretical tools of investigation allowing to choose the adequate binder and to formulate the right coating solution. In this study we chose materials widely used in food and pharmaceutical industries, including; coating agents such as Hydroxypropyl-methylcellulose (HPMC) and Ethyl cellulose (EC), binders such as Polyvinylpyrrolidone (PVP) and Microcrystalline cellulose (MCC), hydrophobic filler such as Stearic acid (SA) and plasticizer such as Polyethylene glycol (PEG). A successful granulation requires good affinity between host and guest particles. In this context, in the first part of this work, two approaches to predict the binder-substrate affinity in dry and in aqueous media were compared; one based on the work of adhesion and the other based on the ideal tensile strength. The concept of ideal tensile strength was extended to ternary systems and applied for granulation in aqueous media. The developed approaches were thereafter tested for various systems (composed of PVP, MCC, HPMC, SA, EC, PEG and water) and compared to experimental observations. Approaches yielded results in good agreement with the experimental observations, but the work of adhesion approach might give more accurate affinity predictions on the particles affinity than the ideal tensile strength approach. Both approaches predicted that HPMC is a good binder for MCC. Results also indicated that PEG has a good affinity with HPMC and SA. In a second part of our work, we used mesoscale simulations and experimental techniques to investigate the structure of agglomerates formed in aqueous colloidal formulations used in coating and granulation processes. For the simulations, dissipative particle dynamics (DPD) and a coarse-grained approach were used. In the DPD method, the compounds were described as a set of soft beads interacting according to the Flory-Huggins model. The repulsive interactions between the beads were evaluated using the solubility parameter (δ) as input, where, δ was calculated by all-atom molecular simulations. The mesoscale simulation results were compared to experimental results obtained by Cryogenic-SEM, particle size distribution analysis and DSC technique. According to the DPD simulations, HPMC polymer is a better stabilizing agent for SA than PVP and MCC. In addition, HPMC is able to cover the SA particle with a thick layer ant to adsorb in depth into its inner core, preventing SA agglomeration and crystal growth. But, for high amounts of SA (above 10% (w/w)), HPMC is unable to fully stabilize SA. We also found that PEG polymer diffuses inside HPMC chains thereby extending and softening the composite polymer. Experimental results presented similar trends; particle size distribution analysis showed that in the presence of HPMC, for low percentages of SA (below 10% (w/w)), the majority of SA particles are below 1 μm in diameter. SEM images revealed that HPMC surrounds SA crystals with a hatching textured film and anchors on their surface.

Depuis quelques décennies, les flavonoïdes sont de plus en plus utilisés dans différents domaines d’applications alimentaires et non alimentaires. Cet engouement est dû principalement à leurs activités antioxydantes. Cependant, la solubilisation, la dispersion et la stabilisation de ces molécules sont variables et constituent un frein à leur utilisation. L’objectif de ce travail est de pallier cet inconvénient en visant à comprendre les interactions entre ces composés et leur milieu en absence et en présence de polymères, par une double approche expérimentale et par modélisation et mésomodélisation moléculaire. Dans un premier temps les interactions entre 3 flavonoïdes la quercétine et ses deux formes glycosilées la rutine et l’isoquercétine dans différents solvants organiques ont été étudiées. Les résultats obtenus (paramètre de Flory Huggins et fonction de la distribution radiale) ont montré que la partie B2 commune aux trois flavonoïdes avec des valeurs de paramètres de Flory Huggins proche de 0.5 dans le M2B2 et plus importantes dans l’acétonitrile est la responsable du comportement des flavonoïdes dans le solvant. Les simulations par DDFT ont montré une agrégation de la quercétine dans le M2B2 contre une dispersion dans l’acétonitrile. Toutes ces observations ont été validées expérimentalement (étude de la solubilité et observations microscopiques). Dans un deuxième temps on a étudié la quercétine en présence d’un bioploymère le PLGA dans l’eau. Des nanoparticules ont été formées en variant la concentration des différents composés et le ratio acide lactique / acide glycolique du PLGA. Les méthodes de la modélisation moléculaire et de la mésomodélisation (calcul du paramètre de solubilité par dynamique moléculaire et observation de la dispersion ou de la séparation de phase par DDFT) ainsi que l’approche expérimentale (DSC, MET …) nous ont menées à la même constatation. En effet la taille des particules augmente avec la concentration du PLGA et le taux d’acide lactique dans le polymère. Aussi la concentration de l’émulsifiant dans le milieu joue un rôle important dans la formation d’agrégats PLGA-Q. Plus sa concentration est importante, plus la formation des particules est difficile comme il joue un rôle sur la viscosité du milieu et par conséquence la diffusivité des molécules dans l’eau. Tous les résultats obtenus par modélisation moléculaire et par mésomodélisation ont été validés expérimentalement. On peut donc conclure que la méthodologie adoptée en simulation peut constituer un outil d’aide à la prédiction du comportement des flavonoïdes / Over the past few decades, flavonoids have become increasingly used in different food and non-food applications due to their important antioxidant activities. However, the solubilization, dispersion and stabilization of these molecules are variable and constitute a brake on their use in different formulations. The objective of this work is to overcome those limitations by understanding the interactions between these compounds and their environment without and with the add of polymers, by a multi-scale approach approach (molecular modeling and mesoscale modeling and experimental study). Initially, interactions between 3 flavonoids (quercetin, rutin and isoquercetin) in various organic solvents, were studied. The obtained results (mainly Flory Huggins parameter and radial distribution function RDF) showed that the B2 part common to the three flavonoids (For example Flory Huggins parameter values were close to 0.5 in the M2B2 and much more important in acetonitrile) is responsible for the miscibility behavior of the flavonoids in the solvent. DDFT simulations showed aggregation of quercetin in M2B2 against dispersion in acetonitrile. All these observations were confirmed experimentally (study of solubility and microscopic observations). Then, quercetin was studied in the presence of a biopolymer, PLGA in water. Nanoparticles were formed by varying the concentration of the various compounds and the lactic acid / glycolic acid ratio in the PLGA. The tools of molecular modeling and mesoscale modeling (calculation of the solubility parameter by molecular dynamics and observation of the dispersion or the phase separation by DDFT) as well as the experimental approach (DSC, MET ...) led us to the same conclusions. Indeed, the particle size increases with the concentration of PLGA and the rate of lactic acid in the polymer. Also the concentration of the emulsifier in the medium has an important role in the formation of PLGA-Q aggregates. The higher its concentration, the more difficult the formation of the particles as it affects the viscosity of the medium and consequently the diffusivity of the molecules in the water. All the results obtained by molecular modeling and by mesoscale modeling have been confirmed experimentally. We can therefore conclude that the methodology adopted in the simulations can be considered as a tool to help on predicting the behavior of flavonoids in different medium

<div>Radio Frequency Power Amplifiers (PA) are essential components of wireless systems and nonlinear in a permanent way. So, high efficiency and linearity at a time are imperative for power amplifiers. However, it is hard to obtain because high efficiency Power Amplifiers are nonlinear and linear Power Amplifiers have poor efficiency. To meet both linearity and efficiency, the linearization techniques such as Digital Predistortion (DPD) has arrested the most attention in industrial and academic sectors due to provide a compromising data between efficiency and linearity. This thesis proposed on digital predistortion techniques to control nonlinear distortion in radio frequency transmitters. </div><div>By using predistortion technique, both linearity and efficiency can obtain. In this thesis a new generic Saleh model for use in memoryless nonlinear power amplifier (PA) behavioral modelling is used. The results are obtained by simulations through MATLAB and experiments. We explore the baseband 13.56 MHz Power Amplifier input and output relationships and reveal that they apparent differently when the Power Amplifier shows long-term, short-term or memory less effects. We derive a SIMULINK based static DPD design depend on a memory polynomial. A polynomial improves both the non-linearity and memory effects in the Power Amplifier. As PA characteristics differs from time to time and operating conditions, we developed a model to calculate the effectiveness of DPD. We extended our static DPD design model into an adaptive DPD test bench using Indirect Learning Architecture (ILA) to implement adaptive DPD which composed of DPD subsystem and DPD coefficient calculation. By this technique, the output of PA achieves linear, amplitude and phase distortions are eliminated, and spectral regrowth is prevented. </div><div>The advanced linearity performance executed through the strategies and methods evolved on this thesis can allow a higher usage of the capability overall performance of existing and emerging exceptionally performance PAs, and therefore an anticipated to have an effect in future wireless communication systems. </div>

Base teórica: As fluoropirimidinas possuem significativa variabilidade na resposta terapêutica e na ocorrência de toxicidade, o que tem sido relacionado à deficiência na depuração metabólica mediada pela enzima diidropirimidina desidrogenase (DPD). Mutações nos genes codificadores da enzima, bem como fatores ambientais podem levar à baixa ou nula expressão enzimática, provocando efeitos adversos graves devido ao acúmulo destes fármacos. Até o presente, nenhum teste reconhecidamente valido para a identificação de indivíduos em risco de toxicidade severa está estabelecido na prática oncológica. A genotipagem para o gene DPYD apresenta poder preditivo limitado, pois é capaz de rastrear somente as mutações já conhecidas, que apresentam baixa frequência populacional. Por esta razão, ensaios funcionais baseados na avaliação da redução fisiológica do uracil (U) para diidrouracil (UH2), igualmente medidada pela DPD, têm sido propostos na identificação de pacientes predispostos à toxicidade. Nesta abordagem são estimadas as razões plasmáticas [UH2]/[U] em níveis basais ou após uma dose oral do U. Recentemente, foi sugerida a realização do teste funcional em saliva como amostra alternativa ao plasma, com maior estabilidade dos analitos. Entretanto, a associação entre as razões metabólicas nesta matriz e a toxicidade não foi validada em amostras clínicas. Objetivos: Avaliar a efetividade dos métodos de determinação da razão metabólica [UH2]/[U] em plasma e saliva e a genotipagem para o gene DPYD como preditores de toxicidade por fluoropirimidinas em pacientes com neoplasias gastrointestinais. Adicionalmente, o trabalho propôs o desenvolvimento de um método bioanalítico para a determinação de U e UH2 por cromatografia líquida de alta eficiência. Métodos: Foram obtidas amostras pareadas de plasma e saliva de 60 pacientes diagnosticados com neoplasia gastrointestinal e com indicação de tratamento com fluoropirimidinas. As concentrações de U e UH2 foram determinadas nas duas matrizes através de LC-MS/MS. Os efeitos adversos do primeiro ciclo de quimioterapia foram classificados de acordo com o NCI-CTCAE versão 4. A genotipagem da DYDP foi realizada por PCR tempo real e incluiu os alelos *2A; *13, Y186C; I560S, *7 Y186C. Resultados: 35% dos pacientes apresentaram toxicidade severa (graus 3/4), sendo a neutropenia a mais frequente (n=11). A genotipagem da DYDP não foi capaz de identificar pacientes em risco de toxicidade, uma vez que não foram encontrados portadores de alelos variáveis. As razões [UH2]/[U] variaram amplamente entre os pacientes, de 0,09 a 26,73 no plasma e de 0,08 a 24 na saliva. As razões [UH2]/[U] no plasma e na saliva demonstraram correlação elevada (rs=-0,575; P<0,01), porém, a saliva demonstrou maior correlação com o grau de toxicidade quando comparada ao plasma (rs=-0,515; P<0,01 vs rs=-0,282 P<0,05). Pacientes com grau de toxicidade 3/4 (n=21) apresentaram menor razão metabólica em comparação a pacientes com grau 1/2 (n=26) ou com ausência de toxicidade (n=13) (média 0.59 vs 2.22 e 2.83 no plasma e 1.62 vs 6.88 e 6.75 na saliva, P<0.01). A partir de curva ROC foi determinado o valor de corte de 1,16 para a razão em saliva com 86% de sensibilidade e 77% de especificidade para a identificação de pacientes com toxicidade severa. Nas amostras de plasma o valor de corte foi 4.0 com 71% de sensibilidade e 76% de especificidade. Adicionalmente, foi desenvolvido e validado um método bioanalítico para a dosagem de U e UH2 com exatidão (98.4–105.3%) e precisão precisão intra-ensaio (5.1–12.1%) e inter-ensaios (5.3–10.1%) satisfatórios. Conclusão: Neste grupo de pacientes a genotipagem dos alelos *2A; Y186C; I560S, Y186C e *7 da DPYD não mostrou-se útil na identificação de indivíduos com deficiência severa da DPD. Entretanto, as razões metabólicas [UH2]/[U] demonstraram ser um promissor teste para avaliar a funcionalidade da enzima e identificar a maioria dos casos de pacientes com sujeitos a toxicidade grave à fluoropirimidinas, com sensibilidade superior da saliva. / Background: Variation on therapeutic response to fluoropirimidines and toxicity have been related to impaired dihydropyrimidine dehydrogenase (DPD) mediated metabolism. Mutations in genes encoding the enzyme as well as environmental factors can lead to reduced or absent enzyme expression, causing serious adverse effects due to the accumulation of these drugs. To date, there is no clinically recognized valid assay, for the identification of individuals at risk of severe toxicity in oncological practice. DPYD genotyping has a limited prediction power, since it is able to identify only the already known mutations, which have low frequency in population. Therefore, functional DPD assays based on the assessment of uracil (U) to dihydrouracil (UH2) metabolism, which is also dependent on DPD, have been proposed to identify patients prone to toxicity. Thus, endogenous metabolic ratios of [UH2]/[U] or after an oral dose of U are determined in plasma. Recently, the use of saliva has been suggested as alternative matrix to plasma, with higher stability of analytes. However, the association between salivary metabolic ratios and toxicity has not been validated in clinical samples. Objective: To evaluate the use of plasma and saliva uracil (U) to dihydrouracil (UH2) metabolic ratios and DPYD genotyping, as a means to identify patients with dihydropyrimidine dehydrogenase (DPD) deficiency and fluoropyrimidine toxicity. Additionally, the work proposed the development of a bioanalytical method for the determination of U and UH2 by high-performance liquid chromatography. Methods: Paired plasma and saliva samples were obtained from 60 patients with gastrointestinal cancer before fluoropyrimidine treatment. U and UH2 concentrations were measured by LC-MS/MS. DPYD was genotyped for alleles *2A; Y186C; I560S, Y186C and *7. Results: 35% of the patients had severe toxicity. There was no variant allele carrier for DPYD. The [UH2]/[U] metabolic ratios were 0.09-26.73 in plasma and 0.08-24.0 in saliva, with higher correlation with toxicity grade in saliva compared to plasma (rs=-0.515 vs rs=-0.282). Median metabolic ratios were lower in patients with severe toxicity as compared to those with absence of toxicity (0.59 vs 2.83 plasma; 1.62 vs 6.75 saliva, P<0.01). A cut-off of 1.16 for salivary ratio was set with 86% sensitivity and 77% specificity for the identification of patients with severe toxicity. Similarly, a plasma cut-off of 4.0 revealed a 71% sensitivity and 76% specificity. Additionally, a bioanalytical method for the quantification of U and UH2, with adequate accuracy (98.4–105.3%) and precision (intra-assay CV 5.1–12.1% and inter-assay CV 5.3–10.1%) was develop and validated. Conclusions: DPYD genotyping for alleles *2A; Y186C; I560S, Y186C and *7 was not helpful in the identification of patients with severe DPD deficiency in this series of patients. The [UH2]/[U] metabolic ratios, however, proved to be a promising functional test to identify the majority of cases of severe DPD activity, with saliva performing better than plasma.

Background/Aim: The British questionnaire ECLiPS (Evaluation of Children’s Listening and Processing Skills; Barry &amp; Moore, 2015) aims to profile listening difficulties in children. The questionnaire consists of 38 statements, divided into five subscales, and a short survey with six questions about general health. The purpose of this study was to investigate whether children diagnosed with Developmental Phonological Disorder (DPD) in their preschool years (3-6 years of age) show listening difficulties in early school years (7-10 years). Methods: 113 guardians of children with Developmental Phonological Disorder (DPD) and 44 guardians of typically developing (TD) Swedish children participated in the study. Comparisons were made between the Swedish TD-group and the English reference (ER) group presented in the ECLiPS technical manual. Mean scores between the DPD-group and the TD-group were compared, as well as were the responses on the short survey. The groups were categorized by age and gender. Results: In general, high consistency was found between the ER- and TD-group in half of the comparisons; 7-, 9- and 10-year-old boys and 8-year-old girls. The DPD-group tended to show more symptoms of listening difficulties than the TD-group. However, the symptoms varied in relation to age and gender. For example, girls in the DPD-group had more symptoms in younger than in older ages. The short survey revealed that the DPD-group had a higher prevalence of ear infections than the TD-group. The difficulties seen in the DPD-group were most profound in statements concerning language, literacy and laterality. Conclusion: Our results indicate that listening difficulties as measured with the ECLiPS are more common in children diagnosed with DPD in preschool years, than typically developing children. The strongest indication for difficulties is seen in the statements regarding language and literacy.   Bakgrund/syfte: Det brittiska frågeformuläret ECLiPS (Evaluation of Children’s Listening and Processing Skills; Barry &amp; Moore, 2015) syftar till att kartlägga lyssningssvårigheter hos barn. Frågeformuläret består av 38 påståenden, indelade i fem underkategorier, samt en kort enkät med sex frågor rörande generell hälsa. Syftet med den här studien var att undersöka huruvida barn som hade diagnosticerats med fonologisk språkstörning (FS) i förskoleåldern (3-6 år) uppvisade lyssningssvårigheter i tidig skolålder (7-10 år). Metod: 113 vårdnadshavare till barn med fonologisk språkstörning och 44 vårdnadshavare till typiskt utvecklade (TU) svenska barn deltog i studien. Jämförelser gjordes mellan TU barn och den engelska referensgruppen (ER) som presenteras i ECLiPS tekniska manual. Barn med FS och barn med TU delades upp i mindre grupper baserat på kön och ålder, och medelvärdesjämförelser gjordes mellan grupperna. Jämförelser gjordes även av svaren på den medicinska enkäten. Resultat: Generellt sett hade ER-gruppen och barn med TU en hög grad av samstämmighet i hälften av jämförelserna; 7-, 9-, och 10-åriga pojkar samt 8-åriga flickor. Barn med FS tenderade att uppvisa en högre grad av lyssningssvårigheter än barn med TD. Dock varierade graden av symptom beroende på ålder och kön. Till exempel tenderade flickor med FS att uppvisa mer symptom i lägre än i högre åldrar. Svaren från den medicinska enkäten visade att barn med FS hade en högre prevalens av öroninfektioner än barn med TU. De svårigheter som barn med FS uppvisade var tydligast inom påståenden som rör språk, läs- och skrivförmåga och lateralitet. Slutsats: Våra resultat indikerar att de lyssningssvårigheter som mäts med ECLiPS är vanligare hos barn som diagnostiserats med fonologisk språkstörning i förskoleåldern än hos typiskt utvecklade barn. Den starkaste indikationen för svårigheter syns i de påståenden som rör språk-, läs- och skrivförmåga.

Base teórica: As fluoropirimidinas possuem significativa variabilidade na resposta terapêutica e na ocorrência de toxicidade, o que tem sido relacionado à deficiência na depuração metabólica mediada pela enzima diidropirimidina desidrogenase (DPD). Mutações nos genes codificadores da enzima, bem como fatores ambientais podem levar à baixa ou nula expressão enzimática, provocando efeitos adversos graves devido ao acúmulo destes fármacos. Até o presente, nenhum teste reconhecidamente valido para a identificação de indivíduos em risco de toxicidade severa está estabelecido na prática oncológica. A genotipagem para o gene DPYD apresenta poder preditivo limitado, pois é capaz de rastrear somente as mutações já conhecidas, que apresentam baixa frequência populacional. Por esta razão, ensaios funcionais baseados na avaliação da redução fisiológica do uracil (U) para diidrouracil (UH2), igualmente medidada pela DPD, têm sido propostos na identificação de pacientes predispostos à toxicidade. Nesta abordagem são estimadas as razões plasmáticas [UH2]/[U] em níveis basais ou após uma dose oral do U. Recentemente, foi sugerida a realização do teste funcional em saliva como amostra alternativa ao plasma, com maior estabilidade dos analitos. Entretanto, a associação entre as razões metabólicas nesta matriz e a toxicidade não foi validada em amostras clínicas. Objetivos: Avaliar a efetividade dos métodos de determinação da razão metabólica [UH2]/[U] em plasma e saliva e a genotipagem para o gene DPYD como preditores de toxicidade por fluoropirimidinas em pacientes com neoplasias gastrointestinais. Adicionalmente, o trabalho propôs o desenvolvimento de um método bioanalítico para a determinação de U e UH2 por cromatografia líquida de alta eficiência. Métodos: Foram obtidas amostras pareadas de plasma e saliva de 60 pacientes diagnosticados com neoplasia gastrointestinal e com indicação de tratamento com fluoropirimidinas. As concentrações de U e UH2 foram determinadas nas duas matrizes através de LC-MS/MS. Os efeitos adversos do primeiro ciclo de quimioterapia foram classificados de acordo com o NCI-CTCAE versão 4. A genotipagem da DYDP foi realizada por PCR tempo real e incluiu os alelos *2A; *13, Y186C; I560S, *7 Y186C. Resultados: 35% dos pacientes apresentaram toxicidade severa (graus 3/4), sendo a neutropenia a mais frequente (n=11). A genotipagem da DYDP não foi capaz de identificar pacientes em risco de toxicidade, uma vez que não foram encontrados portadores de alelos variáveis. As razões [UH2]/[U] variaram amplamente entre os pacientes, de 0,09 a 26,73 no plasma e de 0,08 a 24 na saliva. As razões [UH2]/[U] no plasma e na saliva demonstraram correlação elevada (rs=-0,575; P<0,01), porém, a saliva demonstrou maior correlação com o grau de toxicidade quando comparada ao plasma (rs=-0,515; P<0,01 vs rs=-0,282 P<0,05). Pacientes com grau de toxicidade 3/4 (n=21) apresentaram menor razão metabólica em comparação a pacientes com grau 1/2 (n=26) ou com ausência de toxicidade (n=13) (média 0.59 vs 2.22 e 2.83 no plasma e 1.62 vs 6.88 e 6.75 na saliva, P<0.01). A partir de curva ROC foi determinado o valor de corte de 1,16 para a razão em saliva com 86% de sensibilidade e 77% de especificidade para a identificação de pacientes com toxicidade severa. Nas amostras de plasma o valor de corte foi 4.0 com 71% de sensibilidade e 76% de especificidade. Adicionalmente, foi desenvolvido e validado um método bioanalítico para a dosagem de U e UH2 com exatidão (98.4–105.3%) e precisão precisão intra-ensaio (5.1–12.1%) e inter-ensaios (5.3–10.1%) satisfatórios. Conclusão: Neste grupo de pacientes a genotipagem dos alelos *2A; Y186C; I560S, Y186C e *7 da DPYD não mostrou-se útil na identificação de indivíduos com deficiência severa da DPD. Entretanto, as razões metabólicas [UH2]/[U] demonstraram ser um promissor teste para avaliar a funcionalidade da enzima e identificar a maioria dos casos de pacientes com sujeitos a toxicidade grave à fluoropirimidinas, com sensibilidade superior da saliva. / Background: Variation on therapeutic response to fluoropirimidines and toxicity have been related to impaired dihydropyrimidine dehydrogenase (DPD) mediated metabolism. Mutations in genes encoding the enzyme as well as environmental factors can lead to reduced or absent enzyme expression, causing serious adverse effects due to the accumulation of these drugs. To date, there is no clinically recognized valid assay, for the identification of individuals at risk of severe toxicity in oncological practice. DPYD genotyping has a limited prediction power, since it is able to identify only the already known mutations, which have low frequency in population. Therefore, functional DPD assays based on the assessment of uracil (U) to dihydrouracil (UH2) metabolism, which is also dependent on DPD, have been proposed to identify patients prone to toxicity. Thus, endogenous metabolic ratios of [UH2]/[U] or after an oral dose of U are determined in plasma. Recently, the use of saliva has been suggested as alternative matrix to plasma, with higher stability of analytes. However, the association between salivary metabolic ratios and toxicity has not been validated in clinical samples. Objective: To evaluate the use of plasma and saliva uracil (U) to dihydrouracil (UH2) metabolic ratios and DPYD genotyping, as a means to identify patients with dihydropyrimidine dehydrogenase (DPD) deficiency and fluoropyrimidine toxicity. Additionally, the work proposed the development of a bioanalytical method for the determination of U and UH2 by high-performance liquid chromatography. Methods: Paired plasma and saliva samples were obtained from 60 patients with gastrointestinal cancer before fluoropyrimidine treatment. U and UH2 concentrations were measured by LC-MS/MS. DPYD was genotyped for alleles *2A; Y186C; I560S, Y186C and *7. Results: 35% of the patients had severe toxicity. There was no variant allele carrier for DPYD. The [UH2]/[U] metabolic ratios were 0.09-26.73 in plasma and 0.08-24.0 in saliva, with higher correlation with toxicity grade in saliva compared to plasma (rs=-0.515 vs rs=-0.282). Median metabolic ratios were lower in patients with severe toxicity as compared to those with absence of toxicity (0.59 vs 2.83 plasma; 1.62 vs 6.75 saliva, P<0.01). A cut-off of 1.16 for salivary ratio was set with 86% sensitivity and 77% specificity for the identification of patients with severe toxicity. Similarly, a plasma cut-off of 4.0 revealed a 71% sensitivity and 76% specificity. Additionally, a bioanalytical method for the quantification of U and UH2, with adequate accuracy (98.4–105.3%) and precision (intra-assay CV 5.1–12.1% and inter-assay CV 5.3–10.1%) was develop and validated. Conclusions: DPYD genotyping for alleles *2A; Y186C; I560S, Y186C and *7 was not helpful in the identification of patients with severe DPD deficiency in this series of patients. The [UH2]/[U] metabolic ratios, however, proved to be a promising functional test to identify the majority of cases of severe DPD activity, with saliva performing better than plasma.

Base teórica: As fluoropirimidinas possuem significativa variabilidade na resposta terapêutica e na ocorrência de toxicidade, o que tem sido relacionado à deficiência na depuração metabólica mediada pela enzima diidropirimidina desidrogenase (DPD). Mutações nos genes codificadores da enzima, bem como fatores ambientais podem levar à baixa ou nula expressão enzimática, provocando efeitos adversos graves devido ao acúmulo destes fármacos. Até o presente, nenhum teste reconhecidamente valido para a identificação de indivíduos em risco de toxicidade severa está estabelecido na prática oncológica. A genotipagem para o gene DPYD apresenta poder preditivo limitado, pois é capaz de rastrear somente as mutações já conhecidas, que apresentam baixa frequência populacional. Por esta razão, ensaios funcionais baseados na avaliação da redução fisiológica do uracil (U) para diidrouracil (UH2), igualmente medidada pela DPD, têm sido propostos na identificação de pacientes predispostos à toxicidade. Nesta abordagem são estimadas as razões plasmáticas [UH2]/[U] em níveis basais ou após uma dose oral do U. Recentemente, foi sugerida a realização do teste funcional em saliva como amostra alternativa ao plasma, com maior estabilidade dos analitos. Entretanto, a associação entre as razões metabólicas nesta matriz e a toxicidade não foi validada em amostras clínicas. Objetivos: Avaliar a efetividade dos métodos de determinação da razão metabólica [UH2]/[U] em plasma e saliva e a genotipagem para o gene DPYD como preditores de toxicidade por fluoropirimidinas em pacientes com neoplasias gastrointestinais. Adicionalmente, o trabalho propôs o desenvolvimento de um método bioanalítico para a determinação de U e UH2 por cromatografia líquida de alta eficiência. Métodos: Foram obtidas amostras pareadas de plasma e saliva de 60 pacientes diagnosticados com neoplasia gastrointestinal e com indicação de tratamento com fluoropirimidinas. As concentrações de U e UH2 foram determinadas nas duas matrizes através de LC-MS/MS. Os efeitos adversos do primeiro ciclo de quimioterapia foram classificados de acordo com o NCI-CTCAE versão 4. A genotipagem da DYDP foi realizada por PCR tempo real e incluiu os alelos *2A; *13, Y186C; I560S, *7 Y186C. Resultados: 35% dos pacientes apresentaram toxicidade severa (graus 3/4), sendo a neutropenia a mais frequente (n=11). A genotipagem da DYDP não foi capaz de identificar pacientes em risco de toxicidade, uma vez que não foram encontrados portadores de alelos variáveis. As razões [UH2]/[U] variaram amplamente entre os pacientes, de 0,09 a 26,73 no plasma e de 0,08 a 24 na saliva. As razões [UH2]/[U] no plasma e na saliva demonstraram correlação elevada (rs=-0,575; P<0,01), porém, a saliva demonstrou maior correlação com o grau de toxicidade quando comparada ao plasma (rs=-0,515; P<0,01 vs rs=-0,282 P<0,05). Pacientes com grau de toxicidade 3/4 (n=21) apresentaram menor razão metabólica em comparação a pacientes com grau 1/2 (n=26) ou com ausência de toxicidade (n=13) (média 0.59 vs 2.22 e 2.83 no plasma e 1.62 vs 6.88 e 6.75 na saliva, P<0.01). A partir de curva ROC foi determinado o valor de corte de 1,16 para a razão em saliva com 86% de sensibilidade e 77% de especificidade para a identificação de pacientes com toxicidade severa. Nas amostras de plasma o valor de corte foi 4.0 com 71% de sensibilidade e 76% de especificidade. Adicionalmente, foi desenvolvido e validado um método bioanalítico para a dosagem de U e UH2 com exatidão (98.4–105.3%) e precisão precisão intra-ensaio (5.1–12.1%) e inter-ensaios (5.3–10.1%) satisfatórios. Conclusão: Neste grupo de pacientes a genotipagem dos alelos *2A; Y186C; I560S, Y186C e *7 da DPYD não mostrou-se útil na identificação de indivíduos com deficiência severa da DPD. Entretanto, as razões metabólicas [UH2]/[U] demonstraram ser um promissor teste para avaliar a funcionalidade da enzima e identificar a maioria dos casos de pacientes com sujeitos a toxicidade grave à fluoropirimidinas, com sensibilidade superior da saliva. / Background: Variation on therapeutic response to fluoropirimidines and toxicity have been related to impaired dihydropyrimidine dehydrogenase (DPD) mediated metabolism. Mutations in genes encoding the enzyme as well as environmental factors can lead to reduced or absent enzyme expression, causing serious adverse effects due to the accumulation of these drugs. To date, there is no clinically recognized valid assay, for the identification of individuals at risk of severe toxicity in oncological practice. DPYD genotyping has a limited prediction power, since it is able to identify only the already known mutations, which have low frequency in population. Therefore, functional DPD assays based on the assessment of uracil (U) to dihydrouracil (UH2) metabolism, which is also dependent on DPD, have been proposed to identify patients prone to toxicity. Thus, endogenous metabolic ratios of [UH2]/[U] or after an oral dose of U are determined in plasma. Recently, the use of saliva has been suggested as alternative matrix to plasma, with higher stability of analytes. However, the association between salivary metabolic ratios and toxicity has not been validated in clinical samples. Objective: To evaluate the use of plasma and saliva uracil (U) to dihydrouracil (UH2) metabolic ratios and DPYD genotyping, as a means to identify patients with dihydropyrimidine dehydrogenase (DPD) deficiency and fluoropyrimidine toxicity. Additionally, the work proposed the development of a bioanalytical method for the determination of U and UH2 by high-performance liquid chromatography. Methods: Paired plasma and saliva samples were obtained from 60 patients with gastrointestinal cancer before fluoropyrimidine treatment. U and UH2 concentrations were measured by LC-MS/MS. DPYD was genotyped for alleles *2A; Y186C; I560S, Y186C and *7. Results: 35% of the patients had severe toxicity. There was no variant allele carrier for DPYD. The [UH2]/[U] metabolic ratios were 0.09-26.73 in plasma and 0.08-24.0 in saliva, with higher correlation with toxicity grade in saliva compared to plasma (rs=-0.515 vs rs=-0.282). Median metabolic ratios were lower in patients with severe toxicity as compared to those with absence of toxicity (0.59 vs 2.83 plasma; 1.62 vs 6.75 saliva, P<0.01). A cut-off of 1.16 for salivary ratio was set with 86% sensitivity and 77% specificity for the identification of patients with severe toxicity. Similarly, a plasma cut-off of 4.0 revealed a 71% sensitivity and 76% specificity. Additionally, a bioanalytical method for the quantification of U and UH2, with adequate accuracy (98.4–105.3%) and precision (intra-assay CV 5.1–12.1% and inter-assay CV 5.3–10.1%) was develop and validated. Conclusions: DPYD genotyping for alleles *2A; Y186C; I560S, Y186C and *7 was not helpful in the identification of patients with severe DPD deficiency in this series of patients. The [UH2]/[U] metabolic ratios, however, proved to be a promising functional test to identify the majority of cases of severe DPD activity, with saliva performing better than plasma.

Amyloidos uppstår när amyloid, felveckade proteiner, ackumuleras extracellulärt i vävnaden. Det finns två typer av amyloid som ger upphov till hjärtamyloidos: lätt immunoglobulinkedja amyloidos (AL-amyloidos) och transtyretinamyloidos (TTR-amyloidos). TTR-amyloidos delas in i hereditär transtyretinamyloidos (hATTR) och wild-type transtyretinamyloidos (wtATTR). Beroende på vilken typ av amyloidos ges olika behandlingar. Idag används alltmer en icke-invasiv nuklearmedicinsk undersökning, för diagnostik och differentiering vid hjärtamyloidos. Bedömningen är visuell och utgår från ett utarbetat graderingssystem av Perugini. Vid klinisk fysiologi och nuklearmedicin på Skånes universitetssjukhus har efterfrågan av den nuklearmedicinska undersökningen för hjärtamyloidos ökat. Syftet med denna studie är att optimera hjärtscintigrafi amyloidos med 99mTc-DPD genom beräkning av antalet counts i SPECT/CT och i planara bilder, och därefter beräkna aktiviteten per kilogram kroppsvikt för att undersöka om stråldosen till patienten kan minskas. Dessutom kommer en jämförelse mellan manuella och cirkulära ROI att utföras för att undersöka om någon skillnad föreligger. Denna retrospektiva studie omfattade tidigare genomförda undersökningar och hitintills har 17 patienter genomgått hjärtamyloidos scintigrafi med 99mTc-DPD. Resultaten av studien visade ingen signifikant skillnad mellan cirkulära och manuella ROI (p = 0,504), vilket talade för en god överensstämmelse. För att reducera stråldosen till patienten kan viktbaserad dosmängd vara ett alternativ i framtiden. / Amyloidosis occurs when amyloid, misfolded proteins, accumulate extracellularly in the tissue. There are two types of amyloid that cause cardiac amyloidosis: immunoglobulin light chain amyloidosis (AL amyloidosis) and transthyretin amyloidosis (TTR amyloidosis). TTR amyloidosis is divided into hereditary transthyretin amyloidosis (hATTR) and wild-type transthyretin amyloidosis (wtATTR). Different treatments are available depending on what type of amyloidosis. Today, a non-invasive nuclear medicine examination is increasingly being used for the diagnosis and differentiation of cardiac amyloidosis. The assessment of cardiac amyloidosis involves a visual assessment based on the Perugini grading system. In the Clinical Physiology and Nuclear medicine Department at Skåne University Hospital, there is an increasing demand for cardiac amyloidosis scintigraphy with 99mTc-DPD. The aim of this study is to optimize 99mTc-DPD scintigraphy for cardiac amyloidosis by identifying the number of counts in planar and in SPECT/CT images. The patient’s body weight will also be studied to determine if the patient’s radiation dose can be reduced. In addition, a comparison between manual and circular region of interest (ROI) will be performed to examine whether a difference exists. The study involved a retrospective analysis of 17 patients that have undergone cardiac amyloidosis scintigraphy with 99mTc-DPD. The results showed no significant difference between circular and manual ROI (p = 0.504), which indicated good agreement. Weight-based radiation dose may be an alternative in the future, in order to reduce the radiation dose to the patient.

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