Thiol-ene chemistry is versatile and efficient and can be used as a powerful tool in polymer synthesis. In this thesis, the concept of thiol-ene chemistry has been central, where it has been explored as a tool for the synthesis of well-defined hydrogels and dopa-functional materials towards biomedical applications; such as hydrogels, primers for adhesive fixation of bone fractures, self-healing gels, and micelles for drug-delivery. Using thiol-ene chemistry, well-defined hydrogels were realized in order to study how the structure influences properties such as swelling, stiffness and hydrolytic degradation. It was found that all these characteristics are related to each other, as a more loosely crosslinked hydrogel experiences higher swelling, lower stiffness and higher degradation rates. Dopa-functional materials have gained a lot of interest throughout the years due to the remarkable adhesive properties they possess in wet environments. In the pursuit of new primers towards thiol-ene functional crosslinked bone adhesives, compounds with dopa moieties were proposed. Primers derived from dopamine were found to enhance the adhesion towards bone, and it was concluded that addition of NaOH was essential to achieve good adhesion. The strongest adhesion was achieved when thiol and ene-functional primers were used in combination. Most synthetic routes to dopa-functional polymers involve several protection and deprotection steps and a more simplistic synthetic route is therefore desired. The possibility of using UV-initiated thiol-ene chemistry to produce dopa-functional polymers was therefore investigated. The resulting polymers were shown to exhibit self-healing properties upon complexation with Fe3+ ions. Finally, the developed synthetic route was used to produce dopa and allyl-functional triblock-co-polymers. These triblock-co-polymers were then used to form micelles and evaluated as drug-delivery vehicles for the cancer-drug doxorubicin. The micelles were found to have high drug-loading capacities and slow release profiles and showed promising results when evaluated against breast-cancer cells. / Reaktioner mellan tioler och omättade kemiska föreningar utgör ett mångsidigt och effektivt redskap inom polymersyntes. I denna avhandling har begreppet tiol-en kemi varit centralt och kemin har använts för syntes av såväl väldefinierade hydrogeler som dopa-funktionella material. Dessa material har sedan utvärderats mot biomedicinska tillämpningar såsom hydrogeler, primers för fixering av benfrakturer, självläkande geler och kontrollerad läkemedelsleverans. Tiol-en-kemi har i denna avhandling använts för att framställa väldefinierade hydrogeler som sedan utvärderats med avseende på hur strukturen påverkar egenskaper såsom svällningsgrad, styvhet och nedbrytningshastighet. Det visade sig att alla dessa egenskaper är relaterade till varandra och att lösare tvärbundna hydrogeler uppvisar högre svällning, lägre styvhet och högre nedbrytningshastigheter. Marina musslor har en exceptionell förmåga att fästa mot olika ytor och på grund av detta har det visats en hel del intresse för dopa-funktionella material genom åren. På jakt efter en primer för att öka vidhäftningen hos benlim proponerades därför föreningar med dopafunktionella grupper. Det visade sig att dopaminderivat kunde förbättra vidhäftningen mot ben och det visade sig även att tillsats av natriumhydroxid var viktigt för att uppnå god vidhäftningsförmåga. Den starkaste vidhäftning uppnåddes när derivat med tiol och omättade bindningar användes i kombination. Syntes av dopafunktionella material involverar ofta flera reaktionssteg och en förenklad syntesväg är därför att eftersträva. UV-initierad tiol-en-kemi undersöktes därför som en möjlig syntesväg för att framställa dopafunktionella polymerer. Polymererna visade sig ha självläkande egenskaper vid komplexbildning med järnjoner. Slutligen användes denna syntesväg för att framställa blocksampolymerer. Dessa blocksampolymerer användes sedan för att bilda miceller med lovande resultat vid utvärdering för leverans av läkemedel mot bröstcancer. / <p>QC 20160125</p>

Parkinson's disease (PD) is a debilitating neurodegenerative disorder, affecting roughly 2% of those over the age of 80. Though most cases of PD are "sporadic", arising without a family history, a minority of cases have a clear autosomal dominant or recessive inheritance pattern. The most common known cause of autosomal recessive PD is homozygous inactivation of PARK2, which codes for the E3 ubiquitin ligase parkin. In addition, there is evidence that parkin inactivation may play a role in the pathogenesis of sporadic PD as well. As such, strategies aimed at increasing parkin activity hold therapeutic promise for sporadic PD. Though much work has examined the functions of parkin, and, more recently, the mechanisms by which it is activated, substantially less is known about how parkin levels are regulated in the cell. This is particularly true for activated parkin. Understanding these regulatory mechanisms is critical for the effective development of therapeutic strategies based on upregulation of parkin activity. The work presented in this dissertation provides new insights into these regulatory mechanisms. We show that relatively high doses of the dopamine precursor L-3,4-dihydroxyphenylalanine (L-DOPA) decrease parkin protein levels in vitro, analogously to previous findings using other cellular stressors. Characterizing this effect, we show that L-DOPA increases parkin degradation and that this occurs independently of L-DOPA's conversion to dopamine and of L-DOPA-induced cell death. Furthermore, we define two distinct pathways by which L-DOPA decreases parkin: an oxidative stress-dependent pathway and an oxidative stress-independent pathway. We show that the former overlaps with the previously defined mechanism of PINK1-mediated parkin activation. Specifically, parkin's association with PINK1-generated phosphorylated ubiquitin (phospho-Ub) leads to its proteasomal degradation downstream of oxidative stress, but not via autoubiquitination. Despite the involvement of PINK1 in parkin loss from L-DOPA treatment, we do not observe evidence of mitochondrial parkin activity after L-DOPA treatment, indicating that, surprisingly, parkin loss does not depend on this activity. Additionally, we provide evidence against the involvement of Trib3 and NADPH oxidases in parkin loss from L-DOPA. Finally, we provide preliminary evidence that parkin knockdown does not sensitize cells to L-DOPA-induced death. Taken together, the findings in this dissertation contribute to the understanding of parkin regulation. Our observation that parkin's association with phospho-Ub leads to its degradation following L-DOPA treatment is of particular interest because it may represent the steady-state mechanism by which levels of activated parkin are kept in check. In light of this, an attractive therapeutic strategy may involve uncoupling parkin's activation by phospho-Ub from its phospho-Ub-dependent degradation.

Thèse de doctorat : Médecine. Neurosciences : Université de Nantes : 2005. / Bibliogr.

1 v. (various paging) : ill. ; 26 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.1976) from the Dept. of Human Physiology and Pharmacology, University of Adelaide

A doença de Parkinson (DP) é a segunda doença neurodegenerativa mais comum do mundo, e seu tratamento atual se baseia principalmente no uso de medicações que facilitam a transmissão dopaminérgica nos núcleos da base. A L-DOPA é a principal medicação usada no tratamento da DP, contudo seu uso crônico está associado ao surgimento de complicações motoras, como as discinesias induzidas por L-DOPA (DIL). As DIL ocorrem em cerca de 50% dos pacientes com DP que usaram LDOPA por cerca de 4 a 6 anos, e podem causar uma série de impactos negativos aos pacientes. Pela incapacidade adicional que esta complicação traz aos pacientes com DP, a definição de fatores que possam predizer o surgimento das DIL é de importância direta para o clínico que prescreve L-DOPA rotineiramente. O objetivo deste estudo foi determinar os principais fatores de risco clínicos e genéticos para o desenvolvimento de DIL em uma casuística de pacientes brasileiros com DP. Um estudo transversal foi realizado em pacientes brasileiros de dois centros (Ribeirão Preto e São Paulo) como parte do projeto \"Latin American Research Consortium on the Genetics of PD\" (LARGE-PD), incluindo apenas pacientes com DP e em uso de L-DOPA. A avaliação foi baseada em um exame neurológico completo e em uma entrevista semi-estruturada feita por um médico neurologista especialista em Distúrbios de Movimentos. A presença de DIL foi considerada se a pontuação fosse >= 1 no item 32 da Parte IV na MDS-UPDRS. Baseados em estudos prévios, nós escolhemos oito polimorfismos tipo single nucleotide polymorphism (SNP) nestes genes: COMT, MAOB, ANKK1, DRD3, DAT1, BDNF, ADORA2A and NOS1. A genotipagem foi realizada através de ensaios TaqMan SNP. Foram realizados modelos de regressão logística e análises de sobrevivência para identificarmos os fatores de risco clínicos e genéticos associados ao surgimento de DIL. Ao todo, foram analisados 199 pacientes (sexo masculino - 59%; idade média 61.8 anos), sendo 96 indivíduos (48.2%) com DIL. Através de um modelo de regressão logística multivariado com 7 variáveis independentes, o fenótipo clínico motor do tipo postural instability with gait disorder (PIGD) (OR 0.17, IC 95% 0.07-0.39; p < 0.001), longos períodos de tratamento com L-DOPA (OR 1.31, IC 95% 1.17-1.47; p < 0.001), altas doses diárias equivalentes de L-DOPA (OR 1.00, IC 95% 1.000-1.002; p = 0.04) e a início precoce dos sintomas da 11 DP (OR 1.04, IC 95% 1.01-1.07; p = 0.009) foram os fatores de risco clínicos mais associados ao surgimento de DIL. Dentre os fatores de risco genéticos, apenas o alelo T do SNP rs1799836 no gene da MAOB esteve associado ao aumento na chance de surgimento de DIL (OR 1.51, IC 95% 1.00-2.28; p = 0.05). Nossos resultados mostraram que a predição de surgimento de DIL em pacientes com DP em uso de L-DOPA pode ser feita através de alguns fatores de risco clínicos (fenótipo clínico motor, duração do tratamento com L-DOPA, dose diária equivalente de L-DOPA e idade de início de sintomas) e genéticos, como o SNPrs1799836 no gene da MAOB. Novos estudos com amostras maiores e desenhos prospectivos longitudinais são necessários para se explorar a associação entre estes preditores e o surgimento de DIL. / Parkinson\'s disease (PD) is the second most common neurodegenerative disease in the world, and its treatment is mainly based on drugs involved on dopaminergic neurotransmission in basal ganglia. L-DOPA is the major medication used on the management of PD, but its chronic use is associated with the onset of motor complications, as L-DOPA-induced dyskinesias (LID). LID occur in approximately 50% of patients using L-DOPA over 4 and 6 years, and they cause negative impacts on the quality of life of patients PD. To predict the onset of LID based on clinical and genetic risk may be an useful tool for clinicians which prescribe L-DOPA. The aim of this study was to determinate the main clinical and genetic risk factors for the onset of LID in Brazilian PD patients. A cross-sectional study was conducted with Brazilian PD patients from two centers (Ribeirao Preto and Sao Paulo) as part of the Latin American Research consortium on the Genetics of PD (LARGE-PD), which enrolled only PD patients using L-DOPA. PD patients were submitted to neurological examination and semi-structured interviews performed by movement disorders specialists. Presence of LID was confirmed if UPDRS Part IV had a score >= 1 on item 32. Based on previous studies, we chose eight Single Nucleotide Polymorphisms (SNP) in the following genes: COMT, MAOB, ANKK1, DRD3, DAT1, BDNF, ADORA2A and NOS1. Genotyping was performed using TaqMan SNP genotyping assays. We performed logistic regression and survival analysis to identify clinical and genetic risk factors associated with LID onset We enrolled 199 PD patients (males - 59%; mean age 61.8 years), and 96 patients (48.2%) had LID. At a multivariate model with 7 independent variables, postural instability with gait disorder (PIGD) clinical phenotype (OR 0.17, CI 95% 0.07-0.39; p < 0.001), longer duration of L-DOPA therapy (OR 1.31, Cl 95% 1.17-1.47; p < 0.001), higher L-DOPA equivalent daily doses (OR 1.00, CI 95% 1.000-1.002; p = 0.04), as also as early onset of PD (OR 1.04, CI 95% 1.01-1.07; p = 0.009) were the clinical risk factor more associated with onset of LID. Regarding genetic risk factors, only MAOB SNP rs1799836 was associated with LID, with the T allele increasing the risk of developing LIDs (OR 1.51, CI 95% 1.00-2.28; p = 0.05). Our results showed onset of LID can be predicted based on some clinical (motor clinical phenotype, duration of L-DOPA therapy, L-DOPA equivalent daily dose and age at PD onset) and genetic risk factors, as MAOB SNP rs1799836. Further studies in larger samples, using longitudinal and prospective designs, are needed to explore the association between these predictors and onset of LID.

Parkinson’s disease is a disorder mainly characterized by progressive degeneration of dopamine producing neurons in the substantia nigra of the midbrain. The most commonly used treatment strategy is to pharmacologically restore the lost function by the administration of the dopaminergic precursor L-DOPA. Another treatment strategy is to replace the degenerated neurons with immature fetal ventral mesencephalic tissue, or ultimately stem cell-derived tissue. Grafting trials have, however, revealed poor reinnervation capacity of the grafts, leaving much of the striata dopamine-denervated. An additional drawback is the upcoming of dyskinesia (involuntary movements), a phenomenon also observed during L-DOPA treatment of Parkinson’s disease patients. Attempts to characterize nerve fiber formation from dopamine neurons have demonstrated that the nerve fibers are formed in two morphologically diverse outgrowth patterns, one early outgrowth seen in the absence of astrocytes and one later appearing outgrowth seen in co-existence with astrocytes. The overall objective of this thesis has been to study the dopaminergic outgrowth including guidance of nerve fiber formation, and to look into the mechanisms of L-DOPA-induced dyskinesia. The first paper in this thesis characterizes the different outgrowth patterns described above and their relation to different glial cells. The study demonstrated the two different outgrowth patterns to be a general phenomenon, applying not only to dopamine neurons. Attempts of characterization revealed no difference of origin in terms of dopaminergic subpopulations, i.e. A9 or A10, between the outgrowth patterns. Furthermore, the “roller-drum” technique was found optimal for studying the dual outgrowth sequences. The second and the third paper also utilized the “roller-drum” technique in order to promote both patterns of neuronal fiber formation. The effects of glial cell line-derived neurotrophic factor (GDNF) on the formation of dopamine nerve fibers, was investigated. Cultures prepared from gdnf knockout mice revealed that dopaminergic neurons survive and form nerve fiber outgrowth in the absence of GDNF. The dopaminergic nerve fibers exhibited an outgrowth pattern consistent with that previous observed in rat. GDNF was found to exert effect on the glial-associated outgrowth whereas the non-glial-associated was not affected. Astrocytic proliferation was inhibited using cytosine β-D-arabinofuranoside, resulting in reduced glial-associated outgrowth. The non-glial-associated dopaminergic outgrowth was on the other hand promoted, and was retained over longer time in culture. Furthermore, the non-glial-associated nerve fibers were found to target the fetal frontal cortex. Different developmental stages were shown to promote and affect the outgrowths differently. Taken together, these data indicate and state the importance of astrocytes and growth factors for neuronal nerve fiber formation and guidance. It also stresses the importance of fetal donor age at the time for transplantation. The fourth and fifth studies focus on L-DOPA dynamics and utilize in vivo chronoamperometry. In study four, 6-OHDA dopamine-depleted rats were exposed to chronic L-DOPA treatment and then rated as dyskinetic or non-dyskinetic. The electrochemical recordings demonstrated reduced KCl-evoked release in the intact striatum after chronic L-DOPA treatment. Time for maximal dopamine concentration after L-DOPA administration was found to be shorter in dyskinetic animals than in non-dyskinetic animals. The serotonergic nerve fiber content in the striatum was evaluated and brains from dyskinetic animals were found to exhibit significantly higher nerve fiber density compared to non-dyskinetic animals. Furthermore, the mechanisms behind the conversion of L-DOPA to dopamine in 6-OHDA dopamine-depleted rats were studied. Local administration of L-DOPA in the striatum increased the KCl-evoked dopamine release in the intact striatum. Acute application of L-DOPA resulted sometimes in a rapid conversion to dopamine, probably without vesicle packaging. This type of direct conversion is presumably occurring in non-neuronal tissue. Furthermore, KCl-evoked dopamine releases were present upon local application of L-DOPA in the dopamine-depleted striatum, suggesting that the conversion to dopamine took place elsewhere, than in dopaminergic nerve fibers. In conclusion, these studies state the importance of astrocytes for neuronal nerve fiber formation and elucidate the complexity of L-DOPA conversion in the brain.

Parkinson’s disease (PD) is a progressive neurodegenerative movement disorder characterized by a large number of motor and non-motor deficits, which significantly contribute to reduced quality of life. Despite the definition of the broad spectrum of clinical characteristics, mechanisms triggering illness, the nature of its progression and a character of therapeutic effects still remain unknown. The enormous advances in magnetic resonance imaging (MRI) in the last decades have significantly affected the research attempts to uncover the functional and structural abnormalities in PD and have helped to develop and monitor various treatment strategies, of which dopamine replacement strategies, mainly in form of levodopa, has been the gold standard since the late seventies and eighties. Motor, task-related functional MRI (fMRI) has been extensively used to assess the pathological state of the motor circuitry in PD. Several studies employed motor paradigms and fMRI to review the functional brain responses of participants to levodopa treatment. Interestingly, they provided conflicting results. Wide spectrum of symptoms, variability and asymmetry of the disease presentation, several treatment approaches and their divergent outcomes make PD enormously heterogeneous. In this work we hypothesized that not considering the disease heterogeneity might have been an adequate cause for the discrepant results in aforementioned studies. We show that not accounting for the disease variability might indeed compromise the results and invalidate the consequent interpretations. Accordingly, we propose and formalize a statistical approach to account for the intra and inter subject variability. This might help to minimize this bias in future motor fMRI studies revealing the functional brain dysfunction and contribute to the understanding of still unknown pathophysiological mechanisms underlying PD.

A doença de Parkinson (DP) é a segunda doença neurodegenerativa mais comum do mundo, e seu tratamento atual se baseia principalmente no uso de medicações que facilitam a transmissão dopaminérgica nos núcleos da base. A L-DOPA é a principal medicação usada no tratamento da DP, contudo seu uso crônico está associado ao surgimento de complicações motoras, como as discinesias induzidas por L-DOPA (DIL). As DIL ocorrem em cerca de 50% dos pacientes com DP que usaram LDOPA por cerca de 4 a 6 anos, e podem causar uma série de impactos negativos aos pacientes. Pela incapacidade adicional que esta complicação traz aos pacientes com DP, a definição de fatores que possam predizer o surgimento das DIL é de importância direta para o clínico que prescreve L-DOPA rotineiramente. O objetivo deste estudo foi determinar os principais fatores de risco clínicos e genéticos para o desenvolvimento de DIL em uma casuística de pacientes brasileiros com DP. Um estudo transversal foi realizado em pacientes brasileiros de dois centros (Ribeirão Preto e São Paulo) como parte do projeto \"Latin American Research Consortium on the Genetics of PD\" (LARGE-PD), incluindo apenas pacientes com DP e em uso de L-DOPA. A avaliação foi baseada em um exame neurológico completo e em uma entrevista semi-estruturada feita por um médico neurologista especialista em Distúrbios de Movimentos. A presença de DIL foi considerada se a pontuação fosse >= 1 no item 32 da Parte IV na MDS-UPDRS. Baseados em estudos prévios, nós escolhemos oito polimorfismos tipo single nucleotide polymorphism (SNP) nestes genes: COMT, MAOB, ANKK1, DRD3, DAT1, BDNF, ADORA2A and NOS1. A genotipagem foi realizada através de ensaios TaqMan SNP. Foram realizados modelos de regressão logística e análises de sobrevivência para identificarmos os fatores de risco clínicos e genéticos associados ao surgimento de DIL. Ao todo, foram analisados 199 pacientes (sexo masculino - 59%; idade média 61.8 anos), sendo 96 indivíduos (48.2%) com DIL. Através de um modelo de regressão logística multivariado com 7 variáveis independentes, o fenótipo clínico motor do tipo postural instability with gait disorder (PIGD) (OR 0.17, IC 95% 0.07-0.39; p < 0.001), longos períodos de tratamento com L-DOPA (OR 1.31, IC 95% 1.17-1.47; p < 0.001), altas doses diárias equivalentes de L-DOPA (OR 1.00, IC 95% 1.000-1.002; p = 0.04) e a início precoce dos sintomas da 11 DP (OR 1.04, IC 95% 1.01-1.07; p = 0.009) foram os fatores de risco clínicos mais associados ao surgimento de DIL. Dentre os fatores de risco genéticos, apenas o alelo T do SNP rs1799836 no gene da MAOB esteve associado ao aumento na chance de surgimento de DIL (OR 1.51, IC 95% 1.00-2.28; p = 0.05). Nossos resultados mostraram que a predição de surgimento de DIL em pacientes com DP em uso de L-DOPA pode ser feita através de alguns fatores de risco clínicos (fenótipo clínico motor, duração do tratamento com L-DOPA, dose diária equivalente de L-DOPA e idade de início de sintomas) e genéticos, como o SNPrs1799836 no gene da MAOB. Novos estudos com amostras maiores e desenhos prospectivos longitudinais são necessários para se explorar a associação entre estes preditores e o surgimento de DIL. / Parkinson\'s disease (PD) is the second most common neurodegenerative disease in the world, and its treatment is mainly based on drugs involved on dopaminergic neurotransmission in basal ganglia. L-DOPA is the major medication used on the management of PD, but its chronic use is associated with the onset of motor complications, as L-DOPA-induced dyskinesias (LID). LID occur in approximately 50% of patients using L-DOPA over 4 and 6 years, and they cause negative impacts on the quality of life of patients PD. To predict the onset of LID based on clinical and genetic risk may be an useful tool for clinicians which prescribe L-DOPA. The aim of this study was to determinate the main clinical and genetic risk factors for the onset of LID in Brazilian PD patients. A cross-sectional study was conducted with Brazilian PD patients from two centers (Ribeirao Preto and Sao Paulo) as part of the Latin American Research consortium on the Genetics of PD (LARGE-PD), which enrolled only PD patients using L-DOPA. PD patients were submitted to neurological examination and semi-structured interviews performed by movement disorders specialists. Presence of LID was confirmed if UPDRS Part IV had a score >= 1 on item 32. Based on previous studies, we chose eight Single Nucleotide Polymorphisms (SNP) in the following genes: COMT, MAOB, ANKK1, DRD3, DAT1, BDNF, ADORA2A and NOS1. Genotyping was performed using TaqMan SNP genotyping assays. We performed logistic regression and survival analysis to identify clinical and genetic risk factors associated with LID onset We enrolled 199 PD patients (males - 59%; mean age 61.8 years), and 96 patients (48.2%) had LID. At a multivariate model with 7 independent variables, postural instability with gait disorder (PIGD) clinical phenotype (OR 0.17, CI 95% 0.07-0.39; p < 0.001), longer duration of L-DOPA therapy (OR 1.31, Cl 95% 1.17-1.47; p < 0.001), higher L-DOPA equivalent daily doses (OR 1.00, CI 95% 1.000-1.002; p = 0.04), as also as early onset of PD (OR 1.04, CI 95% 1.01-1.07; p = 0.009) were the clinical risk factor more associated with onset of LID. Regarding genetic risk factors, only MAOB SNP rs1799836 was associated with LID, with the T allele increasing the risk of developing LIDs (OR 1.51, CI 95% 1.00-2.28; p = 0.05). Our results showed onset of LID can be predicted based on some clinical (motor clinical phenotype, duration of L-DOPA therapy, L-DOPA equivalent daily dose and age at PD onset) and genetic risk factors, as MAOB SNP rs1799836. Further studies in larger samples, using longitudinal and prospective designs, are needed to explore the association between these predictors and onset of LID.

Tesis presentada a la Universidad de Chile para optar al grado de Magíster en Química y Memoria para optar al Título de Químico / En esta Tesis se desarrollaron electrodos de pasta de carbono (PC) modificados con líquidos iónicos (LI): dos de tipo imidazolio (1-butil-3-metilimidazolio tetrafluoroborato y hexafluorofosfato) y dos de tipo piridinio (1-butil-4-metilpiridinio tetrafluoroborato y hexa fluorofosfato), para su empleo con fines analíticos. Las superficies de PC modificadas se caracterizaron por voltamperometría cíclica (con mediadores redox clásicos como ferrocenometanol), microscopía de barrido electrónico y de barrido electroquímico. Con los electrodos modificados seleccionados se realizaron estudios del comportamiento electroquímico de tres fármacos de amplio uso y de reconocida actividad redox: levodopa, acetaminofeno y nitrendipino. En todos los casos, el comportamiento anódico o catódico de cada fármaco en los electrodos modificados fue similar al de los electrodos no modificados, en lo que a reversibilidad y dependencia de potenciales de pico vs pH se refiere, cuando fueron estudiados empleando voltamperometría cíclica (VC) y de pulso diferencial (VPD). La principal diferencia entre el uso de electrodos modificados vs no modificados, fue la mayor respuesta en corriente. Para levodopa el incremento fue de más del doble, y para acetaminofeno y nitrendipino en 20 y 17 veces, respectivamente. De acuerdo al comportamiento electroquímico de cada fármaco, se desarrollaron metodologías analíticas empleando VPD. En términos generales las reproducibilidades presentaron coeficientes de variación < 5,4 % y con límites de detección de 0,63 μg/mL, 51 ng/mL y 25 ng/mL para levodopa, acetaminofeno y nitrendipino, respectivamente. Las metodologías analíticas desarrolladas se aplicaron en la cuantificación de cada fármaco en su forma farmacéutica, sin interferencias de los excipientes de las formulaciones y con porcentajes encontrados acordes con lo declarado por los fabricantes / In this thesis, carbon paste (CP) electrodes modified with ionic liquids (IL) were developed: two IL of imidazolium type (1-butyl-3-methylimidazolium tetrafluoroborate and hexafluoro phosphate) and two of pyridinium type (1-butyl-4-methylpyridinium tetrafluoro borate and hexafluorophosphate), to their use for analytical purposes. The modified CP surfaces were characterized by cyclic voltammetry (with classical redox mediators as ferrocenemethanol), scanning electron and scanning electrochemical microscopy. With the selected modified electrodes, were performed studies of the electrochemical behaviour of three drugs widely used and of recognized redox activity: levodopa, acetaminophen and nitrendipine. In all cases, the anodic or cathodic behaviour of each drug in the modified electrodes was similar to that of non-modified electrodes, in which reversibility and dependence on the peak potential vs. pH refers when they were studied using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The main difference between the uses of modified vs. unmodified electrodes was a higher response in peak current. The response was increased more than twice, 20 and 17 times for levodopa, acetaminophen and nitrendipine, respectively. Based on the electrochemical behaviour of each drug, DPV analytical methodologies were developed, finding in general terms reproducibilities with coefficient of variations < 5.4% and detection limits of 0.63 μg/mL, 51 ng/mL and 25 ng/mL for levodopa, acetaminophen and nitrendipine, respectively. Finally, the developed analytical methods were tested in the quantification of each drug in pharmaceutical form, finding on the one hand that the excipients of the formulations do not interfere in the determination of each drug and on the other hand, values consistent with the percentages found declared by the manufacturer / Fondecyt Nº 1110182

A model with human melanoma xenografts transplanted to athymic mice has been adopted for in vivo studies of 5-S-cysteinyldopa (an intermediate pigment metabolite), glutathione, and cysteine. L-Dopa is an intermediate metabolite in pigment formation and is also important in the treatment of Parkinson's disease, and therefore 1 have also studied the pharmacokinetics of this compound. We were first to describe in vivo microdialysis in melanoma tissue and showed that dialysis membranes of cuprophane or polyamide are suitable for studies of interstitial 5-S-cysteinyldopa and selected thiols. Analytical procedures were also improved for quantitation of 5-S-cysteinyldopa, L-dopa, glutathione, cysteine, and N-acetylcysteine (NAC). In the melanoma xenografts the interstitial concentration of 5-S-cysteinyldopa reflected the high intracellular production of this intermediate metabolite. For in vivo manipulation of glutathione in the melanoma tissue we gave intraperitoneal injection of buthionine sulphoximine to the animals and thus reduced the glutathione concentrations substantially. We showed that restitution of glutathione in melanoma tissue occurs spontaneously and is not much improved by treatment with the cysteine deliverers NAC and L-2-oxothiazolidine-4-carboxylate (OTC). 5-S-Cysteinyldopa was not substantially affected by great variations in glutathione concentrations. Transport of NAC from intraperitoneal injection to melanoma tissue occurred rapidly and deacetylation to cysteine in vivo could be detected soon after NAC injection. In vivo formation of cysteine was slower from OTC than from NAC. Pharmacokinetic studies of L-dopa in human subjects indicated a slight to moderate protein binding. Plasma free L-dopa had similar elimination T½ as interstitial L-dopa, but in some cases the elimination of total L-dopa was slower. Difficulties in intestinal absorption of L-dopa were revealed by microdialysis in blood and subcutaneous tissue. Studies showed that this was due to delayed emptying of the stomach. L-Dopa intake increased 5-S-cysteinyldopa concentrations in blood within 30 min in patients with Parkinson's disease and a history of melanoma. No melanoma activation occurred during long-term treatment with L-dopa. Microdialysis is thus a safe and easily applied method for in vivo studies of both pigment metabolites from human melanoma tissue transplanted to nude mice and for pharmacokinetic studies of L-dopa. / On the day of the public defence the status of the articles IV, V and VI was: Submitted.