Porphyria is a rare metabolic disease which occurs as a result of accumulation of endogenous porphyrins due to specific enzyme deficiency in the biosynthetic pathway of heme. Chloroquine is currently used in the treatment of cutaneous porphyria, although its mechanism of action is not yet well understood. It is believed that chloroquine works in porphyria by forming complexes with excess porphyrin molecules and thus enhancing their elimination from the body. Previous reports of porphyrin-chloroquine complexes have been done mostly in aqueous models. In this study, UV/Visible optical absorbance difference spectroscopy was used to study the complexation of protoporphyrin IX with chloroquine and a range of acceptor molecules in hydrophobic models. The results show that chloroquine, mefloquine, amodiaquine, quinacrine, and pyronaridine formed relatively stronger complexes compared to other molecules such as quinine, duroquinone and caffeine. Therefore, relative to chloroquine, some of the molecules with comparable or greater binding affinity to protoporphyrin IX might also be useful in the treatment of porphyria.

The feasibility of most gene therapy strategies depends on the efficient delivery of DNA to target cells and tissues. Current gene delivery carriers can be divided into two classes: viral and non-viral delivery systems. Although the viral carriers are highly efficient due to their invasive nature, safety concerns may restrict their application in clinical settings. Synthetic non-viral carriers attract increasing attention because they are less toxic and allow readily modification. Non-viral carrier mediated gene delivery involves several processes. They must condense DNA into small particles, allow membrane penetration and protect DNA from extracellular and intracellular degradative enzymes. In the present study, a small library of carriers containing various combinations of cell penetrating peptide TAT, SV40 large T protein nuclear localisation signal (NLS) and cationic dendrimer of 7 lysine residues (DEN) was synthesised and tested for their ability to deliver DNA to mammalian cells. We evaluated the contribution of each component as well as the combination of the components on DNA condensation, uptake and gene expression. It was found that all carriers condensed DNA and protected DNA from DNase degradation. We showed that the TAT peptide was essential, but not sufficient, for uptake of exogenous DNA. The addition of either NLS or DEN significantly enhanced uptake. The most efficient carrier contained all three components (DEN-NLS-TAT). The carriers were able to deliver DNA in the presence of serum and were non-toxic to cells at up to 30 μM. However, for those peptides that facilitated DNA uptake, the complexes were targeted to intracellular compartments that required a fusogenic agent, such as chloroquine, before gene expression was observed. Modifications were introduced to the initial carrier library in order to circumvent the chloroquine dependence. The addition of cell penetrating peptide penetratin, virus derived fusogenic peptide or lipoamino acid C12 enhanced either DNA uptake or endosomal release. However, none of the modified carriers were able to produce high level transgene expression in the absence of chloroquine. We also found that the carriers containing lipid components were able to deliver DNA to T-lymphocytes derived cells, which are usually resistant to transfection. However, the toxicity of the lipid-based carriers needs to be reduced before further application. We also evaluated the function of chloroquine as a gene expression enhancer. We demonstrated that chloroquine did not enhance expression solely by promoting endosomal release. This was supported by the fact that fusogenic peptide and endosomal disruptive reagents (bafilomycin A1 and monensin) did not improve gene expression. Other properties of chloroquine, such as DNA protection and transcription enhancement, may also contribute to gene expression. We characterised the uptake mechanism of DEN-NLS-TAT in HeLa cell lines. We found that the uptake of DEN-NLS-TAT/DNA complex in HeLa cell line was mainly via receptor-mediated endocytosis and caveolae endocytosis. Moreover, various intracellular processes, such as intact cytoskeleton and microtubule network, tyrosine and PI 3 kinase activity, and membrane cholesterol were also required for the uptake of the carrier/DNA complex. In conclusion, the results from the present study demonstrated that multi-component peptide-based carriers are versatile carriers for the delivery of plasmid DNA in human cells. The results have improved our understanding of the role of chloroquine as a widely used gene expression enhancer which may be useful in the future improvement of non-viral gene delivery carriers. A strategy to overcome the dependence on chloroquine for gene expression or reduce the toxicity of chloroquine will be necessary for further in vivo applications. The current carrier library may also be used to delivery other cargos such as siRNA or protein to human cells.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Magister Pharmaceuticae - MPharm / Malaria is an infectious disease which continues to kill more than one million people every year and the African continent accounts for most of the malaria death worldwide. New classes of medicine to combat malaria are urgently needed due to the surge in resistance of the Plasmodium falciparum (the parasite that causes malaria in humans) to existing antimalarial drugs. One approach to circumvent the problem of P. falciparum resistance to antimalarial drugs could be the discovery of novel compounds with unique scaffolds and possibly new mechanisms of action. Natural products (NP) provide a wide diversity of compounds with unique scaffolds, as such, a library of virtual compounds (VC) designed from natural products with antiplasmodial activities (NAA) can be a worthy starting point.

In spite of great effort aimed at eradication, the malaria epidemic still claims over 600,000 lives each year, and 50% of the world is at risk of contracting the disease. The most deadly form of malaria is caused by Plasmodium falciparum, which is spread from human to human via the female Anopheles mosquito. P. falciparum's lifecycle, which includes both sexual and asexual reproduction, facilitates rapid evolution in response to drug pressure, resulting in the emergence of resistant strains against every antimalarial medication that has been deployed. There is a great need for new antimalarial drugs. Chloroquine (CQ), an aminoquinoline drug deployed in the 1940s, was an inexpensive, effective and safe drug but now has been rendered ineffective throughout much of the tropical regions due to the emergence of CQ-resistant strains of P. falciparum. A new class of hybrid drugs, called Reversed-CQs, has been developed by linking a molecule with a CQ-like moiety to a molecule with a reversal agent (RA) moiety; an RA is a chemosensitizer that can reverse CQ-resistance. The prototype Reversed-CQ, PL01, was shown to be effective in vitro against sensitive and resistant P. falciparum cell cultures, with IC50 values of 2.9 and 5.3 nM, respectively, in comparison to IC50 values for CQ which were 6.9 and 102 nM, respectively. In the course of the Reversed-CQ research, PL74 was synthesized with a pyridine ring replacing the quinoline ring. It was expected that PL74 would display reversal agent activity but would not display antimalarial activity. However PL74 showed antimalarialactivity with IC50 values of 185 and 169 nM in vitro against CQ-sensitive and CQ-resistant strains, respectively. In the investigation of PL74 it has been found that this molecule has a pyridinium salt structure, novel to the Reversed-CQ compounds, and through a structure-activity relationship (SAR) study, it was shown to have activity that may indicate a mode of action different from the Reversed-CQ compounds. A study of the literature revealed that pyridinium salt compounds, with some similarity to PL74, were found to operate as choline analogs inhibiting the biosynthesis of phosphatidylcholine as their main antimalarial mode of action.

No description available.

Le virus chikungunya est un arbovirus, transmis par les moustiques du genre Aedes, qui provoque des arthralgies invalidantes et parfois des rhumatismes chroniques. Dans une première partie nous avons décrit les aspects ambulatoires cliniques, biologiques et virologiques du chikungunya (CHIK) de la phase aiguë jusqu'au 300ème jour lors de l’épidémie de la Réunion en 2006. Des scores d’aide au diagnostic ont été élaboré et une étude de la diversité virale intra-hôte a été réalisée. Pour compléter nos premiers résultats nous avons étudié une épidémie survenue en République du Congo en 2011. La description clinique était similaire à celle identifiée lors de l’épidémie de la Réunion. L’évaluation du score clinique ne permettait pas de le proposer comme outil diagnostique à l’échelle individuelle mais apparaissait comme un bon marqueur pour le suivi de la courbe épidémique. Une étude de séroprévalence et une analyse phylogénétique complètent ce travail. Le dernier travail porte sur l’utilisation de la chloroquine à la phase aiguë du CHIK lors d’une prise prophylactique chez le singe et lors d’un essai clinique chez l’homme. Le principal effet de ce type de traitement semble lié son action immuno-modulatrice ; en prise préventive il provoque une exacerbation de la symptomatologie aiguë tandis qu’en prise à la phase précoce de la maladie il augmente le risque d’évolution vers des arthralgies chroniques. En conclusion nous avons réalisé une description des formes ambulatoires du CHIK, identifié des facteurs de risques de formes chroniques, proposé des scores d’aide au diagnostic et argumenté la contre-indication de l’utilisation de la chloroquine à la phase aiguë du CHIK. / Chikungunya virus (CHIKV) is an arthropod-borne virus transmitted by Aedes mosquitoes that cause debilitating arthralgia and possible chronic rheumatism. In the first part we describe the clinical, biological and virological presentation of outpatients with chikungunya disease (CHIK) from the acute stage to the chronic stage at day 300, during the outbreak in the Reunion Island in 2006. We elaborated scores for CHIK diagnosis and we also analysed the intra-host genetic diversity.To complete our first results, we investigated a CHIKV outbreak, which occurred in the Republic of Congo in 2011. The clinical presentation was similar to the first description of the Reunion island outbreak. We assessed the clinical score which appeared to be unusable at the individual level but was still relevant to follow the epidemic curve. This work was completed by seroprevalence and phylogenetic analyses.The last study presented in this thesis focused on the use of chloroquine during the acute stage of CHIK in a non-human primate (NHP) model (prophylactic use) and during a clinical trial (therapeutic use). The main effect of chloroquine treatment at the acute stage of CHIK appeared to be related to its immuno-modulatory action; in prophylactic taking, chloroquine exacerbated acute symptoms while treatment during the early stages of the disease increased the risk of acquiring chronic arthralgia.In conclusion, we provide a detailed description of CHIK outpatients and identify risk factors for the chronic stage of the disease. We propose tentative diagnostic scores and we firmly establish that the use of chloroquine at the acute phase of CHIK is contraindicated.

MSc (Chemistry) / Department of Chemistry / See the attached abstract below

Thesis (Ph.D.). / Written for the Institute of Parasitology. Title from title page of PDF (viewed 2008/02/12). Includes bibliographical references.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Horse breeding industry is an activity in ascension worldwide, and is responsible for generating jobs and income. In Brazil, especially in Rio Grande do Sul state, there are several horse breeding farms with high-standard equines. Although these herds are under strict sanitary control, the occurrence of respiratory diseases is an important cause of mortality in foals and reduced athletic performance. Among the respiratory diseases, equine rhodococcosis, caused by the bacterium Rhodococcus equi, is the major cause of pneumonia in foals. Rhodococcus equi is worldwide distributed, and have emerged as an important cause of economic losses due to pneumonia in young animals. However, preventive measures and effective control of the disease are still challenges to be reached. In R. equi infection, iron (Fe) is classified as an essential element not only for the bacterium multiplication, but also as a key for the expression of virulence factors. Studies have shown the presence of specific Fe uptake mechanisms in R. equi, which have been determining its survival in both saprophytic and pathogenic life styles. However, as a type of nutritional immunity, mammals, including horses, reduce the plasmatic concentration of Fe through its binding in proteins, including the transferrin (Tf). In this context, the present thesis was developed to study aspects related to metabolism and acquisition of Fe by R. equi and Fe importance in the pathogenesis of equine rhodococcosis (manuscript 1), control and treatment of infections caused by R. equi through drugs with capability to reduce the availability of intracellular Fe (manuscript 2), and genetic susceptibility to R. equi pneumonia (manuscript 3), including the assessment of polymorphisms in the equine Tf gene as risk factors related to susceptibility and/or resistance to equine rhodococcosis (manuscript 4 ). We conclude that R. equi is evolving to specialize it in the acquisition and utilization of Fe from the host, skills that should be considered as key points for the development of chemotherapeutic agents. Once R. equi encodes redundant mechanisms of acquisition and utilization of Fe, it is likely that chemotherapeutic agents will need act on multiple cellular mechanisms or be used in combination. Furthermore, the term "nutritional immunity" may be considered an important strategy to minimize antimicrobial resistance observed in R. equi. As an example of chemotherapy associated with iron metabolism, we observed that chloroquine inhibits the intracellular multiplication of R. equi, most likely due to intracellular iron deprivation. However, further studies are necessary to evaluate the chloroquine therapeutic potential against R. equi infections. We also observed important chromosomal regions positively associated with R. equi pneumonia, which seem to possess genes associated with immune response against intracellular pathogens. This observation allows us to classify the equine rhodococcosis as a disease of polygenic basis, as postulated by previous studies. Finally, we found that polymorphisms in the Tf gene, including some not described yet in the literature, occur in Brazilian Sport Horses and Brazilian Thoroughbred Horses. There is the occurrence of two alleles between the breeds studied, including heterozygosis for these alleles. We believe that there is a relationship between equine Tf variants, and genetic susceptibility to R. equi pneumonia in the breeds evaluated. Summarizing, we have demonstrated that the modulation of Fe availability may be a useful approach to control the disease. / A equideocultura é uma atividade em ascensão mundial, responsável pela geração de empregos e renda. No Brasil, em especial no Rio Grande do Sul, encontram-se diversos locais de criação de equinos de alto padrão zootécnico. Embora estes rebanhos estejam sob rigoroso controle sanitário, a ocorrência de doenças respiratórias é causa importante de mortalidade em potros e redução de seu desempenho atlético. Dentre as doenças respiratórias, a rodococose equina, causada pela bactéria Rhodococcus equi, é a principal causa de pneumonia nesta categoria animal. R. equi está distribuído mundialmente, e cresce como causa de perdas econômicas devido à pneumonia observada em animais jovens. No entanto, medidas preventivas e efetivo controle da enfermidade são ainda desafios a serem alcançados. Na infecção por R. equi, o ferro (Fe) apresenta-se como um elemento fundamental não somente para multiplicação da bactéria, mas também, como um determinante para a expressão de fatores de virulência. Estudos têm demonstrado a presença de mecanismos específicos de captação de Fe em R. equi, os quais determinam sua sobrevivência tanto durante seu estilo de vida saprófito quanto patogênico. Em contrapartida, como uma forma de imunidade nutricional, mamíferos, entre eles os equinos, diminuem a concentração plasmática de Fe através de sua ligação em proteínas, entre elas, a transferrina (Tf). Neste contexto, esta tese foi elaborada visando contemplar aspectos relacionados ao metabolismo e aquisição de Fe por R. equi e sua importância para patogenia da rodococose equina (manuscrito 1), controle e tratamento de infecções por R. equi através de drogas com capacidade de modular a disponibilidade de Fe intracelular (manuscrito 2), e susceptibilidade genética à pneumonia por R. equi (manuscrito 3), incluindo a avaliação de polimorfismos no gene da Tf equina como fatores de risco relacionados à susceptibilidade e/ou resistência genética à rodococose equina (manuscrito 4). Concluímos que R. equi está evoluindo de forma a especializar-se na aquisição e utilização de Fe a partir do hospedeiro, habilidades que devem ser consideradas como pontos chave no desenvolvimento de agentes quimioterápicos. Uma vez que R. equi codifica redundantes mecanismos de aquisição e utilização de Fe, é provável que agentes quimioterápicos deverão inibir múltiplos mecanismos ou ser utilizados em combinação. Além disso, o conceito de imunidade nutricional pode considerado uma importante estratégia para minimizar a resistência antimicrobiana observada em R. equi. Como um exemplo de quimioterápicos associados ao metabolismo de Fe, observados que chloroquine inibir a multiplicação intracelular de R. equi, muito provavelmente devido à deprivação de Fe intracelular. No entanto, ainda são necessários estudos avaliando o potencial terapêutico de chloroquine como tratamento alternativo de infecções por R. equi. Observou-se, também, importantes regiões cromossômicas positivamente associadas à pneumonia por R. equi, as quais parecem possuir genes associados à resposta imune contra patógenos intracelulares. Esta observação nos permite classificar a rodococose equina como uma enfermidade de base poligênica, como postulado por estudos anteriores. Por fim, verificamos que polimorfismos no gene da Tf, inclusive polimorfismos ainda não descritos na literatura, ocorrem em equinos das raças Brasileiro de Hipismo e Puro Sangue de Corrida, criados no Brasil. Existe a ocorrência de dois alelos entre as raças estudas, incluindo animais heterozigotos para estes alelos. Acredita-se que exista uma relação entre variantes de Tf equina e susceptibilidade genética à pneumonia por R. equi nas raças analisadas. Em suma, demonstrou-se através de diferentes estudos que a modulação da disponibilidade de Fe pode ser uma forma de controle da rodococose equina.