Os neutrófilos desempenham um papel fundamental na defesa do organismo contra microrganismos invasores, através da fagocitose, da degranulação e da produção de espécies reativas de oxigênio (EROs), sendo que esta última é decorrente da ativação do metabolismo oxidativo dos neutrófilos. As EROs produzidas, tais como O2∙-, H2O2, HO∙, HOCl, 1O2 e NO, que são essenciais para matar os microrganismos fagocitados, podem também ser liberadas para o meio extracelular e causar lesões oxidativas aos tecidos do hospedeiro. Essas lesões têm sido implicadas na patogênese de doenças como aterosclerose, câncer, artrite reumatóide, pneumonia e enfisema pulmonar. Com o intuito de prevenir essas lesões oxidativas, tem-se investigado a atividade antioxidante de diversos compostos, especialmente os derivados de plantas. Neste trabalho foram avaliadas as atividades de oito cumarinas simples (compostos 1 a 8) sobre o metabolismo oxidativo de neutrófilos de coelho estimulados com Zimosan opsonizado, empregando-se os ensaios de quimioluminescência (QL) dependente de lucigenina (QLluc) e de QL dependente de luminol (QLlum). A QLluc reflete a produção apenas de O2∙- , enquanto que a QLlum é resultante da oxidação do luminol pelas diversas EROs produzidas pelos neutrófilos estimulados. Assim, a inibição da QLluc e da QLlum será decorrente da redução da concentração das EROs responsáveis pela oxidação da lucigenina e do luminol no meio de reação, respectivamente. Foram avaliadas também a atividade dos antioxidantes a-tocoferol, butil-hidroxitolueno (BHT) e quercetina sobre a QLlum e a QLluc, e a toxicidade das cumarinas 1 a 8 e dos três antioxidantes citados sobre os neutrófilos de coelho. Determinou-se o coeficiente de partição e o volume molecular das cumarinas 1 a 8. Dentre os três antioxidantes, observou-se que a quercetina inibiu tanto a QLlum quanto a QLluc, e não foi tóxica para os neutrófilos, na concentração de 50 mmol/L. O a-tocoferol não inibiu a QLlum nem a QLluc, enquanto o BHT foi tóxico para os neutrófilos de coelho. Para as cumarinas simples, observou-se que: (1) a cumarina 1 não apresentou atividade inibitória sobre a QLluc e sobre a QLlum; (2) as cumarinas 3, 4, 5 e 6 tiveram atividades inibitórias sobre a QLluc semelhantes à da quecetina, que foram maiores que as das cumarinas 2, 7 e 8; (3) as atividades inibitórias das cumarinas 2 a 8 sobre a QLluc não estão relacionadas à lipofilicidade e ao volume molecular desses compostos, nem à toxicidade dos mesmos sobre os neutrófilos de coelho; (4) as cumarinas 3 e 4 provocaram aumento da QLlum medida, enquanto as demais não tiveram atividade neste sistema. / Neutrophils play a crucial role in the defense of the organism against invading microorganisms, thorough phagocytosis, degranulation and generation of reactive oxygen species (EROS). The last one is a consequence of activation of neutrophils\' oxidative metabolism. The produced EROs, such as O2∙-, H2O2, HOCl, HO∙, 1O2 and NO, which are essential for killing phagocyted microorganisms, may also be released to the extracellular milieu and inflict oxidative damage to host tissues. This damage has been implicated in the pathogenesis of diseases, such as arteriosclerosis, cancer, rheumatoid arthritis, pneumonia and lung emphysema. The antioxidant activity of many compounds, especially those obtained from plants, has been investigated with the aim of avoid such oxidative damage. In this work, we studied the activity of eight simple coumarins (compounds 1 to 8) on the oxidative metabolism of rabbit neutrophils upon stimulation by opsonized zymosan, using luminol- (QLlum) and lucigenin (QLluc)-enhanced chemiluminescence (QL) assays. QLluc reflects generation of O2∙- only, whereas QLlum is a result of luminol oxidation by the various EROs produced by the stimulated neutrophils. Inhibition of QLluc and of QLlum will be a consequence of the decrease of the concentration of that EROs which are involved in the oxidation of lucigenin and luminol, respectively, in the reaction medium. We also evaluated the activity of the antioxidants a-tocopherol, butyl-hydroxytoluene (BHT) and quercetin on QLlum and QLluc, and the toxicity of coumarins 1 to 8 and the three antioxidants on rabbit neutrophils. Partition coefficient and molecular volume values of coumarins 1 to 8 were determined. We observed that, among the three antioxidants, quercetin inhibited QLlum and QLluc, and was not toxic to the neutrophils at concentration of 50 mmol/L. BHT was toxic to the rabbit neutrophils whereas a-tocopherol did not inhibit QLlum nor QLluc. For the simple coumarins we observed that: (1) coumarin 1 did not inhibit QLlum nor QLluc; (2) coumarins 3, 4, 5 and 6 had inhibitory effects, which were similar to the quercetin and higher than coumarins 2, 7 and 8; (3) the inhibitory activities of coumarins 2 to 8 on QLluc were not related to the lipophilicity and to the molecular volume of these compounds, nor to their toxicity upon the rabbit neutrophils; (4) coumarins 3 and 4 increased the QLlum, whereas the others had no effect on this system.

Le chondrosarcome (CHS), cancer du cartilage est une tumeur chimio- et radiorésistance dont le seul traitement efficace reste la chirurgie. Une prodrogue vectorisée et activable en hypoxie, ICF05016, est actuellement développée par l’UMR 1240, et évaluée en préclinique comme potentielle alternative théra-peutique pour ce cancer. La structure de cette molécule regroupe i) une moutarde cytotoxique, ii) un vecteur ammonium quaternaire chargé positivement possédant un tropisme pour l’aggrécane, protéoglycane majoritaire de la matrice extracellulaire tumorale, iii) une gâchette de type 2-nitroimidazole permettant une activation sélective en situation d’hypoxie, une des caractéristiques principales du CHS.Ces travaux de thèse ont consisté à pharmaco-moduler cette prodrogue bispécifique ICF05016 en modifiant la position du vecteur ainsi que la nature de l’agent cytotoxique. Ainsi sept prodrogues vectorisées ont été synthétisées présentant une chaine vectrice N,N,N-triméthylpropylaminium soit en C-4, soit en N-1 du cycle imidazole. Leur activation par réduction chimique, mimant l’hypoxie, ainsi que leur affinité pour l’aggrécane ont été confirmées in tubo par des analyses de RMN 31P et de SPR, toutefois elles se sont avérées non sélectives en termes de cytotoxicité (CI50 comprises entre 15 et 1 µM, et ce, quelles que soient les conditions d’oxygénation) et faiblement sensibles à une bio-réduction enzymatique. La fonctionnalisation par un vecteur ammonium quaternaire de la gâchette 2-nitroimidazole annihile donc l’activation en hypoxie des prodrogues.Cette stratégie a ensuite été étendue au cancer de la prostate en remplaçant le vecteur ammonium quaternaire par un ligand de type urée affin pour l’antigène membranaire spécifique de la prostate (PSMA). La première molécule synthétisée, qui possède un espaceur triazole, a démontré une affinité pour le récepteur PSMA, par une étude de compétition avec un radioligand, ainsi qu’une activation in tubo par bioréduction enzymatique. Toutefois aucune cytotoxicité n’a été constatée sur les lignées LNCaP-Luc et PC3-Luc. Une seconde molécule combinant un espaceur triazole avec une séquence peptidique identifiée pour la molécule PSMA-617, actuellement en cours d’essai clinique, est actuellement développée mais sa synthèse doit être optimisée, notamment au niveau de l’étape de cycloaddition 1,3-dipolaire. / Chondrosarcoma (CHS), the malignant tumor of the cartilage, is a chemo- and radio-resistant cancer. Surgical resection is still considered the mainstay of treatment of this pathology. A dual targeted hypoxia-activated prodrug, ICF05016 was developed by the UMR 1240 and evaluated in preclinical studies as a potential therapeutic alternative for CHS. The latter is a nitroheteroaryl-based compound designed as follows: a phosphorodiamidic mustard functionalized with a quaternary ammonium (QA) used as targeting function, and a 2-nitroimidazole group to trigger fragmentation and then release the bis-alkylating mustard anion by bioreduction under hypoxic conditions, chemical hallmark of CHS.This project deals with the pharmacomodulation of ICF05016, more specifically by modification of the position of the targeting moiety as well as the nature of the cytotoxic agent. Seven QA-targeted prodrugs have been synthesized with N,N,N-trimethylpropylaminium tethered to the imidazole either in the C-4, or the N-1 position. These prodrugs were cleaved in vitro under chemical reductive conditions, which mimic in vivo hypoxia conditions. In addition, the binding of these derivatives to aggrecan was highlighted by surface plasmon resonance. In vitro assays on human CHS cells (H-EMC-SS) demonstrated quite equivalent cytotoxicities, whatever the oxygen conditions used and their evaluation as substrate of an oxygen-insensitive nitroreductase revealed the almost total lack of activation. A QA targeting moiety grafted on the trigger seems to alter hypoxia activation.New prodrugs with prostate specific membrane antigen (PSMA)-targeting ligand were synthesized to extend this HAP strategy to prostate cancer. The first tested compound, having a triazole spacer, presented selective affinity for PSMA in an in vitro binding experiment as well as activation under enzymatic reduction. However, no cytotoxicity was observed on LNCaP-Luc and PC3-Luc cells. The synthesis of a prodrug combining the spacer of PSMA-617, currently in clinical trial, and a propyltriazole moiety, was initiated but the 1,3-dipolar cycloaddition still need to be optimized.

Plant viruses have been instrumental in our understanding of the biophysical properties pertaining to non-enveloped icosahedral virus particles. A substantial amount of research has been performed over five decades on Turnip yellow mosaic virus (TYMV), arguably one of the most extensively studied icosahedral plant viruses and the type-member of the Tymovirus plant virus genus. Even with a substantial body of published scientific literature, little is known about the role of specific coat protein (CP) residues in TYMV assembly, disassembly and disencapsidation. We have shown through our mutagenesis studies that the N-terminal region of the CP that is involved in the formation of an annulus structure and is disordered in A-subunit pentamers is not essential in vivo, but annulus-forming residues are critical in ensuring virion stability and low accessibility after virus is purified (Chapter 2). We have shown that a range of amino acid residue types is tolerated within the CP N-terminus in vivo, although they can greatly affect the stability of virions and empty particles, most notably at low pH (Chapter 3). Unlike full-length CP, N-terminal deletion and substitution mutants fail to reassemble into particles in vitro (Chapter 2, 3) suggesting a critical determinant for the N-terminus in reassembly (discussed Chapter 7). This is the first documented in vitro reassembly reported for a member of the Tymoviridae family and should provide a framework for further studies. We have identified a new way to create empty artificial top component (ATC)-particles through treatment with EDTA (Chapter 6) and we also show that tymoviruses can be engineered with altered pH-dependent enhanced stability (Chapter 4). In collaboration with the Qian Wang laboratory from the University of South Carolina we have shown that an RGD (Arg-Gly-Asp) motif can be genetically engineered within the CP of TYMV, resulting in infectious particles with attractive stem-cell adhesion properties (Chapter 5). With focus on basic viral mechanisms, we have crystallized the TYMV virion and ATC particle at pH 7.7 and collected data to less than 5 Å resolution (Chapter 4, supplementary). These structures represent the first tymovirus-based structures solved above pH 5.5 and will provide insight into the N-terminal conformations within the TYMV particle. Finally, we have characterized an N-terminal CP cleavage seen after ATC formation (Chapter 4) suggesting an additional and yet uncharacterized feature associated with decapsidation. / Graduation date: 2012

Very little is known about why and how the Parkinson's disease (PD) neurodegenerative process begins and progresses. In the course of developments for treatment of PD, the discovery of the inhibition of monoamine oxidase (MAO B) was a conceptual breakthrough, and has now been firmly established. MAO B has also been implicated in the neurodegenerative processes resulting from exposure to xenobiotic amines. For example, MAO B catalyzes the first step of the bioactivation of the parkinsonian inducing pro-neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Additional insight into the mechanism of catalysis of MAO B and the mechanism of neurotoxicity by MPTP is therefore very valuable in the pursuit of the treatment of PD. / Thesis (Ph.D. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2008.

Cyclotides are small cyclic plant proteins, and this thesis addresses their cytotoxic structure-activity properties and their mode of action on human cancer cell lines. Cyclotides were isolated from Viola odorata and Viola tricolor; three novel cyclotide sequences and two known sequences, but of new origin, were identified using mass spectrometry, amino acid analysis, and Edman degradation. The cyclotide structure includes three disulphide bonds in a knotted arrangement, which forces hydrophobic amino acid residues to be exposed on the surface of the molecule; 3-D homology models of cyclotides have revealed an amphipathic surface and charged residues located at similar positions in the molecules. The charged amino acid residues were shown to play a key role in the cytotoxicity of the cyclotide cycloviolacinO2 on a human lymphoma cell line. Methylation of Glu caused a dramatic change in cytotoxicity, lowering the potency 48 times, whereas concealing the charge of Arg with 1,2-cyclohexanedione caused virtually no change in potency. Acetylation of the two Lys caused a 3-fold reduction in potency, and masking all positive charges caused a 7-fold reduction. Additionally, disturbing the amphipathic structure by reducing and alkylating the disulphide bonds abolished the cytotoxicity. The time dependency of cytotoxicity and cell gross morphology after cyclotide exposure were investigated on the lymphoma cell line. Cells exposed to 4 µM of cycloviolacinO2 showed necrotic characteristics, such as membrane disintegration, within 5 min; a membrane disruptive effect of cycloviolacinO2 was also observed in a functional assay based on liposomes at a peptide-to-lipid molar ratio of 6.5. The anti-tumour properties of cycloviolacinO2 were evaluated on three human cancer cell lines using the hollow fibre assay in vitro and in vivo. The cyclotide exhibited potent anti-tumour activity in the micro-molar concentration range on all cell lines in vitro, but no effect on tumour growth could be established in vivo.

Cyclotides are a family of small and macrocyclic proteins that have been found in Violacaee and Rubiaceae plant species. These proteins contain a cystine knot: two disulfides bonds together with their connecting peptide backbone form an embedded ring which is penetrated by a third disulfide bond. The cyclotides have been attributed a wide range of biological activities, which in combination with their chemical stability and structural plasticity have made them attractive tools for pharmaceutical applications. The sequence of eleven novel cyclotides, vibi A-K, from Viola biflora was determined by the use of both chemical (extraction and characterization) and molecular biology (cDNA analyses) approaches. A clear discrepancy in the results from the two methods was observed. Additionally, one novel cyclotide, vodo O, was isolated from Viola odorata. To correlate cytotoxic potency to sequence, vodo O and vibi D, E, G and H were tested on a lymphoma cell line. Based on the presence or absence of a cis-Pro bond, the cyclotides are divided into the Möbius and bracelet subfamilies. The bracelet proteins have a higher net charge and are more cytotoxic potent than the Möbius ones. To explore these differences, charged and hydrophobic residues in varv A (Möbius) and cycloviolacin O2 (bracelet) were chemically modified and tested for their cytotoxicity. The net-charge of the two proteins was not important for the potency. The Glu residue in cycloviolacin O2 was crucial, while this residue was of minor importance in varv A. Oxidation of the single Trp residue declined the potency significantly in both proteins. To evaluate how the surface properties correlate to the degree of cytotoxic potency, models of all cyclotides hitherto tested were constructed by homology modelling. Calculations showed that the membrane orientation of varv A and cycloviolacin O2 differed significantly, which might explain their difference in potency

The large pine weevil, Hylobius abietis L., is a major insect pest on conifer seedlings in northern Europe. Due to its feeding newly planted trees get girdled, resulting in high seedling mortality (up to 80%). As a consequence great financial losses to the forest industry occur. Today the seedlings are protected with the pyrethroid insecticide cypermethrin. This insecticide is toxic to aquatic organisms and is, from 2010, prohibited for use in Sweden by the Swedish Chemicals Inspectorate. An alternative to insecticides is to protect the seedlings with antifeedants, compounds that, either through taste or smell or both, deter the weevils from feeding. This thesis describes the search for and the synthesis of such antifeedant compounds. Bark extracts of several woody species, known to be non-palatable to the weevil, were prepared and found to display antifeedant activity against H. abietis. The major chemical constituents of the extracts were tested for antifeedant activity. Antifeedants such as eugenol, 2-phenylethanol and benzylalcohol, but also feeding stimulants such as β-sitosterol and linoleic acid, were identified. An extract of linden bark, Tilia cordata, was shown to contain nonanoic acid, a highly active antifeedant. Other aliphatic carboxylic acids were also found to display high antifeedant activities against the weevil, both in laboratory and in field tests. The enantiomers of dihydropinidine, a piperidine alkaloid present in several conifer species, were prepared by dimethylzinc mediated allylation of 2- methyltetrahydropyridine-N-oxide. When tested in micro feeding assays, no difference in antifeedant activity was found for the enantiomers. In a field test high antifeedant activity, comparable with that of the presently used insecticide cypermethrin, was found for (±)-dihydropindine. Other naturally occurring piperidine alkaloids were synthesised and also found to display high antifeedant activities in laboratory tests. Structure-activity relationships were evaluated for methoxy substituted benzaldehydes, benzoic acids and cinnamic aldehydes, -acids, -esters and -alcohols. While the carboxylic acids were inactive or even feeding stimulants, the aldehydes were the most active antifeedants / QC 20110124

Aquesta tesi és una continuació dels estudis iniciats en l'equip en el camp de les relacions estructura-activitat (SAR i QSAR) dels brassinoesteroides (BRs) mitjançant mètodes computacionals. L'objectiu general és centrar l'atenció en la cadena lateral, ja que la influència dels hidroxils depenia del tipus d'estudi (quantitatiu o qualitatiu) i la influència de l'extrem final de la cadena lateral era molt genèrica. El desenvolupament d'aquest objectiu principal ha portat a: 1. Estudiar les cadenes laterals d'anàlegs BRs androstànics: Basant-se en l'aproximació a l'anàleg actiu (AAA) prenent com a referència l'estructura de la brassinolida, s'ha vist que els anàlegs α-hidroxiester i α-aminoester són computacionalment bons candidats per presentar activitat brassinoesteroide. Un cop sintetitzats (en una tesi paral·lela), tres anàlegs amb la funcionalitat lliure han donat inactius mentre que quatre anàlegs amb la funcionalitat protegida han donat actius o moderadament actius. Basant-se novament en l'AAA, no ha estat possible explicar computacionalment i de forma inequívoca l'activitat i/o inactivitat d'aquests anàlegs. 2. Revisar i redefinir la conformació activa dels BRs: S'ha conclòs que la conformació activa in silico és l'anomenada HIP. Aquesta és la que explica amb més coherència la distribució tridimensional tan dels hidroxils com de l'extrem final de la cadena lateral de cara a explicar la unió de les cinc cadenes laterals tipus dels BRs amb el receptor. La raó per la qual s'han trobat diverses conformacions actives es troba en l'anàlisi conformacional dels BRs i no pas en els processos de selecció de la conformació activa. 3. Estudiar la influència de la conformació activa dels BRs sobre els models de QSAR: Conformacions actives estructuralment diferents han donat lloc a models quantitati-vament similars, però qualitativament diferents. Quantitativament similars perquè les parts dels BRs que correlacionen amb l'activitat són les mateixes. Qualitativament diferents perquè la contribució a l'activitat d'aquestes parts, especialment de cadena lateral, i la variació de la predictibilitat en funció de l'estructura són diferents en cada cas. Els models reduïts i el model HOMO han posat de manifest que no es pot extreure més informació dels models degut als desequilibris estructurals del conjunt de BRs que formen part del data set. El model a 1 μg/planta explica els requeriments estructurals que fan que un BR sigui actiu o inactiu. El model HIP explica els requeriments estructurals que determinen el grau d'activitat dels BRs actius. Fora de l'objectiu principal, però íntimament relacionat amb els estudis de QSAR s'ha volgut: 4. Determinar l'error experimental de la resposta i de les dades d'activitat: Comparant-los amb els errors dels models, s'observa que el model a 1 μg/planta està força ben ajustat i no té gaire marge de millora. En canvi, que el model HIP pot millorar considerablement sobretot en la predictibilitat, sempre i quan s'arreglin els desequilibris estructurals. D'altre banda, s'ha vist que els diferents tractaments estadístics realitzats en el bioassaig no afecten significativament al valor d'activitat. / Esta Tesis es una continuación de los estudios iniciados por el equipo en el campo de las relaciones estructura-actividad (SAR y QSAR) de los brasinoesteroides (BRs) mediante métodos computacionales. El objetivo general es centrar la atención en la cadena lateral, ya que la influencia de los hidroxilos dependía del tipo de estudio (cuantitativo o cualitativo) y la influencia del extremo final de la cadena lateral era muy genérica. El desarrollo de este objetivo principal ha llevado a: 1. Estudiar las cadenas laterales de los análogos BRs androstánicos: Basándose en la apro-ximación al análogo activo tomando como referencia la estructura de la brasinolida, se ha observado que los análogos α-hidroxiester i α-aminoester son computacionalmente buenos candidatos para presentar actividad brassinoesteroide. Una vez sintetizados (en una tesis paralela), tres análogos con la funcionalidad libre han resultado inactivos mientras que cuatro análogos con la funcionalidad protegida han resultado activos o moderadamente activos. Basándose nuevamente en la AAA, no ha sido posible explicar computacionalmente y de forma inequívoca la actividad o inactividad de estos análogos. 2. Revisar y redefinir la conformación activa de los BRs: Se ha llegado a la conclusión que la conformación activa in silico es la llamada HIP. Esta es la que explica con más coherencia la distribución tridimensional tanto de los hidroxilos como del extremo final de la cadena lateral a fin de explicar la unión de las cinco cadenas laterales tipo de los BRs con el receptor. La razón por la cual se han encontrado diversas conformaciones activas se encuentra en el análisis conformacional y no en los procesos de selección de la conformación activa. 3. Estudiar la influencia de la conformación activa de los BRs en los modelos de QSAR: Conformaciones activas estructuralmente diferentes han dado lugar a modelos cuantita-tivamente similares, pero cualitativamente diferentes. Cuantitativamente similares porque las partes de los BRs que correlacionan con la actividad son las mismas. Cualitativamente diferentes porque la contribución a la actividad de dichas partes, especialmente de la cadena lateral, y la variación de la predictibilidad en función de la estructura son diferentes en cada caso. Los modelos reducidos y el modelo HOMO han puesto de manifiesto que no se puede extraer más información de los modelos debido a los desequilibrios estructurales del conjunto de BRs que conforman el "data set". El modelo a 1 μg/planta explica los requisitos estructurales que hacen que un BR sea activo o inactivo. El modelo HIP explica los requisitos estructurales que determinan el grado de actividad de los BRs activos. Fuera del objetivo principal, pero íntimamente relacionado con los estudios de QSAR se ha querido: 4. Determinar el error experimental de la respuesta y de los datos de actividad: Compa-rándolos con los errores de los modelos, se observa que el modelo a 1 μg/planta está bastante bien ajustado y tiene poco margen de mejora. En cambio, el modelo HIP puede mejorar considerablemente sobretodo en la predictibilidad, siempre y cuando se solucionen los desequilibrios estructurales. Por otro lado, se ha observado que los diferentes tratamientos estadísticos realizados en el bioensayo no afectan significativamente al valor de actividad. / This Thesis is the continuation of the studies started by our laboratory in the field of brassinosteroids (BRs) structure activity relationships (SAR and QSAR) using computational methods. The main aim is to focus the study on the side chain, due to the influence of hydroxyl groups depends on the study (quantitative or qualitative) and the influence of the side chain end is very generic. The development of this goal has leaded to: 1. Study the side chain of androstanic BRs analogues: Based on active analogue approach (AAA) taking brassinolide as the reference structure, it has been shown that α-hydroxyester and α-aminoester analogues are computationally good candidates to elicit brassinosteroid activity. Once synthesized (in a parallel thesis), three analogues with free functionality have result inactive but four analogues with protected functionality have result active or mild active. Based once again on AAA it has not been possible explain computationally and unequivocally the activity and/or inactivity of these analogues. 2. Revise and redefine the active conformation of BRs: It has been concluded that in silico active conformation is the named as HIP. This explains more consistently the tridimensional distribution of both the hydroxyls and the end of the side chain in order to explain the union of the five side chain types with BRs receptor. The reason for having found several active conformations is in BRs conformational analysis not in the active conformation selection procedures. 3. Study the influence of BRs active conformation in QSAR models: Active conformations structurally different has lead to models which are quantitatively similar but qualitatively different. Quantitatively similar due to the parts of BRs that correlate with activity are the same. Qualitatively different due to this parts contribution, especially the side chain, and the structure depending variation of predictability are different on each model. Reduced models and HOMO model has shown that get more information from the models is not possible due to a structural imbalance in BRs data set. The 1 μg/plant model explains the structural requirements that make BRs active or inactive. The HIP model explains the structural requirements that determine the activity degree of active BRs. Out of the main aim, but close related to QSAR studies I wanted to: 4. Determine the experimental error of both the response and the activity data: Compared with models error, it is observed that the 1 μg/plant model is really well adjusted and has little improvement margin, but the HIP model can be considerably improved, overall in predictability. On the other hand, it has been shown that the different statistical treatments done in bioassay do not affect in a significant way the activity value.

<p>The large pine weevil, Hylobius abietis L., is a major insect pest on conifer seedlings in northern Europe. Due to its feeding newly planted trees get girdled, resulting in high seedling mortality (up to 80%). As a consequence great financial losses to the forest industry occur. Today the seedlings are protected with the pyrethroid insecticide cypermethrin. This insecticide is toxic to aquatic organisms and is, from 2010, prohibited for use in Sweden by the Swedish Chemicals Inspectorate. An alternative to insecticides is to protect the seedlings with antifeedants, compounds that, either through taste or smell or both, deter the weevils from feeding. This thesis describes the search for and the synthesis of such antifeedant compounds.</p><p>Bark extracts of several woody species, known to be non-palatable to the weevil, were prepared and found to display antifeedant activity against H. abietis. The major chemical constituents of the extracts were tested for antifeedant activity. Antifeedants such as eugenol, 2-phenylethanol and benzylalcohol, but also feeding stimulants such as β-sitosterol and linoleic acid, were identified. An extract of linden bark, Tilia cordata, was shown to contain nonanoic acid, a highly active antifeedant. Other aliphatic carboxylic acids were also found to display high antifeedant activities against the weevil, both in laboratory and in field tests.</p><p>The enantiomers of dihydropinidine, a piperidine alkaloid present in several conifer species, were prepared by dimethylzinc mediated allylation of 2- methyltetrahydropyridine-N-oxide. When tested in micro feeding assays, no difference in antifeedant activity was found for the enantiomers. In a field test high antifeedant activity, comparable with that of the presently used insecticide cypermethrin, was found for (±)-dihydropindine. Other naturally occurring piperidine alkaloids were synthesised and also found to display high antifeedant activities in laboratory tests.</p><p>Structure-activity relationships were evaluated for methoxy substituted benzaldehydes, benzoic acids and cinnamic aldehydes, -acids, -esters and -alcohols. While the carboxylic acids were inactive or even feeding stimulants, the aldehydes were the most active antifeedants</p>

The debilitating effects of spinal cord injury can be attributed to a lack of regeneration in the central nervous system. Identification of growth-promoting pathways, particularly ones that can be controlled by small molecules, could provide significant advancements in regenerative science and lead to potential treatments for spinal cord injury. The biological investigations of neuroregenerative small molecules, specifically the natural products clovanemagnolol and vinaxanthone, have been expanded to a whole organism context using the nematode Caenorhabditis elegans (C. elegans) as a tool for these studies. A straightforward assay using C. elegans was developed to screen for compounds that promote neuronal outgrowth in vivo. This outgrowth assay was then used to guide the design of chemically edited analogs of clovanemagnolol that maintained biological activity while possessing structures amenable to further modification for mechanism of action studies. Pull-down experiments using affinity reagents synthesized from a neuroactive structural derivative, clovanebisphenol, and the C. elegans proteome combined with mass spectrometry-based protein identification and genetic recapitulation using mutant C. elegans identified the putative protein target of the small molecule as a kinesin light chain, KLC-1. Furthermore, the small molecule-promoted regeneration of injured neurons in vivo was studied using laser microsurgery to cut specific axons in C. elegans followed by treatment with a library of analogs of the growth-promoting natural product vinaxanthone. Enhanced axonal regeneration was observed following small molecule treatment and the results were used to determine the structure-activity relationship of vinaxanthone, which may guide future development of potential drug candidates for the treatment of spinal cord injury. / text