Dissertations / Theses on the topic 'Biological activity'

In 1963 the study of marine natural products was just beginning and there were only a handful of public records with only one of these publications reporting a new compound. There has since been 9220 papers published, reporting on 24 662 new compounds. Annually a variety of cembrane diterpenoids with a range of biological activities are reportedly isolated from marine soft coral, and most notably these compounds possess anticancer properties. The investigation of a marine soft coral, Sinularia notanda, for novel bioactive compounds is presented here. The aim was to identify compounds active against key HIV enzymes as well as against cervical cancer, an opportunistic malignancy affecting many HIV positive women in Sub-Saharan Africa. A methanol extract of S. notanda was prepared and the cytotoxicity of the crude extract tested against a cervical cancer (HeLa) cell line using sodium 2,3-bis-(2-methoxy-4-nitro- 5-sulfophenyl)-2H-tetrazolium- 5-carboxanilide (XTT). The crude extract was also tested for HIV-1 protease inhibition using a direct enzyme assay, and antioxidant activity determined using 1,1-diphenyl-2picrylhydrazyl (DPPH). Bioassay guided fractionation, column chromatography and thin layer chromatography were used to identify active fractions from the total extract as well as to isolate biologically active compounds. The isolated compounds were tested for cytotoxic activity against four different cell lines. Nuclear magnetic resonance spectroscopy and x-ray crystallography were used for structure determination. The crude ethyl acetate fraction of S. notanda had a 50% cytotoxic concentration (CC50) of 33.82 ?g/ml, exhibited moderate inhibition of HIV-1 protease (between 40 and 60% inhibition), was unable to inhibit reverse transcriptase and showed antioxidant activity at a concentration (IC50) of 76.15 ?g/ml. In comparison, the crude methanol fraction had a CC50 of 145.80 ?g/ml, showed significantly lower protease inhibition (p < 0.05), and showed antioxidant activity at an IC50 of 27.16 ?g/ml. Extracts from natural sources including soft coral are routinely screened for free radical scavenging ability and the DPPH assay is one of the fastest methods to do so. Free radicals are known to induce oxidative damage to biomolecules which can eventually lead to diseases such as cancer. Oxidative damage can also be caused by HIV infection and oxidative stress levels may be exacerbated by antiretroviral treatment. Free radical scavenging antioxidants can provide protection against the damage caused by reactive oxygen species and are therefore considered as important nutraceuticals. Structural elucidation of a crystal isolated from S. notanda using NMR and x-ray crystallography confirmed a unique cembrane diterpenoid structure. The identified compound [(1R,3R,5S,12R,13S,E)-12-hydroxy-5,9,13-trimethyl-16-methylene-4,14-dioxatricyclo[11.3.2.03,5]octadec-8-en-15-one] showed moderate HIV-1 protease inhibition. This compound was abbreviated as CPD1. A second isolated compound designated E7 was found to be toxic to human leukemic monocyte lymphoma (U937) cells at 25 ?g/ml resulting in cell viability of less than 10%. Structure elucidation of E7 by 1D and 2D NMR as well as mass spectrometric analysis confirmed this compound to be structurally identical to the one isolated as a crystal and it also exhibited similar cytotoxic behaviour. Although this is not the first time CPD1 has been isolated from a coral of the Sinularia genus, data presented in this dissertation represents the first time that the compound was isolated from S. notanda. In Sinularia flexiblis CPD1 was isolated as a ketone (carbon-oxygen double bond at C12) while in the current study it was isolated from S. notanda as an alcohol (hydroxyl group at C12). Ketones are produced from the oxidation of secondary alcohols and the change in functional groups at C12 of CPD1 could be attributed to different organic solvents being used for initial extraction. A third compound was isolated and showed < 50% inhibition of HeLa cell growth and > 90% inhibition of U937 cell growth at 50 ?g/ml. Structure elucidation data identified the compound as 3-caffeoylquinic acid. This compound is not synthesised by the coral itself but is produced by algae that the coral ingested. The cembrane diterpenoid isolated from S. notanda in this study showed moderate inhibition of HIV-1 protease and selective cytotoxicity towards the U937 lymphoma cell line (selectivity index > 2). These responses are not unusual as cembrane diterpenoids with anti-cancer potential are increasingly being isolated from soft corals.
Dissertation (MSc)--University of Pretoria, 2015.
Biochemistry
MSc
Unrestricted

Cancer is a group a diseases that involves abnormal cell growth with potential to invade or spread to other parts of the body, and it represents the second leading cause of death in developed countries. Cisplatin is one of the most chemotherapeutic drug. In spite of its great efficacy, it shows several side effects and most patients develop a resistance to cisplatin. To overcome the cisplatin resistance, drugs are often administered in combination in order to exploit the drug synergy. After discovery of cisplatin, the research focused on metal complexes less toxic, more effective and that exploit synergistic effect when used in combination. In this work I studied new copper, zinc and vanadium complexes with biological activity. I tested in vitro the studied compounds alone and in combination with drug currently in use against a panel of wild type tumour cell lines and their cisplatin-resistant sublines. I applied chemometric tools such as experimental design (ED) and artificial neural networks (ANNs) to the biochemical data collected. Finally, I used the artificial neural networks to evaluate the cell culture cross-contamination. I selected a new family of copper(II) complexes with 1,10-phenanthroline (phen), 1,10-phenanthrolin-5,6-dione (phendione), and 1,10-phenanthrolin-5,6-diol (phendiol) for the synthesis of new antiproliferative agents. Considering that the DNA is an important target for several cytotoxic metal complexes, I studied the interaction of these Cu(II) complexes with DNA. I tested the ligands and complexes against normal and tumour derived human cell lines. I tested combinations of the studied complexes and cisplatin for their potential synergistic effect against a panel of wild type tumour cell lines and their cisplatin-resistant sublines. I evaluated the selectivity of drug combinations testing the compounds also against ex vivo cultures of human normal cell lines. Considering that the synergy may arise from a chemical reaction among the drugs, I studied the possible formation of new adducts between cisplatin, copper(II) complexes and glutathione. I studied the phospholipid profile of wild type human cancer cell lines and their cisplatin-resistant sublines, given that changes in lipid composition and distribution on the cell membranes have been observed in cancer cells. The in vitro cultured cell lines are widely used as model in biomedical research and the cross-contamination of cell lines represents a highly relevant problem. The ex-post discovery of erroneous results and conclusions led to paper retraction and many high-impact journals started to adopt a zero-tolerance policy requiring confirmation of cell line identity as prerequisite for publication. On the base of these considerations, I decided to develop and validate a method for evaluation of cell culture cross-contamination. I also studied zinc and vanadium complexes. Zinc is an essential metal ion involved in a wide variety of biological processes and several proteins bind zinc for their proper functioning. I studied zinc complexes with the drug methimazole (MeImHS) and its anion (MeImS) in order to provide information for the structure prediction and reactivity of Zn-metalloproteins and -metalloenzymes. Vanadium plays a number of roles in biological systems and vanadocene dichloride was the first discovered vanadium species with antitumour activity. Considering that the mechanism of the anticancer agent vanadocene dichloride is closely related to the biotransformation in the blood plasma, I studied the speciation of vanadocene dichloride in the plasma under physiological conditions. In order to prepare new metal complexes, I also synthesized and characterized a new group of Schiff base ligands derived from salicyladehyde and six natural amino acids. For the analysis of the collected data, I used the ED to set up the experiments for the evaluation of the synergistic effect of drug combinations, and for the study of the possible formation of new adducts between cisplatin, glutathione and studied complexes. I used ANNs for predict and quantify the synergism of drugs, and for the evaluation of cell culture cross-contamination levels.

Influenza is a major health problem worldwide, as the periodic pandemics of the 20th century have highlighted. Some countermeasures have been developed and have indeed diminish the devastating effects of the disease, such as modern health care, vaccination and novel medicines. Yet, the threat of a recombinant mutant virus that could affect millions of people and the recently discovered resistance of some virus strains to the current available treatments, render the need for new ways of fighting influenza A virus urgent. In the present dissertation we have taken amantadine as a model, an antiviral drug in disuse at the moment due to the appearance of resistant strains, that targets a viral proton channel named M2. Thus, we have synthesized and evaluated several polycyclic amines as potential wild-type M2 channel blockers and amantadine-resistant mutants, like V27A, as well. We have succeeded in the obtention of highly potent wild-type and V27A inhibitors and most remarkable, some of them exhibited a dual activity on both M2 channels. Noteworthy, among the prepared compounds, a polycyclic guanidine presented the higher activity ever recorded against the V27A mutant. Again working with polycyclic molecules but from a more theoretical point of view, the monomer, dimer and dihydrodimer of a highly strained pyramidalized alkene were synthesized and fully characterized. Importantly, the dimer featured four cyclohexanes in a frozen boat conformation and possessed hydrogen flagpole interactions that were relevant to theoretical organic chemists.
La grip presenta un greu problema arreu del món, com les pandèmies del segle XX han demostrat. S’han pres algunes mesures per lluitar-hi que han realment disminuït els efectes devastadors de la malaltia, com son la hospitalització moderna, la vacunació i les noves medicines. Tot i així, l’amenaça d’un virus recombinant mutant que pugui afectar a milions de persones i la recentment descoberta resistència d’algunes soques del virus als tractaments actuals, han provocat que la necessitat per a noves maneres de lluitar contra la grip A sigui urgent. En la present Tesi, hem pres amantadina com a model, un medicament antiviral actualment en desús degut a l’aparició de soques resistents, que té com a diana un canal de protons del virus anomenat M2. Així doncs, hem sintetitzat i avaluat diverses amines policícliques com a potencials blocadors del canal salvatge M2 i mutants resistents a amantadine, com el V27A, també. Hem tingut èxit en l’obtenció de potents inhibidors del canal salvatge i del V27A i lo més destacable és que alguns han mostrat una activitat dual en ambos canals M2. Cap remarcar que, entre els compostos preparats,una guanidina policíclica va presentar l’activitat més alta mai enregistrada contra el mutant V27A. Seguint amb les molècules policícliques però des d’un punt de vista més teòric, el monomer, dimer i dihidrodimer de un alqué altament piramidalitzat van ser sintetitzats i completament caracteritzats. Cal subratllar que, el dimer posseïa quatre ciclohexans en conformació bot congelat i mostrava interaccions entre els hidrògens flagpole que eren rellevants per als químics orgànics teòrics.

Diets rich in cruciferous vegetables· are associated with a reduced risk of cancer. This reduction in risk may be attributed to isothiocyantes (lTCs), which are the degradation products of glucosinolates. Sulforaphane (4-methylsulfinylbutyl isothiocyanate, SF) is the predominant ITC formed from heading broccoli ('calabrese') and iberin (3-methylsulphinyl propyl isothiocyanate) is the predominant ITC formed from purple sprouting broccoli. While, there are many studies regarding the biological activity of SF, there are few studies associated with iberin. Moreover, the majority of studies have been undertaken with transformed cells, usually epithelial in origin, that have cancerous phenotypes. In this study, primary epithelial and fibroblast cells derived from benign prostatic hyperplasia tissue (BPH) were used as a more appropriate model of normal prostate tissue. There are thought to be. complex interactions between epithelial and fibroblast .cells during the initiation and progression of prostate cancer. Affymetrix array was used to report global gene expression changes in both primary fibroblast and epithelial cells on exposure to physiologically appropriate concentrations ofSF and iberin. The majority of genes altered between iberin and SF treated cells were different; however, according to GenMAPP analysis, these genes were involved in some ofthe same pathways. Genes altered by both iberin and SF fitted into four main gene ontology categories: xenobiotic metabolism, cell cycle arrest, apoptosis and oxidative stress. A number ofgenes from these categories were selected for real time RT-PCR analysis, which showed a novel increase in gene expression on iberin and SF exposure. These genes were eyelin-dependent kinase inhibitor lA (p21wafllCipl), kruppel-like factor 4 (KLF4), pleiomorphic adenoma gene-like 1 (PLAGLl), tumour necrosis factor receptor super family, member lOb (TNFRSFlOb) and thioredoxin reductase 1 (TrxRl). This study is also the first evidence that iberin and SF reduce the mRNA expression of interferon induced transmembrane protein (lFITMl), chondroitin sulfate proteoglycan 2 (CSPG2) and vimentin (VIM) in prostate epithelial cells. Histone acetylation was investigated as a mechanism of global gene control by SF and iberin, however there was inconsistent evidence that either isothiocyante controls gene expression through histone deacetylase inhibition.

Acylcarnitines are intermediates of fatty acid oxidation and accumulate as a result of a metabolic defect. Accumulation of palmitoylcarnitine (palcar), a long-chain acylcarnitine, has been observed in diabetes mellitus type II, obesity and kidney cancer. Certain dietary intervention studies have been shown to reduce serum and urinary levels of acylcarnitines. It was shown that palcar accumulates in prostate cancer tissue, possibly indicative of the metabolic changes associated with cancer development, but that palcar at high concentrations may have activities that could be associated with cancer development. Through the use of cancerous (PC3, DU145) and non-cancerous (PNT1A, BPH) cell models, it was shown that high levels of palcar could induce gene expression of the inflammatory cytokine IL-6, and induce its secretion in PC3 cells. This was associated with the rapid influx of Ca2+. Through the use of various metabolic inhibitors, it was shown that Ca2+ influx includes the activation of G-protein coupled receptors, L-type Ca2+ channels and PI3K pathway. However, it was shown through the use of global gene arrays that lower levels of palcar with uncorrected P value induced many changes in gene expression in PNT1A cells. A comparison with the global changes induced by DHT, an androgen linked to prostate cancer progression, revealed a significant overlap in activity between palcar and DHT, suggesting that palcar may have a potential role in promoting cancer progression. In PC3 cells through the use of real time RT-PCR palcar was shown to induce glycolysis. In conclusion, it is suggested that palcar may represent a potential biomarker of the metabolic dysfunction associated with prostate cancer. At physiological levels palcar had no effects on the prostate cancer cells, however, at high levels palcar drive tumour development through inducing key inflammatory cytokine and inducing changes in gene expression associated with glycolysis.

The ability of glucosinolates to act as host recognition cues and oviposition stimulants for root flies has been previously established. To further investigate the interactions between pest and glucosinolate a number of simple and complex glucosinolates were synthesised and tested by contact chemoreception. A crude structure-activity relationship was identified whereby the stimulatory activity of the glucosinolate increased as the alkyl side chain was increased from propyl to pentyl, heptyl and nonyl. Comparison of the novel synthetic glucosinolate, naphthylmethyl glucosinolate, with glucobrassicin, a naturally occurring indole derivative, showed the former to have little or no activity whereas the latter is the most active natural stimulant. The synthetic glucosinolates were also demonstrated to act as substrates for the enzyme myrosinase, being hydrolysed to ?-D-glucose and the corresponding isothiocyanate. In addition, (7-methoxycarbonylheptyl) glucosinolate, prepared as a precursor to (7-carboxyheptyl) glucosinolate, was found to be a substrate. High resolution NMR studies of the latter compound showed this acidic glucosinolate and indeed alkyl glucosinolates to adopt an unexpected conformation in aqueous solution. Furthermore, a number of alkyl thiohydroximates were synthesised and used as HPLC and LC-MS standards to aid glucosinolate identification. Isothiocyanates have been identified as chemopreventative agents which inhibit carcinogen activation mediated by cytochrome P450 enzymes. The postulated oxidation of isothiocyanates to isocyanates by these enzymes, was studied using a number of chemical model systems. Oxidation of isothiocyanates was efficiently achieved using dimethyl dioxirane (DMD). Although, the resulting isocyanates could not be isolated, their production was confirmed by GC/MS and FT-IR analysis of reaction solutions. A number of ureas were also prepared by trapping the isocyanates in situ. These compounds were demonstrated to arise from the isocyanate and not oxidation of the corresponding thiourea. In addition, peracids were found to produce isocyanates, although less efficiently.

Doutoramento em Química
O uso inapropriado de antibióticos no tratamento de doenças infeciosas tem levado a um aumento da resistência de diversos microrganismos patogénicos, o que apresenta atualmente um problema de saúde pública e tem motivado a procura de estratégias alternativas para o controlo destes microrganismos. Por outro lado, a procura de novas moléculas ou novas combinações de moléculas para o combate ao cancro é um assunto em constante desenvolvimento. Na presente dissertação descreve-se o trabalho desenvolvido para a obtenção de conjugados de ftalocianina–sulfonamida e a avaliação da atividade dos novos compostos como fotossensibilizadores. Os conjugados foram idealizados com o intuito de promover um possível efeito sinérgico das suas unidades constituintes, nomeadamente propriedades antitumorais e/ou antimicrobianas. Para tal, desenvolveram-se métodos de síntese de sulfonamidas e de conjugados de ftalocianina–sulfonamida. Algumas das ftalocianinas obtidas foram testadas como fotossensibilizadores na eliminação fotodinâmica de células tumorais e de bactérias, utilizando-se as linhas celulares de carcinoma de células escamosas orais (HSC3) e de queratinócitos orais (HaCaT), e as bactérias Escherichia coli (Gram-negativo) Staphylococcus aureus (Gram-positivo) como modelos biológicos. Os resultados destes estudos revelaram que as ftalocianinas estudadas são muito promissoras como fotossensibilizadores para a inativação fotodinâmica de células tumorais e de microrganismos. Por outro lado, foi também desenvolvido um ensaio enzimático para avaliar a atividade dos novos compostos como inativadores da enzima anidrase carbónica, em particular a isoforma IX que se encontra sobre-expressa em células tumorais e é bem conhecida como reguladora do pH em processos de hipoxia e acidose metabólica. Este estudo vem dar mais um passo no conhecimento científico das ftalocianinas e evidencia o potencial das ftalocianinas sulfonadas na perspetiva do controlo de infeções e da tumorogénese.
The inappropriate use of antibiotics in the treatment of infections has led to an increase in the resistance of several pathogenic microorganisms, which represents a major public health issue and triggered the search for novel antimicrobial drugs. On the other hand, the search for new molecules or new combinations of molecules for the fight against cancer is a subject in constant development. The present dissertation describes the work developed to obtain phthalocyanine–sulfonamide conjugates and the biological evaluation of these compounds as photosensitizers. These conjugates were designed to explore their antimicrobial and/or antitumor properties. Methods for the synthesis of sulfonamides and phthalocyanine–sulfonamide conjugates were developed. Some of the phthalocyanines obtained were tested as photosensitizers for the photodynamic inactivation of tumor cells and bacteria. HSC3 oral squamous cell carcinoma, HaCaT 'normal' keratinocytes, and the bacteria Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) were used as biological models. The phthalocyanines studied proved to be very promising to be considered in future studies in the perspective of the photodynamic inactivation of tumor cells and microorganisms. On the other hand, an enzymatic assay was also developed to evaluate the activity of the compounds obtained as inactivators of the enzyme carbonic anhydrase, in particular the IX isoform that is overexpressed in tumor cells and is well-known as pH regulator in processes of hypoxia and metabolic acidosis. This study represents a contribution to the application of phthalocyanines, and in particular sulfonated phthalocyanines, in the control of infections and tumorigenesis.

Lipopolysaccharide (LPS), a major constituent of Gram-negative bacteria, is implicated as the key factor in the development of the Systemic Inflammatory Response Syndrome (SIRS). LPS can arise from an underlying bacteraemia, but given that the majority of patients with SIRS have no detectable bacteraemia, then LPS derived from the gut must be considered. Bacteroides species outnumber the enterobacteria such as E.coli in the gut by approximately 1000-fold. Although Bacteroides LPS is less endotoxic, by simple arithmetic there must be as much biological potential from the LPS of Bacteroides as from E.coli. This thesis re-examines the biological activity of Bacteroides LPS and its possible role in the development of SIRS. LPSs were extracted from seven Bacteroides species by three different techniques: the phenol-water (PW), the phenol-chloroform-petroleum (PCP) and Triton-Mg²⁺. The biological activity of these Bacteroides LPSs was compared to that of an E.coli O18K^- LPS control. In general, Bacteroides LPSs prepared by the PW method were found to have a significantly higher activity in a mouse lethality model, LAL assay, TNF and IL-8 induction assays, than LPS extracted by the PCP or Triton methods. Bacteroides LPS extracted by the PCP method had consistently low activity in all assays. LPS from B.fragilis NCTC 9343 and B.caccae had a consistently higher activity than LPS from B.vulgatus and B.thetaiotaomicron in most assays. Differences in activity between B.fragilis NCTC 9343 LPS grown in different media was seen. The PW method selected for greater amounts of carbohydrate and KDO and the PCP the least. Further information from sub-population studies, Percoll profiles, chemotype on PAGE and chemical analysis failed to account for differences in biological activity between extraction methods and Bacteroides species.

The improved synthesis and resolution of the first naturally occurring polyarsenical, Arsenicin A [As₄0₃(CH₂)₃] has been achieved. The putative structure of Arsenicin A resembled that of arsenic(III) oxide (As40 6) , but where three of the oxygen atoms in the inorganic oxide had been replaced by methylene groups in a chiral C2 arrangement. The five-step synthesis involves reduction of methylenebis(phenylarsinic acid) to the bis( secondary arsine) (RAs * ,RAs *)-( ± )/(RAs * ,S As *)-CH2[ AsHPh h followed by deprotonation and reaction of the resulting diarsenide with ( chloromethyl)diphenylarsine to give the tetra( tertiary arsine) (RAs * ,RAs *)-( ± )l(RAs * ,S As *)-CH2[ AsPh(CH2AsPh2) )i. Replacement of the six phenyl groups in the tetra(tertiary arsine) with iodine was accomplished by reaction with anhydrous hydrogen iodide to give the hexaiodoarsine ( RAs *, RAs *)-( ± )/ ( RAs * ,S As *)-CH2 [ Asl(CH2Ash) ]i, crystals of the (RAs * ,S As*) diastereomer being characterised by X-ray crystallography. Hydrolysis of the hexaiodoarsine with aqueous ammonia gives (±)-Arsenicin A as colourless air- and moisture-stable crystals in an overall yield of 36% after column chromatography and recrystallisation from benzene. (±)-Arsenicin A exhibits strong absorptions in the UV region, even though the molecule contains no obvious chromophore. A theoretical investigation revealed that the absorption was facilitated by through-space and through-bond interactions between lone pairs on the arsenic and oxygen atoms and the organometallic framework of the molecule. (±)Arsenicin A was resolved with >99% efficiency by preparative chiral HPLC on a Chiralpak IA column with use of dichloromethane as eluent and the structure and absolute configuration of(S)-(- )-Arsenicin A were established by X-ray crystallography. The individual enantiomers of (±)-Arsenicin A racernise in solution in the presence of traces of acid and high-level ab initio calculations have been carried out to examine the mechanism of the process. (±)-Arsenicin A exhibits a 21-fold greater inhibition of the induction of proliferation arrest and induces cell death at a 27-fold lower concentration in the acute promyelocytic leukemia (APL) cell line than the current "arsenical gold standard", arsenic(III) oxide (Trisenox®). Treatment of a benzene solution of (±)-Arsenicin A with aqueous sodium sulfide produces first the trisulfur analogue (±)-Arsenicin A-S3 (AsA-83) (not isolated), which undergoes reductive desulfurisation with excess sulfide to give the disulfide (±)Arsenicin A-S2 and then the monosulfide (±)-Arsenicin A-S1 (AsA-81). The derivative (±)-Arsenicin A-S2 exists as a pair of separable diastereomers, (±)-Arsenicin A-a-S2 (AsA-a-82) and (±)-Arsenicin A-{3-S2 (AsA-iS-82). The crystal structures of AsA-a-82, AsA-Jj-82 and AsA-81 have been determined. The disulfides have the cage-like structure of the mineral uzonite (As4Ss) in which there is a single As-As bond; the disulfide diastereomers each contain four chiral arsenic stereocentres. The monosulfide cage AsA- 81 contains two As- As bonds and has a structure related to the mineral realgar (a-As4S4) in which three of the sulfur atoms have been replaced by methylene groups in a Ci-chiral arrangement. As found for (±)-Arsenicin A, the sulfur derivatives exhibit strong UV absorptions and can be resolved on a Chiralpak IA column. The selenium derivatives(±)Arsenicin A-Se1 (AsA-8e1), (±)-Arsenicin A-a-Se2 (AsA-a-8e2) and (±)-Arsenicin A-{3- Se2 (AsA-Jj-8e2) have been synthesised by the reaction of a benzene solution of(±)Arsenicin A with sodium hydrogen selenide. The crystal structure of the monoselenide AsA-8e1 was determined and shown to have the realgar-type, cage structure. Preliminary results indicate that AsA-81 and AsA-8e1 are considerably more potent against the acute promelocytic leukemia cell line than arsenic(III) oxide and all histotypes of ovarian cancer cell lines tested than the standard chemotherapeutic drugs, cisplatin and carboplatin.

My PhD project has been focused on the design, synthesis and biological activity studies of glicomimetics. During these three years I have been mainly interested in three different arguments (three proteins: API, Akt, CIM6Pr) that I will elucidate in detail in this thesis. Even if the target proteins (enzymes and receptors) are very different, these three arguments have a common element. In all cases, starting from the known mechanistic, structural and biological information of the single protein, I have designed glycomimetics that are potentially able to interact with the target protein. All the studied proteins are biologically relevant, so the final aim of the synthetic effort is always a biological test, in order to check the hypothesis done during the design step and to get new useful information about the biological system. The synthesized compounds are always glycomimetics or, more generally speaking, glycans: they can be arabinose-based mimetic of A5P, glucose-based mimetic of an inisitol structure, or synthetic glycoproteins. The most used analytical instrument for the characterization of all the synthesized compounds and for the study of their interaction with enzymes is NMR spectroscopy. The wide use of this analytical technique led me to study topics that were not related to carbohydrates themselves, but related to NMR techniques. Therefore, I increased my knowledge and applied NMR spectroscopy to the biomaterial field and in particular I have studied the reactivity of reaction systems widely used for biomaterial production using NMR. I will briefly discuss these works in the last part of my thesis.

Cell-penetrating peptides (CPPs) have emerged as a group of remarkable delivery vectors for various hydrophilic macromolecules, otherwise excluded from cells due to the protective plasma membrane. Unbiased conclusions regarding e.g. uptake mechanism, intracellular distribution and cargo delivery efficacy is complicated by the use of different methodological parameters by different laboratories. The first paper in this thesis introduced unifying protocols enabling comparison of results from different research groups. One of these methods, HPLC, was used in paper II to investigate CPP uptake and degradation in yeasts. Both parameters varied depending on peptide and yeast species; however pVEC emerged as a promising delivery vector in yeast since it internalized into both species tested without concomitant degradation. Protein mimicry was another investigated phenomenon and in paper III a 22-mer peptide from the p14Arf protein (Arf (1-22)) was found to be sufficient for retaining its function as a tumor suppressor. This peptide comprised a combination of apoptogenic property and CPP in one unity, thus providing opportunity to conjugate cytotoxic agents boosting the tumoricidal activity. Surprisingly, a partially inverted control peptide to Arf (1-22), called M918, was found to be an extraordinary CPP. In paper IV, it was shown to be superior to well-established CPPs in delivery of both peptide nucleic acids and proteins. Albeit the promising results these two peptides displayed, their utility in vivo, as with all peptides, is hampered by rapid degradation. With the aim of improving their stability, Arf (1-22) and M918 were synthesized with D-amino acids in the reverse order, a modification called retro-inverso (RI) isomerization. Their cell-penetrating ability was retained, but the treated cells displayed unexpected morphological alterations indicative of apoptosis. The presented results demonstrate the versatility of CPPs, functioning as vectors in both yeast and mammalian cells and as protein mimicking peptides with biological activity. Their potential as drug delivery agents is obvious; however, peptide degradation is an issue that requires further improvements before clinical success is in reach.
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 5: In press.

Ultra high molecular weight polyethylene (UHMWPE) wear particles have been implicated as one of the major causes of late aseptic loosening. Particles of a critical and volume promote an inflammatory response from macrophages, primarily. which leads to cytokine release and the resorption of bone at the bone-implant interface.

Propolis or bee glue, is collected by bees and contains secondary metabolites largely derived from trees or shrubs; it has been used traditionally as a natural remedy with a wide range of biological activities. Its chemical composition is highly complex and variable, and it has been studied in detail worldwide except in Africa. This study investigated the chemical composition and activity of African propolis against blood stream form of Trypanosoma brucei, the causative agent for sleeping sickness that threatens a large population of both humans and animals in sub-Saharan Africa. Extracts of propolis samples (n=12) collected from different regions in Nigeria and one sample collected from South Africa were chemically profiled by using various analytical techniques. These included high performance liquid chromatography (HPLC), coupled with different detection systems including evaporative light scattering detection (ELSD), ultraviolet detection (UV), and high resolution mass spectrometry (HRMS), along with gas chromatography- mass spectrometry (GC-MS) and proton-nuclear magnetic resonance (1H-NMR).Principal components analysis (PCA) of the processed LC-MS data collected was used in order to characterize samples according to their chemical composition. PCA demonstrated the uniqueness in chemical composition of some samples that were also active against Trypanosoma brucei. Therefore, the study proceeded to investigate in detail four samples collected mainly from the southern part of Nigeria. An optimized medium pressure chromatographic technique was used to isolate some of the component(s) responsible for the anti-trypanosomal activity. Two samples collected from Rivers State Nigeria had a different appearance from the rest of the propolis samples, being red in colour and had the highest trypanocidal activity (EC50=4.2 and 6.9 μg/mL) respectively. Their chemical composition was comparable to that of Brazilian red propolis. Fractionation work led to the isolation of ten phenolic compounds including calycosin, liquiritigenen, pinocembrin, vestitol, medicarpin, 8-prenylnaringenin, 6-prenylnaringenin, propolin D, macarangin and a new benzofuran. All compounds structurally elucidated by 1D and 2D Nuclear Magnetic Resonance (NMR) spectroscopy and LC-MSn. Some compounds showed strong inhibitory activity against trypanosomes such as medicarpin (MIC=11.5 μM) and propolin D (MIC=7.4 μM), macarangin (EC50 =18.5 μM), 8-prenylnaringenin (EC50= 17.9 μM), and vestitol (EC50= 30.5 μM). The new benzofuran was moderately active with (EC50=58.01 μM). Fractionation of the propolis sample collected from the Ugelli/Delta sample led to isolation of three compounds 1,3,7-trihydroxy-2,8-di-(3-methylbut-2-enyl) xanthone, 1,3,7-trihydroxy-4,8-di-(3-methylbut-2-enyl) xanthone and a new xanthone. These compounds were tested against T. brucei and presented high activities of EC50= 3.9, 11.04, 14.7 μM respectively. Triterpenes were the main fingerprint compounds in a sample collected from Ijebu-Ode/Ogun; three compounds were isolated and elucidated as ambonic acid, mangiferonic acid and α-amyrin. These compounds had EC50 values against T. brucei of 39.5, 25.5 and 20.9 μM respectively. Finally, sample D46SA from South Africa was found to contain mainly flavonols and diterpenic acids; three compounds pinocembrin, acetylimbricatolic acid and (-)-pimara-8 (14), 15-dien-19-oic acid were isolated and tested. All were moderately active against T. brucei.with MIC ranging from 41.4-137.3 μMIn conclusion, this work has proved the variabiliy of propolis collected even from the same region and the widespread activity of propolis against (blood stream form) T. brucei. It is likely that some of the propolis samples contain compounds with even higher activity that have not yet been isolated.

The synthesis and structure of 1,2-quinone mono-oximes have been reviewed. The reaction of 3-hydroxyphenol, 3-hydroxy-2-aethylphenol, 3-hydroxy-5-methylphenol and N-acetyl-3-aminophenol with amyl nitrite/M(OEt) (M - Na or K) has been systematically examined. It has been found that the complex formed depends on the reaction temperature and phenol/M(OEt) ratio. Infra-red spectroscopic studies have shown that in the solid state 5-hydroxy-l,2-benzoquinone 2-oxime (hqoH,), 5-hydroxy-3-methyl-l,2-benzoquinone 2-oxime (3-MehqoH2), 5-hydroxy-6-methyl-l,2-benzoquinone 2-oxime (e-MehqoH,) and H-acetyl-5-amino- 1,2-benzoquinone 2-oxime (N-AcqoH) and their sodium and potassium complexes exist in the oximic form rather than the nitroso form. Nuclear magnetic resonance studies have also shown that in d,-DHSO solution hqoH,, 3-MehqoH, and 6-MehqoH} and their sodium complexes exist in one form only which is oximic in character. However, in DjO the results for the sodium complexes of hqoH, and e-MeqoH, indicate the presence of at least two species. In the case of the sodium 5-hydroxy-6-methyl-l,2-benzoquinone 2-oximate these species are oximic in character. An X-ray crystallographic study of e-HehqoH, has shown that in the solid state this compound exists in the 1,4- rather than the 1,2-quinone 2-oxlmic form. The synthesis of iron(II) complexes of hqoH,, 3-MehqoH] and 6-HehqoH] using the direct and the nitrosation methods was examined. The direct method gave rise to the complexes Fe(hqoH), OHjO, Fe(3-HehqoH), and Fe(6-MehqoH)2 '2H20 whereas the nitrosation method gave rise to ill-defined solids. Na[Fe(N-Acqo), ]-4H20 was obtained by nitrosation of N-acetyl-3-aminopnenol in the presence of Iron(II) ammonium sulphate. Hossbauer and magnetic studies indicate that Na[Fe(N-Acqo)j]-4H20 is a low spin iron(II) complex whereas the bischelates have properties indicative of the S - 1 spin state. In-vlvo assesment of the iron chelating ability of hqoHj, 3-MehqoH2, 6-MehqoH2, N-AcqoH , N,N-dimethyl-5- amino-1,2-benzoquinone 2-oxime and violuric acid was carried out using a normal rat model. The chelators hqoH, and 6-HehqoH2were found to be effective in removing iron when administered intra-muscularly but they also caused the excretion of magnesium. Their activity was lower than that of desferrioxamine and neither was effective when administered orally.

The effects of incubation of CRP with human primary and monocytic cell lines were examined using monocytic cytokine expression, adhesion molecule expression and adhesion to endothelial cells and intracellular peroxide formation, as end points. Monocytic intracellular signalling events were investigated after interaction of CRP with specific CRP receptors on monocytes. These initial signalling events were examined for their role in modulating monocyric adhesipn molecule and cytokine expression. Monocyte recruitment and retention in the vasculature is also influenced by oxidative stress. Therefore the effect of 6 weeks of antioxidant intervention in vivo was examined on monocytic adhesion molecule expression, adhesion to endothelial cells ex vivo and on serum CRP concentrations, pre- and post- supplementation with the antioxidants vitamin C and vitamin E. In summary, CRP is able to bind Fc?RIIa. CRP binding Fc?R initiates an intracellular signalling cascade that phosphorylates the non-receptor ryrosine kinase, Syk, associated with intracellular ryrosine activating motifs on the cytoplasmic tail of Fey receptors. CRP incubations increased phosphatidyl inositol turnover and Syk phosphorylation ultimately led to Ca2+ mobilisation in monocytes. CRP mediated Syk phosphorylation in monocytes leads to an increase in CD1 lb and IL-6 expression. CRP engagement with monocytes also leads to an increase in peroxide production, which can be inhibited in vitro using the antioxidants a-tocopherol and ascorbic acid. CRP mediated CDllb expression is not redox regulated by CRP mediated changes in cytosolic peroxides. The Fc?RIIa polymorphism at codon 131 effects the phenotypic driven changes described in monocytes by CRP, where R/R allotypes have a greater increase in CD1 lb, in response to CRP, which may be involved in promoting the monocytic inflammatory response. CRP leads to an increase in the expression of pro-inflammatory cytokines, which alters the immune phenotype of circulating monocytes. Vitamin C supplementation reduced monocytic adhesion to endothelial cells, but had no effect on serum levels of CRP.

Oxamniquine, 6-hydroxymethyl-2-N-isopropylaminomethyl-7-nitro-1,2,3,4- tetrahydroquinoline, is a potent schistosomicide used clinically in the treatment of infections due to Schistosoma mansoni. Schistosomiasis is the second most important tropical disease after malaria. Although oxamniquine is relatively well tolerated, severe central nervous system (CNS) effects characterized by convulsions, have been reported in a small percentage of the population treated with this drug.

This thesis investigates the biological activity of selected non-steroidal analogues of sex steroid hormones by examining two different effects of analogues on endogenous sex hormone activity. Non-steroidal analogues of sex hormones were synthesised to study their biological interactions with a sex steroid receptor and a sex steroid metabolising enzyme. Chapter One introduces the steroid hormones and their physiology, which leads to a review of the mechanisms by which steroids exert their effects. Their implication in disease is discussed, with particular emphasis on the sex steroids. As the biological activity of steroids is related to their chemical structure, the important features of steroid structure are identified, including the cyclopentanoperhydrophenanthrene nucleus, arrangement of ring substituents and ring junction conformation. The concept of non-steroidal analogues of steroids is introduced, and the harmful or beneficial effects analogues have on endogenous steroid activity are considered. Alteration of steroid activity and its consequences are focussed on two main areas; the potential adverse effects of environmental chemicals which mimic sex steroid activity, and the use of non-steroidal analogues in medicinal chemistry for treating sex steroid related disease. Chapter Two describes an investigation into the 17β-estradiol mimicking activity of non-steroidal analogues. Exogenous chemicals that mimic estradiol are of concern as they may alter endogenous estradiol activity and disrupt endocrine systems. Firstly, an introduction to the field of research concerned with environmental chemicals that mimic steroid hormones is given. The interaction of xenoestrogens with the estrogen receptor is described, as are the methods available for assessing the estrogen mimicking activity of xenoestrogens. The concern for insecticides mimicking estrogen activity is described by reviewing reported activities of insecticides, which leads into a discussion of work carried out as part of this thesis. Metabolites of the pyrethroid insecticides permethrin and cypermethrin, 2.14, 2.15, and 2.16 were synthesised while others were commercially obtained. The interaction of pyrethroid insecticide metabolites with the human estrogen receptor expressed in recombinant yeast (Saccharomyces cerevisiae) was studied, following the establishment and validation of the assay. Metabolites 2.11, 2.12, and 2.14 were found to weakly stimulate estrogen receptor-mediated estradiol responsive gene expression in the yeast assay (105 less active than 17β-estradiol). Since the activity of the metabolites using the yeast assay was greater than for the parent compounds, metabolic pathways need to be considered when assessing the impact of exposure to environmental estrogens. The low estrogenic activity suggests these compounds are not individually contributing significantly to the xenoestrogenic impact on humans, but will add to total xenoestrogen exposure. Chapter Three describes the inhibition of a sex steroid metabolising enzyme, steroid 5a-reductase, by novel non-steroidal compounds. Inhibitors of this enzyme are potentially useful therapeutic agents for regulating the activity of an androgen in prostate disorders. A review of the literature on non-steroidal inhibition of 5a-reductase identified three key structural features known to enhance inhibitor potency; ring substitution, position and nature of ring unsaturation and angular methyl group presence. These features were taken into account in the design of inhibitors synthesised in this thesis (3.55-3.57, 3.59, 3.61, 3.62, 3.110 and 3.111). Inhibitors consisting of non-steroidal 5- or 1-aryl pyridone scaffolds were synthesised to investigate SAR for 4'-substituents. The 5-aryl 1-methyl-2-pyridone/piperidone scaffold of compounds 3.55-3.57 and 3.59 was constructed by Suzuki cross coupling methodology, while the 1-aryl 2-methyl 2,3-dihydro-4-pyridone scaffold of 3.61 and 3.62 was constructed by aza Diels-Alder methodology. Long carbon chain olefin containing tethers 3.107 and 3.108 were synthesised for conjugation to inhibitor 3.57 by cross metathesis to give conjugates 3.110 and 3.111. Compounds 3.55-3.57, 3.59, 3.61, 3.62, 3.110 and 3.111 inhibited the type 1 5a-reductase isozyme expressed by HEK-I cells, with activities comparable to those of related literature compounds. The 1-aryl 2,3-dihydro-4-pyridone 3.62 inhibited both the type 1 and 2 isozymes (expressed by HEK-II cells) of 5a-reductase. The presence of bulky hydrophobic groups (benzoyl, long chain tethers) at the 4' position enhanced the potency of type 1 inhibition by 5-aryl pyridone type compounds in comparison to N,N-diisopropyl- and N-allylacetamide groups. This information provides further understanding of SAR within and across different classes of non-steroidal inhibitors of steroid 5a-reductase towards improved drug design.

The discovery of cisplatin revolutionised the treatment of cancer and opened the door to investigations into the discovery and use of other metallodrugs as chemotherapeutic agents. Both goldI and goldIII complexes have demonstrated promising anticancer properties both in vitro and in vivo. The following work will focus on the synthesis and anticancer activity of cyclometalated goldIII complexes with both tridentate (C^Npz^C) pincer ligands and bidentate (C^N) cyclometalated ligands to improve their physiological stability. Biologically relevant ligands were then incorporated into the complexes via the free coordination sites. These were selected to enhance the cytotoxicity of the complexes towards human cancer cell lines as well as improving the selectivity towards cancer cell lines over healthy cells. Chapter 1 explores the use of goldIII compounds as anticancer agents and their typical cellular and molecular targets. Chapter 2 introduces the synthesis and anticancer activity of the first (C^Npz^C)AuIII complexes of acyclic carbene ligands, decorated with amine and amino ester functional groups. Chapter 3 introduces the synthesis and anticancer properties of cyclometalated goldIII complexes with acridine-decorated functional groups, chosen to promote DNA binding. Chapter 4 discusses the synthesis and biological activity of cyclometalated goldIII complexes with dithiocarbamate ligands. The complexes were all tested for their anticancer activity in vitro towards a panel of human cancer cell lines, including some cell lines that typically show a reduced sensitivity towards cisplatin. Investigations into the possible mechanism of action of these complexes were also undertaken, including DNA binding assays, GSH reactivity and the production of ROS.

Six new compounds were isolated from the aerial and root parts of S. viridis L. cv. Blue Jeans. Two new triterpenoids, lup-20(29)-ene-2α-acetate-3β-ol, and lup-20(29)-ene-2α-ol-3β-acetate were found in the aerial part together with lup-20(29)-ene-2α-3β-diol, ursolic acid, oleanolic acid, β-sitosterol and β-sitosterol glucoside. Three new diterpenoids, 1-oxomicrostegiol, viroxocane, viridoquinone, together with five known diterpenoids, ferruginol, salvinolonyl 12-methyl ether, microstegiol, 7α-acetoxy-14-hydroxy-8,13-abietadiene-11,12-dione and 7α,14-dihydroxy-8,13-abietadiene-11,12-dione were found in roots. 1-Docosyl ferulate, 2'',3''-di-O-acetyl-martynoside and a mixture of 2-(4'-alkoxy-phenyl) ethyl alkanoates were also isolated from roots. Seven caffeic acid derivatives, five flavonoid glycosides, and salidroside were found in the crude aerial fraction. Four caffeic acid derivatives were known phenylpropanoids, i.e. trans-, cis-verbascoside, leucosceptoside A and martynoside, which are now reported in the genus Salvia for the first time. The others were caffeic acid, rosmarinic acid and 6-O-caffeoyl-glucose. A new flavonoid glycoside, luteolin-7-O-α-rhamnopyranosyl-(1→6)-β-galactopyranoside was also identified in the aerial part with four known flavone glycosides: luteolin-7-O-β-glucopyranoside, luteolin-7-O-β-galactopyranoside, luteolin-7-O-rutinoside and apigenin-7-O-β-glucopyranoside. Verbascoside (acteoside), which is a major component in this plant, showed a significant protective effect against UVA induced damage in a human skin fibroblast model in vitro. It exhibited 1.4 fold protective effect against UVA induced necrosis with 1.4 fold higher in cell survival. 50 μM Verbascoside showed the same protective effect as 100 μM DFO at a high intensity UVA dose (500 kJ/m2). Further determination of organelle specific protection suggested a mechanism of action in mitochondria. Two terpenoids, lup-20(29)-ene-2α-acetate-3β-ol and 7α,14-dihydroxy-8,13-abieta-diene-11,12-dione, exhibited antibacterial activity against Enterococcus faecalis with MIC 50 μM. Microstegiol was also active against Staphylococcus aureus with MIC 50 μM. Ursolic acid, oleanolic acid and ferruginol showed appreciable antibacterial activity against three Gram-positive bacteria, Staphylococcus aureus, Enterococcus faecalis, and Bacillus cereus with MIC 12.5-50 μM. The other diterpenoids were active against all three Gram-positive bacteria with MIC 100-200 μM. None of crude fractions was active against three Gram-negative bacteria, Klebsiella pneumoniae, Proteus vulgaris, and Escherichia coli.

Esta tesis aborda la actividad biológica de la fibronectina (FN) como proteína de interfase en la interacción célula-material. La tesis investiga la respuesta de la proteína, en términos de cantidad adsorbida y conformación, ante diferentes propiedades físico-químicas del material. Además, se correlaciona la respuesta celular temprana y la funcionalidad celular con el estado de la proteína adsorbida sobre el material. Para ello se prepararon diferentes series de materiales con propiedades físico-químicas controladas. La distribución de FN sobre las diferentes superficies se caracterizó mediante el uso de la microscopía de fuerza atómica (AFM) y la densidad superficial adsorbida fue cuantificada mediante técnicas de marcado radioactivo y western blot. La respuesta celular se evaluó en términos de la adhesión inicial a las superficies, así como los procesos posteriores de diferenciación, proliferación, reorganización y producción de matriz extracelular. Se investigó el efecto de la nanotopografía en la adsorción de la FN y el comportamiento celular sobre una serie de topografías controladas en la escala nanométrica, obtenidas mediante el spin casting de soluciones de ácido poli(L-láctico)/poliestireno (PLLA/PS) de distintas concentraciones. La migración del PLLA hacia la superficie del film durante el proceso de spin coating proporciona superficies de PLLA con nanopicos de diferentes tamaños (14, 29 y 45 nm). El tamaño de la nanoestrutura afecta a la densidad de FN adsorbida, siendo mayor en la superficie de menor nanotopografía. En cuanto a la respuesta celular inicial, se observan adhesiones focales más desarrolladas y mejor reorganización celular de la capa de FN adsorbida en las superficies de mayor topografía (29 and 45 nm), lo que resulta en una mayor producción y organización de nueva matriz. Por otra parte se empleó una familia de materiales con sutiles variaciones en la composición química: polímeros acrílicos (polimetil, etil y butil acrilato -PMA, PEA y P
González García, C. (2012). BIOLOGICAL ACTIVITY OF FIBRONECTIN AT THE CELL-MATERIAL INTERFACE [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/17701
Palancia

Les nanomatériaux représentent actuellement des systèmes très attractifs grâce à leurs propriétés physiques, chimiques et mécaniques particulières. La possibilité de manipuler ce type de structures est à la base d'une nouvelle science appelée nanotechnologie. En particulier, la recherche développée après la découverte de molécules tubulaires baptisées nanotubes de carbone représente une contribution fondamentale aux nanosciences. Les nanotubes de carbone (CNTs) sont constitués par des couches de graphite enroulées pour former des structures cylindriques. Deux catégories existent : les nanotubes de carbone à plusieurs parois concentriques (MWNTs ou Multi Walled carbon nanotubes) et les nanotubes à simple parois (SWNTs ou Single Walled carbon nanotubes). Leur diamètre est de l'ordre du milliardième de mètre ; pour les SWNTs il varie entre 0. 4 nm et 2 nm, et entre 1. 4 et 100 nm pour les MWNTs. La longueur est encore plus variable et est comprise dans les deux cas entre quelques centaine de nanomètres et quelques centaines de microns. Les CNTs sont considérés comme des matériaux uniques ayant des applications très prometteuses, spécialement en nanotechnologie, nanoélectronique, science des matériaux mais aussi en chimie médicinale. Les applications potentielles des nanotubes de carbone en chimie médicinale sont à l'heure actuelle très prometteuses étant donné leur capacité d'interagir avec des macromolécules comme les protéines, les polysaccharides et les oligonucleotides. Jusqu'à ce jour, les applications biologique des nanotubes ont été très peu explorées. La raison majeure est certainement l'absence de solubilité de ce matériau en solution aqueuse. La solubilisation des CNTs dans les solvants organiques est possible après le greffage de groupes solubilisants sur la structure tubulaires. Différentes techniques ont été explorées et des nombreuses réactions chimiques peuvent être utilisées pour cet objectif. Pour étendre les applications des CNTs en chimie médicinale il était absolument nécessaire de développer des méthodes permettant de les solubiliser dans des milieux aqueux. Nous avons ainsi développé une méthode de fonctionnalisation basée sur la réaction de cycloaddition 1,3-dipolaire d'ylure d'azométhine à la surface externe des nanotubes. Les nanotubes de carbone ont ainsi été fonctionnalisés par des groupements aminés solubles en solution aqueuse pouvant être facilement dérivatisés par des acides aminés. Ce travail a représenté la première étape vers la synthèse de premières conjugués peptides-nanotubes. La synthèse, la caractérisation et les éventuelles applications biologiques des nanotubes de carbone fonctionnalisés avec des acides aminés et des peptides n'ont pas été développées et exploitées de manière systématique. Cependant, nous avons immobilisé des peptides ayant une activité biologique sur les parois externes de ces nanotubes de carbone afin de trouver des applications médicales intéressantes. Le potentiel offert par ces nouvelles structures est de pouvoir être utilisé comme système d'intérêt thérapeutique dans des domaines aussi divers que la vectorisation des molécules bioactives (peptidiques ou non peptidiques), la vaccination, ainsi que la multiprésentation des molécules inhibitrices ou activatrices des récepteurs multimériques. Nous pouvons aussi les utiliser dans le domaine du diagnostique. Pour ce faire, différentes stratégies de synthèse ont été utilisées pour préparer des dérivés peptides-nanotubes de carbone qui ont été ensuite testés pour leur activité biologique. Une étude systématique a été conduit in vitro et in vivo afin de vérifier et d'évaluer l'influence de ces molécules à base de carbone sur l'activité biologique du peptide greffé. Nous avons choisi de lier aux nanotubes des peptides ayant des propriétés immuno-modulatrices pour des maladies comme la fièvre afteuse chez les animaux ou le Lupus Erythemateux Disséminé chez l'homme. Les propriétés antigéniques et la réactivité immunologique de ces nouvelles molécules conjuguées ont été vérifiées à l'aide de différentes techniques comme résonance plasmonique de surface (SPR), le test Elisa et par injection directe chez la souris. Nous avons mis en évidence que la présence des nanotubes augmente la réponse de la production d'anticorps in vivo par rapport à l'injection du peptide isolé et en plus, renforce la capacité de ces anticorps à tuer le virus. Les résultats obtenus à ce stade de notre recherche montrent l'énorme potentialité offerte par ces systèmes en ce qui concerne la biocompatibilité et leurs propriétés de délivrance et présentation. L'aspect lié à la toxicité a également été étudiée par cytométrie de flux. De même la capacité des nanotubes de carbone à pénétrer dans les cellules sans détruire les membranes ou les structures cellulaires à été étudié par microscopie à fluorescence. Nous avons montré qu'un nanotube de carbone fonctionnalisé avec une molécule fluorescente est capable de pénétrer dans des cellules bien qu'à l'heure actuelle nous ne connaissons pas encore en détail le mécanisme d'entrée à l'intérieur de la cellule. Apparemment le mécanisme de pénétration le plus probable semble être dû à des phénomènes d'endocitose aspécifiques passives plutôt que par des mécanismes actifs. Les interactions entre la membrane cellulaire et la structure polaire du nanotube fonctionnalisé constituent la force propulsif (driving force) pour l'internalisation du nanotube. Des expériences effectuées en condition d'absence d'énergie cellulaire ont montré la facilité d'internalisation d'un nanotube comme a été confirmé par l'analyse à l'aide de la microscopie électronique à transmission (TEM) des cellules traitées avec les MWNT. Sur la base de ces résultats, nous avons exploré la possibilité d'utiliser les nanotubes comme nouveaux vecteur thérapeutiques. En effet les nanotubes de carbone ont un gros potentiel dans le transport de molécules comme l'ADN ou autres petits médicaments ayant une faible biodisponibilité. Depuis des années les liposomes représentent les vecteurs normalement utilisés afin de véhiculer l'ADN pour des applications en thérapie génique. Après la synthèse d'un dérivé de CNT riche en groupes fonctionnels positivement chargés, il a été possible d'évaluer les interaction entre un nanotube et une molécule d'ADN plasmidique négativement chargée. L'idée était de comparer les deux systèmes de transport : les liposomes et les nanotubes de carbone. Différentes techniques ont été utilisées pour caractériser les complexes obtenus. La microscopie électronique à balayage, à transmission, les études de SPR, PCS et électrophorèse ont permis de montrer la formation du complexe CNT-ADN et de déterminer sa stabilité à différent rapport de charge négatif : positif. La condensation de matériel génétique à la surface de nanotubes fonctionalisés est donc réalisable et applicable pour le développement de nouveaux vecteurs pour la thérapie génique. L'étude de transfection in vitro a démontré l'efficacité du transport et de l'expression du plasmide véhiculé par les nanotubes à l'intérieur de la cellule. Suite à l'injection du complexe CNT-ADN à différentes doses et concentrations, nous avons ensuite évalué la réponse in vivo chez la souris. Le résultats d'expression des gènes ont montré une toutefois faible efficacité, en comparaison avec les liposomes-ADN. La longue optimisation qui a été nécessaire pour l'utilisation des liposomes comme vecteurs non virales dans la thérapie génique, nous donne fort espoir pour l'amélioration de notre système vecteur basé sur les nanotubes de carbone fonctionnalisés. En conclusion dans ce travail de thèse, nous avons réussi à développer et caractériser une nouvelle architecture chimique macromoléculaire fonctionnelle, utilisable comme nouveau outil pour la délivrance de molécules bioactives (médicaments, peptides antigéniques, acides nucléiques) et pour la reconnaissance supramoléculair. Le développement futur de la chimie des nanotubes de carbone et l'optimisation de leurs interactions avec les systèmes vivants constituent la continuation active de ce projet de recherche innovant
Carbon nanotubes (CNT) consist of graphene sheets rolled-up into a tubular form. Since their discovery, they appeared immediately as an interesting material for technological applications, including for instance the fabrication of nanoelectronic components. Recently, CNT have also attracted much attention for their potential in biological applications. The main difficulty to integrate this material into biological systems derives from its complete lack of solubility in organic solvents and aqueous solutions. The ability to solubilise and separate individual CNT is still a great challenge. A very general way to achieve this is by organic functionalisation, which is a rapidly expanding field. In this thesis, I focused my interests on the synthesis and use of the first water soluble side-wall functionalised carbon nanotubes. I employed the 1,3-dipolar cicloaddition of azomethine ylides to carbon nanotubes. I have demonstrated that it is possible to further derivatise them by coupling single N-protected amino acids. This was the first step towards the preparation of covalently linked peptide-carbon nanotube conjugates. In this context, I have developed a powerful strategy for linking bioactive peptides to carbon nanotubes for immunological applications. Immobilisation of peptides to the external walls of carbon nanotubes may find interesting applications in diagnostics, vaccine and drug delivery or multipresentation of bioactive molecules. For this aim, peptides with immunological properties were selected for their coupling to the external surface of the CNT. The immunological reactivity and the peptide recognition were assessed by a peptide specific antibody using surface plasmon resonance and ELISA test. These experiments showed that the peptide linked to CNT retain its conformational characteristics for antibody recognition. Furthermore, biological studies performed in vivo demonstrated that CNT-peptide conjugates elicited high antibody titers. Significant pathogen neutralising capacity was observed for the antibodies induced by CNT-peptide conjugates. This highlights: 1) the potential of carbon nanotubes for vaccine delivery, and 2) the importance of antigen presentation in vivo for the induction of antibodies with the right specificity. Functionalised carbon nanotubes have been showed able to cross the cell membrane and to accumulate in the cytoplasm or reach the nucleus without being toxic for the cell up to 10 µM concentration. These findings highlight the potential use of peptide-carbon nanotube conjugates for diagnostic purposes and pave the way for their application in vaccine and drug delivery. Although the elucidation of the mechanism of entry requires further investigations, I excluded active ATP dependent endocytosis. This is because inhibitors of endosome-mediated translocation and decrease of the incubation temperature did not prevent cellular uptake of the different functionalised CNT. In addition, TEM images revealed the tubes crossing the cell membrane as nano-needles without any perturbation or disruption of the membrane. Cell viability after treatment with functionalised nanotubes has also been largely investigated. Highly soluble functionalised CNT in aqueous biological media exhibited notably reduced cellular toxicity in vitro. Cell viability was studied using flow cytometry. Following the synthesis of positively charged carbon nanotubes I investigate their interaction with plasmid DNA. The cationic-anionic interaction between CNT and DNA has been characterised by different techniques both qualitatively and quantitatively. TEM, photocorrelation spectroscopy, SPR and electrophoresis allowed to describe the stability of the CNT-DNA complexes. The condensation of genetic material onto the carbon nanotubes was then confirmed and biological test were performed. The excellent ability of the ammonium functionalised carbon nanotubes to enter cells and potentially reach their nuclei was exploited for the delivery of plasmid DNA. In vitro experiments showed a high level of gene expression when mammalian cells were transfected with DNA-CNT complexes. The following success obtained in in vivo treatment of mice, highlighted the possibility to use this system for gene delivery in gene therapy. Preliminary comparative gene expression data between functionalised CNT:DNA and commercially available lipid:DNA delivery systems showed that our first generation CNT-based gene delivery system is less efficient for in vitro transfection than the lipid:DNA system. However, there is a lot of room for further improvement of the carbon nanotube system for gene delivery. In conclusion, in this Thesis it was possible to develop and characterise a new chemical macromolecular architecture exploitable as new tool for molecular delivery, and molecular recognition. The further development of carbon nanotube chemistry, the optimisation of their interaction with biomolecules and their use in biomedical applications represent the future perspectives of this research

The potential use of synthetic hammerhead ribozymes as novel anti-brain tumour agents was investigated in this study. Chimeric 2'-O-methylated hammerhead ribozymes proved to be significantly more stable (>4000-fold) in serum than unmodified RNA ribozymes and exhibited high in vitro catalytic activity. The cellular association of an internally [32P]-labelled 2'-O-methylated chimeric ribozyme in U87-MG human glioma cells was temperature-, energy- and pH-dependent and involved an active process that could be competed with a variety of polyanions. Indications are that the predominant mechanism of uptake is by adsorptive and / or receptor mediated endocytosis. Twenty 2'-O-methylated chimeric ribozymes were designed to cleave various sites along the EGFr mRNA. In vitro, 18 ribozymes exhibited high activity in cleaving a complementary short substrate. Using LipofectAMINETM as a delivery agent, the efficacy of these ribozymes was evaluated in the A431 cell line, which expresses amplified levels of EGFr. Studies revealed that although the ribozymes were taken up by the cells and remained stable over a period of 4 days, no significant reduction in either EGFr expression or cell proliferation was evident. The presence of tolomerase, a ribonucleoprotein responsible for telomere elongation, has been strongly associated with tumour progression. The biological activity of a 2'-O-methylated ribozyme targeted against the RNA component of telomerase was determined. The ribozyme exhibited specific dose-dependent inhibition of telomerase activity in U87-MG cell lysates with an IC50 of ˜4M. When 4M ribozyme was delivered to intact U87-MG cells, complexed to LipofectAMINETM, telomerase activity was significantly reduced to 74.54.17% of the untreated control.

The antioxidant property of myo-inositol hexakisphosphate is important in the prevention of hydroxyl radical formation which may allow it to act as a 'safe' carrier of iron within the cell. Here, the hypothesis that the recently discovered natural product, myo-inositol 1,2,3-trisphosphate represents the simplest structure to mimic phytate's antioxidant activity has been tested. The first synthesis of myo-inositol 1,2,3-trisphosphate has been completed, along with its X-ray structure determination and that of key synthetic intermediates. Iron binding studies of myo-inositol 1,2,3-trisphosphate demonstrated that phosphate groups with the equatorial-axial-equatorial conformation are required for complete inhibition of hydroxyl radical formation. myo-Inositol monophosphatase is a key enzyme in recycling myo-inositol from its monophosphates in the brain and its inhibition is implicated in lithium's antimanic properties. Current synthetic strategies require inositol compounds to be protected (often with more than one group), resolved, phosphorylated and deprotected to produce the desired optically active myo-inositol phosphates. Here, the synthesis of myo-inositol 3-phosphate has been achieved in only 4 steps from myo-inositol. The stereoselective addition of the chiral phosphorylating agent (2R,4S,5R)-2-chloro-3,4-dimethyl-5-phenyl-1,3,2-oxazaphospholidin-2-one to a protected inositol intermediate allowed separation of diastereoisomers and easy deprotection to myo-inositol 3-phosphate. This strategy also allows the possible introduction of labels of oxygen and sulphur to give a thiophosphate of known stereochemistry at phosphorus which would be useful for the analysis of the stereochemical course of phosphate hydrolysis catalysed by inositol monophosphatase.

Nucleophosmin (NPM1) is a multifunctional phosphoprotein localized predominantly within the nucleoli of eukaryotic cells. Mutations within its C-terminal domain are frequently observed in patients with acute myeloid leukemia (AML), are thought to play a key role in the initiation and progression of the disease, and result in aberrant, cytoplasmic localization of the mutant protein. It has previously been demonstrated that the electrophilic antiproliferative natural product (+)-avrainvillamide binds to proteins, including nucleophosmin, by ¬S-alkylation of cysteine residues. In this thesis we report that the biological activity of avrainvillamide is mediated by NPM1 and the nuclear export receptor exprtin-1 (Crm1). Using mass spectrometry, we demonstrate that the antiproliferative activity of a series of avrainvillamide analogs correlates with their ability to bind C-terminal NPM1 truncation constructs; it is also observed that the interaction between avrainvillamide and the C-terminal domain of NPM1 is fully reversible under our experimental conditions. We report that avrainvillamide restores nucleolar localization of certain AML-associated mutant forms of NPM1 and provide evidence that this relocalization is mediated by interactions of avrainvillamide with mutant NPM1 and Crm1. Immunofluorescence and mass spectrometric experiments employing a series of different NPM1 constructs suggest that a specific interaction between avrainvillamide and cysteine-275 of certain NPM1 mutants mediates the relocalization of these proteins to the nucleolus. Avrainvillamide is further shown to inhibit nuclear export of Crm1 cargo proteins, including AML-associated NPM1 mutants; this marks the first evidence that avrainvillamide directly influences the biology of a cellular target other than NPM1. We also observe that avrainvillamide treatment displaces thr199-phosphorylated NPM1 from duplicated centrosomes, leads to an accumulation of supernumerary centrosomes, and causes mitotic defects in vitro. Finally, we show that avrainvillamide treatment increases levels of thr199-phosphorylated NPM1 by inhibiting the action of protein phosphatase 1 beta on phosphorylated NPM1, thereby indirectly displacing NPM1 from nucleoli and destabilizing nucleolar and nuclear structure.
Chemistry and Chemical Biology

碩士
嘉南藥理科技大學
生物科技研究所
93
3,4-dihydroxyphenyl-alanine (DOPA) is one of central nervous system transmission media, a precursor of the adrenalin or the melanin and may treat Parkinson、s disease in human. At present, the knowledge regarding DOPA and its biological activity is still limited. The objective of this study was to investigate the biological activity of DOPA . The study was to investigate the protection of DOPA on ultraviolet ray B (UV-B) illumination, the relation of DOPA and free radical by using the UV-B illumination, the cell survival percentage analysis, superoxide dismutases (SOD) assay, the free radical assay. Results of the study showed that DOPA has 1,1-diphenyl-2-picryl hydrazyl (DPPH), nitric oxide (NO), 2,2-azino-bis(3-ethylbenz- thiazoline-6-sulfonic acid) (ABTS), and liposome free radical scavenging activities, and also protecting fibroblast from UV-B radiation. Data indicated that DOPA with concentration of 25uM was higher scavenging activity of DPPH than 80%, and is similar to that of tocopherol;DOPA (25uM) had the same NO free radical scavenging activity (60%) with quercetin (25uM);in the analysis of ABTS free radical scavenging activity, DOPA and ascorbic acid were good antioxidants;in the liposome system assay, results also demonstrated that DOPA showed stronger scavenging activity than tocopherol with same concentration. In the UV-B illumination, DOPA can protect 3T3 fibroblast from death and prevent the increasing of SOD . The result of this study indicated DOPA in guards against exposes to the sun and to eliminate free radical, anti-oxidation, and anti-aging with further research and clinical practice potential value.

碩士
國立政治大學
會計學系
107
This research tries to apply common biological characteristics to construct a common biological activity-based costing (CBABC) system, which system can apply to all different agricultural, forestry, fishery and husbandry industries. This research also looks into IAS 41, the meaning of biological characteristics, cost accounting framework of agricultural activity, cost of agricultural produce which is collected by Council of Agriculture, and the research on ABC of agricultural activity. This research not only builds the framework theory of CBABC but also applies the CBABC on two cases — tomato planting and pig breeding. CBABC integrates the common biological characteristics with activity-based costing system. Therefore, CBABC could be easily customized for different agricultural activities. With common framework and adjustable system, CBABC should be much more easily to promote than any other agricultural costing systems. This research expects CBABC could improve the competitive advantage of farmers by providing the financial forecast, and integrated with production management system. In the future, A CBABC agriculture resource planning system could help farmers to solve cost calculation problems as well as prepare reasonable financial reports. The most importmant is that with reasonable financial reports, banking stystem could be attracted to invest in agricultural industry.

Thesis (Ph.D.)--University of Alberta, 2010.
A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Department of Chemical and Materials Engineering, Medical Sciences - Biomedical Engineering. Title from pdf file main screen (viewed on January 30, 2010). Includes bibliographical references.

碩士
國立高雄醫學大學
藥學研究所碩士在職專班
90
Abstract 4-[(2,4-dichloropheny)amino]-6,7-dimethoxy-3-quinolinecarbonitrile is a novel heterocycle possessing a wide variety of biological effects, including the inhibition of Src kinase activity (IC50=30 nM). Src inhibition could be efficacious for the treatment of various diseases, including cancer osteoporosis and the brain damage that often follows stroke. Src inhibitor proved effectively in the treatment of osteoporosis. In order to explore structure-activity relationships, a number of difluoro and trifluoro substituted anilinoquinolines were synthesized and evaluated for anticancer activities. Chlorination of 6,7,8-trifluoro-1,4-dihydroquinoline-3-carboxylic acid ethyl ester (2) with phosphorus oxychloride gave 4-chloro-6,7,8-trifluoro-1,4-dihydroquinoline-3-carboxylic acid ethyl ester (3) which was further treated with three 4-substituted anilines to give the compound of 4-anilinoquinolines (4a-c). Hydrolysis of 4a-c afforded 4-anilinoquinolines-3-carboxylic acid (5a-b) respectively. The preliminary anticancer assay show that these compounds places the bioactivities against the growth of NCI-H460 (lung cancer), MCF7 (breast cancer), and SF-268 (CNS cancer). 4-(3-acetylphenylamino)-6,8-difluoro-7-(1-piperazinyl) quinoline-3-carboxylic acid ethyl ester was the most potent, with 100% growth inhibition of NCI-H460, MCF7, and SF-268 at a concentration of 100μM. In addition, the tricyclic analogs of 6-fluoro-4-[3-(1-hydroxyiminoethyl)phenylamino]-7-(1-piperazinyl)quinoline-3-carboxylic acid ethyl ester, were also synthesized for anticancer evaluation.