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Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.

The work presented in this thesis describes the determination of structures of peptides and proteins at atomic resolution. Nuclear Magnetic Resonance (NMR) spectroscopy was used as the principal method of investigation. The thesis is divided into three parts. Part I of the thesis consists of chapters 1 to 4, and deals with structural studies of two novel conotoxins. Part II of the thesis consists of chapter 5 and deals with structural studies of Small MutS Related (Smr) domain from Helicobacter pylori MutS2. Part III of the thesis consists of Appendices A to D. Appendix A describes implementation of a novel pulse sequence for determination of disulfide connectivity using long-range 13 C–13 C scalar couplings across disulfide bonds. Appendices B, C and D contain supplementary infor- mation (acquisition parameters and chemical shifts) for the structural studies presented in parts I and II of the thesis. Part I: Structural studies of novel conotoxins from Conus monile Chapter 1 gives a brief overview of the conotoxins and their structural studies. The first half of the chapter describes biosynthesis, classification schemes, nomenclature, com- monly observed post-translational modifications and applications of conotoxins. The latter half of this chapter summarizes the challenges involved in the structural studies of conotoxins in light of the recent developments in integrated transcriptomic and venomic studies of conotoxins. The key homonuclear and heteronuclear NMR experiments that are employed for structural studies of conotoxins are summarized. Emphasis was laid on describing the spectral features and the structural information that can be gleaned from these experiments. Finally, the current mass spectrometric and NMR methods available for determination of disulfide connectivity are discussed Chapter 2 describes sample preparation and preliminary biophysical characteriza- tion of a conotoxin Mo3964 that contains a hitherto uncharacterized cysteine framework (C–CC–C–C–C). The sequence of Mo3964 was identified at the nucleic acid level as a cDNA clone. Analysis of the signal sequence revealed that the toxin belongs to the M-superfamily, while the cysteine framework bears more resemblance to O- and K- super- family of conotoxins. Structural studies were initiated to determine the disulfide connec- tivity, tertiary structure and biological activity. The gene corresponding to the mature toxin sequence was cloned in a bacterial expression vector pET21a(+) as a C-terminal tag to the cytochrome b5 fusion protein host system. The fusion protein was obtained by recombinant expression using the bacterial expression host E. coli BL21(DE3) and the mature toxin was obtained by either enzymatic or chemical cleavage of the fusion protein followed by size exclusion chromatography and reverse phase HPLC. Proton 1D NMR spectra of the purified peptide exhibited sharp lines and good spec- tral dispersion indicating that molecule was well folded. Formation of disulfide bonds in the mature toxin was ascertained by high resolution mass spectra of intact and chemically modified Mo3964. The peptide toxin exhibited remarkable stability to chemical denatu- ration and proteolytic digestion. Spectroscopic studies clearly showed that Mo3964 pos- sesses a very stable and well defined structure as long as its disulfide bonds are intact. Analytical size exclusion chromatography and Multi Angle Light Scattering (MALS) studies showed that Mo3964 exists in solution as monomer albeit with a non-globular structure. Electrophysiological studies showed that Mo3964 inhibits outward potassium currents in rat Dorsal Root Ganglion (DRG) neurons and increases the reversal potential of rat voltage gated sodium channel rNav 1.2 stably expressed on Chinese Hamster Ovary (CHO) cells at peptide concentrations as low as 10 nM. Chapter 3 describes the determination of disulfide connectivity and tertiary stricture of Mo3964. Initial attempts to determine disulfide connectivity using direct fragmenta- tion of the intact peptide in the mass spectrometer failed due to the relatively large size of the molecule and its resistance to endoproteases. Partial reduction alkylation based methods failed as the first stage of partial reduction gave rise to a mixture of various single disulfide bond reduced species which could not be separated from each other. Subsequently, information about the disulfide connectivity was obtained using a method that does not necessitate separation of such a mixture of single disulfide bond reduced species. This method involves partial reduction, cyanylation of the reduced cysteines and alkali mediated cleavage of the peptide backbone on the N-terminus of cyanylated cysteines. Structural studies were carried out using homonuclear and heteronuclear NMR meth- ods. The hydrogen bond network and hence topology of the molecule was determined with high accuracy using the long-range HNCO-COSY experiment that correlates hydrogen- bond donor-acceptor pairs. This experiment utilizes the three bond heteronuclear scalar coupling, i.e., the h3JN C O′ coupling across the hydrogen bonds. All these restraints proved crucial to the assignment of the disulfide connectivity in Mo3964, given its novel cysteine framework. The structure of Mo3964 was calculated using a total of 549 NOE distance restraints, 84 dihedral angle restraints and 28 hydrogen bond distance restraints. The tertiary structure was constructed from the disulfide connectivity pattern 1–3, 2–5 and 4–6, that is hitherto undescribed for the M–superfamily conotoxins. The ensemble of structures showed a backbone Root Mean Square Deviation of 0.68 ± 0.18 Å, with 87% and 13% of the backbone dihedral (φ, ψ) angles lying in the most favored and additional allowed regions of the Ramachandran map. The remarkable stability and anomalous spectral properties exhibited by Mo3964 could be rationalized using the disulfide connectivity and the tertiary structure. The tertiary structural fold has not been described for any of the known Conus peptides. Further, a search for structures similar to that of Mo3964 using the web server DALI returned no hits indicating that the peptide scaffold of Mo3964 has no structural homologues. Hence, the conotoxin Mo3964 represents a new bioactive peptide fold that is stabilized by disulfide bonds and adds to the existing repertoire of scaffolds that can be used to design stable bioactive peptide molecules. The structure of Mo3964 was submitted to the Protein Data Bank (PDB ID: 2MW7)[1]. Chapter 4 describes the structural studies of a 17 residue, single disulfide containing conopeptide Mo1853. The samples for structural studies were obtained either by chemical synthesis or by recombinant expression methods. Structural studies using homonuclear solution NMR methods revealed that Mo1853 exists as two equally populated cis and trans X–Pro conformers which are in slow exchange regime, compared to the chemical shift timescale. Sequence specific assignments were obtained for both the conformers by analysis of homonuclear 2D 1 H,1H–DQF–COSY,1H,1 H–TOCSY, 1H,1 H–NOESY and 1H,1 H–ROESY spectra. Temperature dependence of chemical shifts was measured and coalescence was observed for two amide protons at 318 K. At this temperature, the rate of exchange and the free energy of activation were determined to be 59 Hz and ≈ 67.2 kJ mol−1 respectively. The evidence for this conformational equilibrium was also observed as exchange correlation peaks in the 2D- NOESY and ROESY spectra. Tertiary structures of both the cis and trans conformers were determined using distance restraints, backbone dihedral angle restraints, the disulfide bond restraint and the cis or trans conformation of the X–Pro peptide bond. Tertiary structures of both the conformers consist of a 29-membered macro-cyclic ring formed by 9 amino acid residues which are cyclized by side chain to side chain disulfide bond. The conformation of the X–Pro peptide bond which is located within this macro-cyclic ring causes the cis structure to be compact and the trans structure to be in an extended form. Analysis of the tertiary structures indicated that the trans conformer is stabilized by hydrogen bonds while the cis conformer is likely to be stabilized by hydrophobic interactions. This was further corroborated by the fact that at lower temperatures, the hydrophobic interactions became weaker reducing the population of the cis conformer with respect to that of the trans conformer. Preliminary electrophysiological studies carried out on rat DRG neurons indicate that Mo1853 transiently reduces late outward potassium currents. Part II: Structural studies of Small MutS Related (Smr) domain from Helicobacter pylori MutS2 Chapter 5 presents the solution NMR studies of the Smr domain from MutS2 of H. pylori , henceforth called as HpSmr. In H. pylori , MutS2 is involved in suppression of homologous recombination and its Smr domain was shown to be necessary for this activity. As of date, in spite of the availability of structural information for the Smr domain, unambiguous identification of the residues involved in metal binding, DNA binding and catalysis remains elusive. Structural studies were carried out on two different constructs of HpSmr viz., HpSmr– (His)6 and GSHM–HpSmr, with and without the hexahistidine tag respectively. Se- quence specific assignments of HpSmr–(His)6 were obtained at two different sample pH conditions viz., pH 8.0 and pH 5.35 using the standard suite of triple resonance NMR experiments. Since, valines and leucines constitute about 25% of the total number of amino acid residues in HpSmr–(His)6 , stereospecific assignments were obtained for di- astereotopic methyl groups of these residues by preparing a fractionally 13C labeled sample of HpSmr–(His)6 . Solution structure of HpSmr–(His)6 at pH 8.0 was determined using 766 NOE restraints, 170 backbone dihedral angle restraints and 70 hydrogen bond distance restraints. The tertiary structure exhibits the canonical α/β sandwich fold ex- hibited by all the other known structures of Smr domains. Further, NMR studies and analytical gel filtration studies indicated the presence of pH dependent conformational exchange in HpSmr that involves strand to coil transition in the C-terminal β-strand. In order ascertain that the conformational equilibrium is not at an artifact caused by the C-terminal hexa-histidine-tag, HpSmr protein construct GSHM–HpSmr, which does not have the hexa-histidine-tag, was prepared. Conformational exchange was observed in this construct as well. The preliminary NMR evidence suggests that the conformational exchange is caused by pH dependent cis–trans isomerization of a semi-conserved Proline residue Pro66 . We have hypothesized that the pH dependent modulation of the activity of Smr domain of MutS2 can be advantageous to H. pylori . Such a regulation could help the bacteria to achieve optimal rate of homologous recombination in response to changes in pH, which is necessary for maintaining homeostasis and tiding over stress conditions. Part III: Appendix Appendix A describes an NMR pulse program LRCC_CH2 that was designed with the aim of determining disulfide connectivity using long-range 13C–13 C (C β –C β ′ ) couplings across the disulfide bond. This experiment is a modification of an earlier experiment pub- lished by Bax and co-workers designed to measure the side-chain χ3 dihedral angle in me- thionines. The experiment described here is optimized for the detection of 3 bond scalar coupled methylene carbons. The details of modifications introduced in LRCC_CH2, its product operator analysis, a representative spectrum acquired on [U-13C,15 N]–Mo3964, short-comings and future directions are described. The C programming code that was used to implement the pulse program is also included in the appendix. Appendices B, C and D contain the supplementary information (acquisition pa- rameters for the NMR experiments and chemical shifts) for the structural studies carried out on Mo3964, Mo1853 and HpSmr.

Submitted by VANESSA GONÇALVES WOLF null (nessa.wolf@hotmail.com) on 2017-07-13T23:39:50Z No. of bitstreams: 1 20170708145234dissertacao_final_vanessa_goncalves_wolf_corrigida_jul_8.pdf: 3123091 bytes, checksum: 0e5f498e0767893a065858a9ae558c1c (MD5) / Approved for entry into archive by Monique Sasaki (sayumi_sasaki@hotmail.com) on 2017-07-14T18:44:45Z (GMT) No. of bitstreams: 1 wolf_vg_me_arafcf.pdf: 3123091 bytes, checksum: 0e5f498e0767893a065858a9ae558c1c (MD5) / Made available in DSpace on 2017-07-14T18:44:45Z (GMT). No. of bitstreams: 1 wolf_vg_me_arafcf.pdf: 3123091 bytes, checksum: 0e5f498e0767893a065858a9ae558c1c (MD5) Previous issue date: 2017-06-22 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Helicobacter pylori é um dos principais causadores de gastrite crônica e úlcera péptica, e embora o mecanismo envolvido na inflamação gástrica por esta bactéria não esteja completamente elucidado, sabe-se do importante papel das espécies reativas de oxigênio (EROs) produzidas por polimorfonucleares neutrófilos, que são atraídos e ativados pelo agente da infecção, sem que, entretanto, consigam debelar a mesma, mas que contribuem fortemente para a lesão tecidual e o processo inflamatório crônico. Diante disso, tem aumentado a busca de novas estratégias de tratamento que possam levar à redução do estresse oxidativo gerado no sítio da infecção, com consequente redução do processo inflamatório. Neste sentido, ácido gálico, juntamente com seus ésteres (galato de metila, propila, hexila e octila), foram utilizados neste estudo, com o objetivo de avaliar suas ações como inibidores da liberação de EROs por neutrófilos ativados, bem como seus efeitos antimicrobianos sobre H. pylori. Para a avaliação da atividade antioxidante dessas substâncias foram realizados ensaios livres de células (efeito supressor sobre o radical DPPH e sobre radicais peroxila), e o efeito anti-EROs foi avaliado utilizando neutrófilos isolados de sangue humano estimulados por H. pylori, Zymosan ou PMA, através de ensaio quimiluminescente dependente de luminol ou lucigenina, ensaio com WST-1, ensaio de inibição da produção de HOCl, e o teste do NBT. Ensaio antimicrobiano foi realizado através da técnica de microdiluição em caldo. A presença da cadeia lateral carbônica levou a um significativo aumento na capacidade dos ésteres em inibir a produção de EROs por neutrófilos ativados, quando comparados ao ácido precursor, com destaque para os galatos de hexila e octila, que inibiram em quase 100% a produção de radical ânion superóxido, bem como de todas as EROs do burst oxidativo de forma geral. Galatos de octila e hexila também mostraram-se ser as moléculas com maior atividade antimicrobiana sobre H. pylori, com um valor de CIM de 125 e 250g/mL, respectivamente, ao passo que as demais substâncias apresentaram valor de CIM acima de 1000 g/mL. Os resultados obtidos mostram o grande potencial dos ésteres do ácido gálico quanto à suas atividades anti-H. pylori e anti-EROs, e além disso demonstram a importância da presença de uma cadeia carbônica lateral, conferindo maior hidrofobicidade à molécula, para obter-se a máxima atividade antimicrobiana in vitro e a máxima atividade antioxidante em modelo ex vivo. Assim, os ésteres do ácido gálico apresentam-se como moléculas promissoras no tratamento da infecção por Helicobacter pylori, apresentando ação antimicrobiana sobre o mesmo, bem como na redução do estresse oxidativo gerado no sítio da infecção. / Helicobacter pylori is one of major cause of chronic gastritis and peptic ulcer disease, and although the mechanism involved in gastric inflammation by this bacterium is not fully understood, it is know the important role of reactive oxygen species (ROS) produced by polymorphonuclear neutrophils (PMNs), which are attracted and activated by infection agent, without, however, to be able to overcome the same, but which contribute strongly to the tissue damage and chronic inflammation. Therefore, it has increased the search for new strategies of treatment that can lead to the reduction of the oxidative stress generated at the infection site, with consequent reduction of the inflammatory process. In this sense, gallic acid, together with its esters (methyl, propyl, hexyl and octyl gallate), were used in this study, with the aim of evaluating their actions as inhibitors of ROS release by activated neutrophils, as well as their antimicrobial effects on H. pylori. Cell-free assays (suppressor effect on the DPPH radical and peroxyl radicals) were performed to evaluate the antioxidant activity of these substances, and the anti-EROs effect was evaluated using neutrophils isolated from human blood, stimulated by H. pylori, Zymosan or PMA, through luminol-dependent or lucigenin-dependent chemiluminescent assay, WST-1 assay, inhibition of HOCl production assay, and the NBT assay. Antimicrobial assay was performed by broth microdilution technique. The presence of the carbonic side chain led to a significant increase in the ability of the esters to inhibit the ROS production by activated neutrophils when compared to the precursor acid, especially hexyl and octyl gallates, which inhibited practically 100% of the superoxide anion radical production, as well as all ROS of the oxidative burst in general. Octyl and hexyl gallates were also shown to be the molecules with the highest antimicrobial activity on H. pylori, with a MIC value of 125 and 250 μg/mL, respectively, while the other substances had a MIC value higher than 1000 g/ml. The results show the great potential of the esters of gallic acid for their anti-H. pylori and anti-EROs activities, and furthermore demonstrate the importance of the presence of a lateral carbonic chain, giving greater hydrophobicity to the molecule, to obtain the maximum antimicrobial activity in vitro and the maximum antioxidant activity in an ex vivo model. Thus, esters of gallic acid are promising molecules in the treatment of Helicobacter pylori infection, presenting antimicrobial action on the same, as well as reducing the oxidative stress generated at the site of infection. / CNPq: 130667/2015-3 / FAPESP: 2015/21693-0

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No. of bitstreams: 1 sposito_l_me_arafcf.pdf: 2393152 bytes, checksum: cd33f70c4383729bdf5dc49ce9d3375f (MD5) Previous issue date: 2017-06-02 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O uso de produtos naturais na terapêutica convencional vem ganhando destaque nos últimos anos com relação a atividade antimicrobiana devido à alta taxa de efeitos tóxicos e ao excessivo crescimento do número de cepas multirresistentes aos antimicrobianos convencionais empregados na prática. Entre as novas triagens terapêuticas frente às afecções bacterianas, as infecções causadas pelo Helicobacter pylori merecem atenção especial, uma vez que estão diretamente relacionadas com quadros clínicos severos, como a gastrite crônica, úlcera péptica e câncer gástrico. A Casearia sylvestris Swartz é uma planta popularmente conhecida como guaçatonga ou erva-de-bugre, utilizada na medicina popular no tratamento de transtornos gástricos que nos últimos anos vem despertando interesse na comunidade científica, por suas importantes propriedades com potencial terapêutico como atividade anti-inflamatório, antiofídico, cicatrizante, citotóxica em linhagens de células tumorais e principalmente antiulcerogênica. Neste contexto, uma excelente alternativa para o aprimoramento dos parâmetros biodisponíveis dos extratos vegetais muito utilizada na atualidade é a incorporação destes em sistemas nanotecnológicos de liberação de fármaco para que o produto ativo tenha uma melhor ação. Deste modo, o presente trabalho teve por objetivo avaliar o potencial biológico antibacteriano in vitro do extrato etanólico, extrato aquoso, fração e subfrações das folhas de Casearia sylvestris Swartz, incorporar em um sistema de liberação de fármacos os derivados mais ativos e a partir dos resultados realizar o experimento in vivo contra a cepa ATCC 43504 de H. pylori. Os extratos foram preparados por infusão e maceração. Para as análises biológicas utilizamos a técnica de microdiluição para determinar a concentração inibitória mínima e o biofilme formado. O estudo foi desenvolvido aplicando-se metodologias in vitro e in vivo para elucidar o perfil antibacteriano pretendido, obtendo-se resultados satisfatórios, deixando esse estudo mais interessante, já que a literatura mostra que a C.sylvestris tem perfil anti-ulcerogênico. A partir desses resultados pudemos concluir que os derivados de C. sylvestris apresentaram estatisticamente atividades antibacterianas tanto in vitro como in vivo, sendo que a maioria dos derivados vegetais com exceção dos extratos aquosos, fração 1 e 3 e a subfração 17, tiveram alguma atividade frente ao micro-organismo. Essa atividade provavelmente esta relacionada aos diterpenos clerodânicos que estão em abundância nos derivados da C. sylvestris. A incorporação no sistema nanoestruturado não potencializou a atividade dos derivados vegetais como esperado na maioria dos teste, isto talvez seja pelo fato de a composição dos derivados vegetais e do sistema não serem compatíveis quimicamente. Desde que a literatura mostrou a atividade antiulcerogênica dos derivados vegetais da C. sylvestris, eles podem ser utilizados tanto na cicatrização das lesões como também para a erradicação do H. pylori. / The use of natural products in conventional therapeutics has been gaining highlight in recent years relative to the antimicrobial activity due to the high rate of toxic effects and the excessive growth of the number of multiresistant strains to he conventional antimicrobials used in practice. Among the new therapeutic screenings for bacterial diseases, infections caused by Helicobacter pylori deserve special attention, since they are directly related to severe clinical conditions, such as chronic gastritis, peptic ulcer and gastric cancer. Casearia sylvestris Swartz is a plant popularly known as guaçatonga or erva-de-bugre, used in popular medicine to the treatment of gastric disorders that in recent years has gained the interest of the scientific community due to its important properties with therapeutic potential as an anti-inflammatory, antiofidic, healing, cytotoxicity against tumor cells and mainly antiulcerogenic. In this context, an excellent alternative for the improvement of the bioavailability parameters of the vegetal extracts that is used today is the incorporation of these in nanotechnological systems of drug release so that the active product has a better action. Thus, the aim of this study was to evaluate the in vitro antibacterial potential of the ethanolic extract, aqueous extract, fraction and subfractions of the leaves of Casearia sylvestris Swartz, and incorporate into a drug delivery system the most active derivatives and from these results carry out the in vivo experiment against the H. pylori ATCC strain. The extracts were prepared by infusion and maceration. For the biological analyzes we use the microdilution technique to determine the minimum inhibitory concentration and the formed biofilm. The study was developed applying in vitro and in vivo methodologies to elucidate the antibacterial profile. From the results, we can conclude that the C. sylvestris derivatives showed statistically antibacterial activities both in vitro and in vivo. Most of the vegetal derivatives with the exception of aqueous extracts, fractions 1 and 3 and subfraction 17, had some activity against to the microorganism. This activity is probably related to clerodan diterpenes that are abundant in C. sylvestris derivatives. The incorporation into the nanostructured system did not potentiate the activity of the vegetable derivatives as expected in most tests, this is perhaps due to the fact that the composition of the plant derivatives and the system are not chemically compatible. Since the literature shows an antiulcerogenic activity of C. sylvestris derivatives, they could be used both for wound healing and for H. pylori eradication.

Philosophiae Doctor - PhD (Biotechnology) / Availability of genome sequences for numerous bacterial species comprising of different bacterial strains allows elucidation of species and strain specific adaptations that facilitate their survival in widely fluctuating micro-environments and enhance their pathogenic potential. Different bacterial species use different strategies in their pathogenesis and the pathogenic potential of a bacterial species is dependent on its genomic complement of virulence factors. A bacterial virulence factor, within the context of this study, is defined as any endogenous protein product encoded by a gene that aids in the adhesion, invasion, colonization, persistence and pathogenesis of a bacterium within a host. Anecdotal evidence suggests that bacterial virulence genes are undergoing diversifying evolution to counteract the rapid adaptability of its host’s immune defences. Genome sequences of pathogenic bacterial species and strains provide unique opportunities to study the action of diversifying selection operating on different classes of bacterial genes. / South Africa

Please read the abstract in the section front of this document / Dissertation (MSc)--University of Pretoria, 2007. / Chemical Pathology / unrestricted

Introduction. The operative link for gastritis assessment (OLGA) and the operative link on gastric intestinal meta-plasia assessment (OLGIM) staging systems have been sug-gested to provide risk of assessment for gastric cancer. Objec-tive. To evaluate the distribution of OLGA and OLGIM staging by age and Helicobacter pylori status. Material and methods. We studied 197 subjects undergoing elective upper gastrointestinal endoscopy. The presence of the H. pylori and histological changes were evaluated using the updated Sydney system. Stages III and IV of OLGA/OLGIM were considered high risk stages. Results. The H. pylori rate was 56.85% (112/197). High-risk OLGA/OLGIM cases were rare: 7/112 (6.5%) cases of OLGA in the H. pylori positive group and 6/85 (7%) in the H. pylori negative group; 5 (4.4%) cases of OLGIM in the H. pylori positive and 6 (7%) in the H. pylori negative. The proportion of advanced stages of OLGA and OLGIM increased with age (p < 0.001). High-risk OLGA was not found before age 40 regardless of the presence of H. pylori, but increased to 16.2%, 10.3%, 17.3% and 40.8% in subjects in the fourth, fifth, sixth and seventh decade of life respectively. The OLGIM high risk showed a similar trend: 0% before 40 years and up to 22.6% in people of 70 years. Conclusions. High-risk OLGA/OLGIM cases are infrequent before age 40 and increase significantly with age. No relation was found with the presence of the H. pylori. According to these protocols, only a fifth of the patients would strictly require endoscopic control. / Revisión por pares

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