No / This review focuses on treatment targets for the most common inflammatory skin diseases, eczema and psoriasis with an emphasis on cytokines expressed in the uppermost layer of the skin which is easily accessible for diagnostic and therapeutic approaches. Recently, a significant body of research has highlighted the influence of the skin barrier and the patients’ microbiome on skin inflammatory responses and we will comment on their impact on mediator regulation. Itch is a prominent dermatology symptom which is influenced by cytokines and can via itch–scratch cycle impact on the skin barrier and mediator expression associated with damage. Taking the contribution of pruritus and superficial skin damage into account, we address cytokines as targets for stratified treatment approaches in subgroups of eczema and psoriasis.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Plant defensin peptides have become promising and attractive candidates to be used as antifungal agents in agricultural biotechnology. These peptides have a broad spectrum antifungal activity and play a vital role in the innate immune system of plants. Plant diseases caused by fungi are a major contributor to the decrease in the quality and safety of agricultural products. Due to the dangerous effects and negative environmental impact of pesticides, an effective, safe, natural and durable method to control crop pathogens has therefore become one of the major concerns in modern agriculture. Although these peptides are promising and attractive candidates, their precise mechanism of action is to date still unknown. Several common observations have been made. These include the antagonistic effect of cations on the activity of plant defensins. It is of vital importance to understand the underlying mechanism of the cation-antagonistic effect on the antifungal potency of defensin peptides in order to evaluate the possible contribution to defence reactions against microorganisms in planta. To this end we set out to characterize the effect of cations in the form of biological salts, NaCl, KCl, MgCl2 and CaCl2 on the structural stability and activity in terms of growth inhibition, morphological effects and permeabilization. In order to perform these characterization experiments, a production method resulting in a greater yield and involving simple and rapid purification methods was required. Heliophila coronopifolia peptides have previously been produced in a bacterial system, however the purification methods were tedious resulting in poor yields. Pichia pastoris was selected as production system as several other plant defensins have been successfully produced in this eukaryotic system. Hc-AFP1 and Hc-AFP3 was successfully produced using the Pichia production system and rendered active peptides. Hc-AFP2 and Hc- AFP4 was, however, not produced correctly, due to a post-translational modification event leading to the cyclization of the N-terminal glutamine to generate pyroglutamic acid. This modification negatively influenced the activity of these peptides. An active Hc-AFP2 could be produced by replacing the production buffer with a reduced ionic buffer. The effect of divalent and monovalent cations on the secondary structure of Hc-AFP1 was evaluated by circular dichroism spectroscopy. These cations induced a conformational change in the secondary structure of Hc-AFP1, with NaCl and MgCl2 inducing a more defined secondary structure and KCl and CaCl2 inducing a less defined secondary structure. Monovalent cations caused a slight reduction in the growth inhibition activity of Hc-AFP1 on Botrytis cinerea, however, characteristic hyperbranching and other morphogentic effects were still visible. Divalent cations had a greater antagonistic effect on the activity of Hc-AFP1, completely abolishing the growth inhibitory activity of the peptide, but the induced morphological effects on hyphae remained present. The activity of Hc-AFP1 to permeabilize B. cinerea hyphae was not influenced by the addition of cations, however it was in fact increased to up to 10-fold. However, since the growth inhibition activity of Hc-AFP1 was reduced in the presence of the biological salts indicates that permeabilization is not the sole activity responsible for growth inhibition caused by Hc-AFP1. This peptide probably has an alternative/primary target and more complex MOA. This is the first known report of the investigation of the influence of cations on the structure of plant defensin peptides. It is clear that cations induce a secondary structural conformational change in Hc-AFP1. This may be linked to the antagonism on the activity of this peptide. This study provides significant progress towards the structure-function analysis of plant defensins. / AFRIKAANSE OPSOMMING: Plantdefensinpeptiede word beskou as belowende en aantreklike kandidate vir gebruik as swammiddles in agribiotegnologie. Hierdie peptiede beskik oor breë spektrum antifungiese aktiwiteit en speel ‘n essensiële rol in die ingebore immuunsisteem van plante. Plant siektes wat deur swamme veroorsaak word dra betekenisvol by tot die afname in die kwaliteit en veiligheid van landbouprodukte. As gevolg van die skadelike effekte en negatiewe omgewingsimpak van plaagdoders, het effektiewe, veilige, natuurlike en duursame metodes om gewaspatogene te beheer, van die belangrikste vraagstukke van moderne landbou geword. Alhoewel hierdie peptiede belowende en aantreklike kandidate is vir die toepassing, is hulle presiese meganisme van aksie tot vandag toe steeds onbekend. Verskeie algemene waarnemings is egter al gemaak. Dit sluit die antagonistiese effek van katione op die aktiwiteit van plantdefensinpeptiede in. Dit is kernbelangrik om die onderliggende meganisme van die katioon-antagonistiese effek op die antifungiese effektiwiteit te verstaan om die moontlike bydrae van die peptiede tot die verdedigingsreaksies teen mikro-organismes in planta te evalueer. Met die doel voor oë het ons gemik om die effek van katione, spesifiek in die vorm van die biologiese soute NaCl, KCl, MgCl2 en CaCl2, op die strukturele stabiliteit en aktiwiteit in terme van groei inhibisie, morfologiese effekte en permeabilisasie te karakteriseer. Om uiteindelik hierdie karakterisasie eksperimente uit te voer was dit nodig om ‘n metode met ‘n groter opbrengs en wat vinnige suiwering van die peptied ondersteun, te optimiseer. Heliophila coronopifolia peptiede was voorheen in ‘n bakteriese sisteem geproduseer, maar die suiweringsmetodes was tydsaam en het gelei tot ‘n swak opbrengs. Pichia pastoris is dus geselekteer as die produksie sisteem aangesien verskeie ander plantdefensinpeptiede al suksesvol geproduseer is in hierdie eukariotiese sisteem. Hc-AFP1 and Hc-AFP3 is suksesvol vervaardig in die Pichia sisteem en het aktiewiteit getoon. Hc-AFP2 and Hc-AFP4 kon egter nie korrek vervaardig word nie as gevolg van ‘n na-vertalingsverandering wat gelei het tot die siklisering van die N-terminale glutamien, om piroglutamiensuur te lewer. Hierdie verandering het die aktiwiteit van die peptied negatief beinvloed. ‘n Aktiewe Hc-AFP2 kon wel vervaardig word deur die produksiebuffer te vervang met ‘n lae-ionise buffer. Die effek van divalente en monovalente katione op die sekondêre struktuur van Hc-AFP1 is ge-evalueer deur van sirkulêre dikroisme spektroskopie gebruik te maak. Hierdie katione het ‘n vouingsverandering in die sekondêre struktuur van Hc-AFP1 geïnduseer, NaCl and MgCl2 het ‘n meer gedefinieërde sekondêre struktuur induseer, terwyl KCl and CaCl2 ‘n minder gedefinieërde sekondêre struktuur geinduseer het. Monovalente katione het ‘n effense vermindering in die groei-inhibisie aktiwiteit van Hc-AFP1 op Botrytis cinerea veroorsaak, alhoewel kenmerkende hife-oorvertakking en ander morfologiese effekte nogsteeds sigbaar was. Divalente katione het ‘n sterker antagonistiese effek gehad op die aktiwiteit van Hc-AFP1, waar dit totaal en al die groei-inhibisie aktiwiteit van die peptied vernietig het, alhoewel die geïnduseerde morfologiese effekte op die hiffes steeds sigbaar was . Die aktiwiteit van Hc-AFP1 om B. cinerea hyphae te permeabiliseer is nie negatief beinvloed deur die byvoeging van katione nie, tewens dit het die aktiwiteit tot 10-voudig verhoog. Aangesien die groei-inhibisie aktiwiteit van Hc-AFP1 nie verminder is in die teenwoordigheid van die biologiese soute nie, dui dit aan dat permeabilisasie nie die enigste aktiwiteit is wat die groei inhibisie veroorsaak het nie. Die peptied het dus moontlik ‘n alternatiewe of primêre teiken en ‘n meer komplekse meganisme van aksie. Dit is die eerste verslag wat die invloed van katione op die struktuur van plantdefensinpeptiede ondersoek het. Dit is duidelik dat katione ‘n sekondêre strukturele vouingsverandering in Hc-AFP1 induseer. Hierdie verandering mag dalk bydra tot die antagonistiese uitwerking op die aktiwiteit van hierdie peptied. Hierdie studie het betekensisvolle vordering gemaak met die analise van die struktuur-funksie interaksie van plantdefensinpeptiede. / The National Research Foundation (NRF), Institute of Wine Biotechnology (IWBT), THRIP and Winetech for financial assistance.

IL-36 gamma is a proinflamatory cytokine which belongs to the IL-1 family of cytokines. It is expressed in the skin and by epithelial cells (ECs) lining lung and gut tissue. We used human 3-D organotypic cells, that recapitulate either in vivo human vaginal or cervical tissue, to explore the possible role of IL-36 gamma in host defense against pathogens in the human female reproductive tract (FRT). EC were exposed to compounds derived from virus or bacterial sources and induction and regulation of IL-36 gamma and its receptor was determined. Polyinosinic-polycytidylic acid (poly I:C), flagellin, and synthetic lipoprotein (FSL-1) significantly induced expression of IL-36 gamma in a dose-dependent manner, and appeared to be TLR-dependent. Recombinant IL-36 gamma treatment resulted in self amplification of IL-36 gamma and its receptor (IL-36R) via increased gene expression, and promoted other inflammatory signaling pathways. This is the first report to demonstrate that the IL-36 receptor and IL-36 gamma are present in the human FRT EC and that they are differentially induced by microbial products at this site. We conclude that IL-36 gamma is a driver for epithelial and immune activation following microbial insult and, as such, may play a critical role in host defense in the FRT.

A detailed description of the characteristics of antimicrobial peptides (AMPs) is highly demanded, since the resistance against traditional antibiotics is an emerging problem in medicine. They are part of the innate immune system in every organism, and they are very efficient in the protection against bacteria, viruses, fungi and even cancer cells. Their advantage is that their target is the cell membrane, in contrast to antibiotics which disturb the metabolism of the respective cell type. This allows AMPs to be more active and faster. The lack of an efficient therapy for some cancer types and the evolvement of resistance against existing antitumor agents make AMPs promising in cancer therapy besides being an alternative to traditional antibiotics. The aim of this work was the physical-chemical characterization of two fragments of LL-37, a human antimicrobial peptide from the cathelicidin family. The fragments LL-32 and LL-20 exhibited contrary behavior in biological experiments concerning their activity against bacterial cells, human cells and human cancer cells. LL-32 had even a higher activity than LL-37, while LL-20 had almost no effect. The interaction of the two fragments with model membranes was systematically studied in this work to understand their mode of action. Planar lipid films were mainly applied as model systems in combination with IR-spectroscopy and X-ray scattering methods. Circular Dichroism spectroscopy in bulk systems completed the results. In the first approach, the structure of the peptides was determined in aqueous solution and compared to the structure of the peptides at the air/water interface. In bulk, both peptides are in an unstructured conformation. Adsorbed and confined to at the air-water interface, the peptides differ drastically in their surface activity as well as in the secondary structure. While LL-32 transforms into an α-helix lying flat at the water surface, LL-20 stays partly unstructured. This is in good agreement with the high antimicrobial activity of LL-32. In the second approach, experiments with lipid monolayers as biomimetic models for the cell membrane were performed. It could be shown that the peptides fluidize condensed monolayers of negatively charged DPPG which can be related to the thinning of a bacterial cell membrane. An interaction of the peptides with zwitterionic PCs, as models for mammalian cells, was not clearly observed, even though LL-32 is haemolytic. In the third approach, the lipid monolayers were more adapted to the composition of human erythrocyte membranes by incorporating sphingomyelin (SM) into the PC monolayers. Physical-chemical properties of the lipid films were determined and the influence of the peptides on them was studied. It could be shown that the interaction of the more active LL-32 is strongly increased for heterogeneous lipid films containing both gel and fluid phases, while the interaction of LL-20 with the monolayers was unaffected. The results indicate an interaction of LL-32 with the membrane in a detergent-like way. Additionally, the modelling of the peptide interaction with cancer cells was performed by incorporating some negatively charged lipids into the PC/SM monolayers, but the increased charge had no effect on the interaction of LL-32. It was concluded, that the high anti-cancer activity of the peptide originates from the changed fluidity of cell membrane rather than from the increased surface charge. Furthermore, similarities to the physical-chemical properties of melittin, an AMP from the bee venom, were demonstrated. / Aufgrund der steigenden Resistenzen von Zellstämmen gegen traditionelle Therapeutika sind alternative medizinische Behandlungsmöglichkeiten für bakterielle Infektionen und Krebs stark gefragt. Antimikrobielle Peptide (AMPs) sind Bestandteil der unspezifischen Immunabwehr und kommen in jedem Organismus vor. AMPs lagern sich von außen an die Zellmembran an und zerstören ihre Integrität. Das macht sie effizient und vor allem schnell in der Wirkung gegen Bakterien, Viren, Pilzen und sogar Krebszellen. Das Ziel dieser Arbeit lag in der physikalisch-chemischen Charakterisierung zweier Peptidfragmente die unterschiedliche biologische Aktivität aufweisen. Die Peptide LL-32 und LL-20 waren Teile des humanen LL-37 aus der Kathelizidin-Familie. LL-32 wies eine stärke Aktivität als das Mutterpeptid auf, während LL-20 kaum aktiv gegen die verschiedenen Zelltypen war. In dieser Arbeit wurde die Wechselwirkung der Peptide mit Zellmembranen systematisch anhand von zweidimensionalen Modellmembranen in dieser Arbeit untersucht. Dafür wurden Filmwaagenmessungen mit IR-spektroskopischen und Röntgenstreumethoden gekoppelt. Circulardichroismus-Spektroskopie im Volumen komplementierte die Ergebnisse. In der ersten Näherung wurde die Struktur der Peptide in Lösung mit der Struktur an der Wasser/Luft-Grenzfläche verglichen. In wässriger Lösung sind beide Peptidfragmente unstrukturiert, nehmen jedoch eine α-helikale Sekundärstruktur an, wenn sie an die Wasser/Luft-Grenzfläche adsorbiert sind. Das biologisch unwirksamere LL-20 bleibt dabei teilweise ungeordnet. Das steht im Zusammenhang mit einer geringeren Grenzflächenaktivität des Peptids. In der Zweiten Näherung wurden Versuche mit Lipidmonoschichten als biomimetisches Modell für die Wechselwirkung mit der Zellmembran durchgeführt. Es konnte gezeigt werden, dass sich die Peptide fluidisierend auf negativ geladene Dipalmitylphosphatidylglycerol (DPPG) Monoschichten auswirken, was einer Membranverdünnung an Bakterienzellen entspricht. Eine Interaktion der Peptide mit zwitterionischem Phosphatidylcholin (PC), das als Modell für Säugetierzellen verwendet wurde, konnte nicht klar beobachtet werden, obwohl biologische Experimente das hämolytische Verhalten zumindest von LL-32 zeigten. In der dritten Näherung wurde das Membranmodell näher an die Membran von humanen Erythrozyten angepasst, indem gemischte Monoschichten aus Sphingomyelin (SM) und PC hergestellt wurden. Die physikalisch-chemischen Eigenschaften der Lipidfilme wurden zunächst ausgearbeitet und anschließend der Einfluss der Peptide untersucht. Es konnte anhand verschiedener Versuche gezeigt werden, dass die Wechselwirkung von LL-32 mit der Modellmembran verstärkt ist, wenn eine Koexistenz von fluiden und Gelphasen auftritt. Zusätzlich wurde die Wechselwirkung der Peptide mit der Membran von Krebszellen imitiert, indem ein geringer Anteil negativ geladener Lipide in die Monoschicht eingebaut wurde. Das hatte allerdings keinen nachweislichen Effekt, so dass geschlussfolgert werden konnte, dass die hohe Aktivität von LL-32 gegen Krebszellen ihren Grund in der veränderten Fluidität der Membran hat und nicht in der veränderten Oberflächenladung. Darüber hinaus wurden Ähnlichkeiten zu Melittin, einem AMP aus dem Bienengift, dargelegt. Die Ergebnisse dieser Arbeit sprechen für einen Detergenzien-artigen Wirkmechanismus des Peptids LL-32 an der Zellmembran.

A range of simulations have been conducted to investigate the behaviour of a diverse set of complex biological membrane systems. The processes of interest have required simulations over extended time and length scales, but without sacrifice of molecular detail. For this reason, the primary technique used has been coarse-grained molecular dynamics (CG MD) simulations, in which small groups of atoms are combined into lower-resolution CG particles. The increased computational efficiency of this technique has allowed simulations with time scales of microseconds, and length scales of hundreds of nm. The membrane-permeabilizing action of the antimicrobial peptide maculatin 1.1 was investigated. This short α-helical peptide is thought to kill bacteria by permeabilizing the plasma membrane, but the exact mechanism has not been confirmed. Multiscale (CG and atomistic) simulations show that maculatin can insert into membranes to form disordered, water-permeable aggregates, while CG simulations of large numbers of peptides resulted in substantial deformation of lipid vesicles. The simulations imply that both pore-forming and lytic mechanisms are available to maculatin 1.1, and that the predominance of either depends on conditions such as peptide concentration and membrane composition. A generalized study of membrane protein aggregation was conducted via CG simulations of lipid bilayers containing multiple copies of model transmembrane proteins: either α-helical bundles or β-barrels. By varying the lipid tail length and the membrane type (planar bilayer or spherical vesicle), the simulations display protein aggregation ranging from negligible to extensive; they show how this biologically important process is modulated by hydrophobic mismatch, membrane curvature, and the structural class or orientation of the protein. The association of influenza hemagglutinin (HA) with putative lipid rafts was investigated by simulating aggregates of HA in a domain-forming membrane. The CG MD study addressed an important limitation of model membrane experiments by investigating the influence of high local protein concentration on membrane phase behaviour. The simulations showed attenuated diffusion of unsaturated lipids within HA aggregates, leading to spontaneous accumulation of raft-type lipids (saturated lipids and cholesterol). A CG model of the entire influenza viral envelope was constructed in realistic dimensions, comprising the three types of viral envelope protein (HA, neuraminidase and M2) inserted into a large lipid vesicle. The study represents one of the largest near-atomistic simulations of a biological membrane to date. It shows how the high concentration of proteins found in the viral envelope can attenuate formation of lipid domains, which may help to explain why lipid rafts do not form on large scales in vivo.

Pour comprendre les mécanismes de l'immunité innée, nous utilisons Caenorhabditis elegans comme un organism model host et coniospora Drechmeria comme un pathogène. D. coniospora adhère à la cuticule de C. elegans pour infecter son épiderme. Le ver répond par une régulation de gènes de défense multiples, y compris des gènes codant pour des peptides antimicrobiens (AMP) comme nlp-29. En utilisant des vers transgéniques portant des constructions rapporteurs fluorescents comme nlp-29p :: gfp, nous pouvons suivre l'expression des gènes in vivo AMP et de chercher des gènes nécessaires à l'induction de gènes AMP à travers les écrans génétiques. Le but de mon projet était de caractériser des mutants qui ont été identifiés dans un nouvel écran génétique saturée, où 57 nouveaux allèles Nipi qui manquent nlp-29 d'induction après l'infection ont été isolés. Adaptation d'une nouvelle cartographie combinée SNP et toute stratégie de séquençage du génome, nous avons pu isoler 15 allèles de gènes nouveaux Nipi déjà connus et 12 allèles de 6 «nouveaux» gènes. Notre travail a confirmé le rôle principal de la MAPK PKCδ/p38 dans la régulation de l'expression nlp-29 AMP après l'infection, ainsi que le facteur de transcription STA-2/STAT et le transporteur SNF-12/SLC6. Nous faire progresser nos connaissances en identifiant NIPI-4 comme un régulateur positif de l'expression des gènes nlp peptide antimicrobien après l'infection. NIPI-4 est un membre de la famille des kinases nématode spécifique et est prévu pour être un pseudokinase. Nous avons montré qu'il agit dans l'épiderme partie aval de la MAPK p38. / To understand the mechanisms of innate immunity, we use Caenorhabditis elegans as a model host and Drechmeria coniospora as a fungal pathogen. D. coniospora adheres to the cuticle of C. elegans to infect its epidermis. The worm responds by an up-regulation of multiple defence genes, including genes encoding anti-microbial peptides (AMP) like nlp-29. Using transgenic worms carrying fluorescent reporter constructs like nlp-29p::gfp, we can follow AMP gene expression in vivo and look for genes required for the induction of AMP genes through genetic screens. The aim of my project was to characterize mutants that have been identified in a new saturated genetic screen, where 57 new Nipi alleles that lack nlp-29 induction after infection were isolated. Adapting a new combined SNP mapping and whole genome sequencing strategy we were able to isolate 15 new alleles of previously known Nipi genes and 12 alleles of 6 “new” genes. Our work confirmed the primary role of the PKCδ/p38 MAPK in the regulation of nlp-29 AMP expression after infection, as well as the STA-2/STAT transcription factor and the SNF-12/SLC6 transporter. We further advance our knowledge by identifying NIPI-4 as a positive regulator of nlp antimicrobial peptide genes expression after infection. NIPI-4 is a member of a nematode-specific kinase family and is predicted to be a pseudokinase. We showed that it acts in the epidermis partially downstream of the p38 MAPK. It also controls the constitutive expression of antimicrobial peptide genes of the cnc family that are targets of TGFß regulation. Together these suggested that NIPI-4 acts with STA-2 and SNF-12 to regulate AMP gene expression in the epidermis.

Sabe-se que a hemoglobina é uma rica fonte de peptídeos antimicrobianos (hemocidinas). A primeira hemocidina derivada da hemoglobina bovina caracterizada em carrapatos foi o peptídeo Hb33-61, que é ativo contra bactérias gram-positivas e fungos. Acredita-se que tais hemocidinas sejam geradas proteoliticamente no intestino do carrapato. Neste trabalho nós caracterizamos bioquimicamente uma catepsina D, designada BmAP. A análise da expressão gênica por qPCR mostrou que ela é expressa predominantemente no intestino. Através de LC-MS/MS, determinamos a especificidade de clivagem da BmAP utilizando Hb bovina, e verificamos que resíduos hidrofóbicos foram preferencialmente clivados nos subsítios P1 e P1. Também investigamos a especificidade de clivagem da catepsina L intestinal BmCL1, utilizando uma biblioteca combinatória de tetrapeptídeos e através de hemoglobinólise in vitro. A BmCL1 preferiu resíduos alifáticos no P2 e polares no P1 e P1. Além disso, hidrolisou a cadeia da Hb bovina entre A63/A64, gerando peptídeos com estrutura primária similar ao Hb 33-61. A hemoglobinólise com a BmAP e/ou BmCL1 resultou na formação de algumas hemocidinas, corroborando a hipótese do seu envolvimento na geração endógena de peptídeos antimicrobianos. / It is known that hemoglobin is a rich source of antimicrobial peptides (hemocidins). The first hemoglobin-derived hemocidin characterized in ticks was the peptide Hb33-61, which is active against Gram-positive bacteria and fungi. It is believed that hemocidins are endogenously generated in the tick gut. In this work we biochemically characterized a cathepsin D, designated BmAP. Expression analysis by qRT-PCR showed that it is expressed predominantly in the gut. Through LC-MS/MS, we determined the cleavage specificity of BmAP using bovine hemoglobin, and we verified that hydrophobic residues were preferentially cleaved at the subsites P1 and P1. We also investigated the cleavage specificity of the intestinal cathepsin L BmCL1, using a positional scanning synthetic combinatorial library and through in vitro hemoglobinolysis. BmCL1 preferred aliphatic residues at P2 and polar residues at P1 and P1. Also, it hydrolysed the subunit of bovine hemoglobin at A63/A64, generating peptides with a primary structure similar to Hb 33-61. Hemoglobinolysis with BmAP and/or BmCL1 resulted in the formation of some hemocidins, corroborating the hypothesis that these proteinases are involved in the endogenous generation of antimicrobial peptides

A hemoglobina possuí um grande potencial antimicrobiano, sendo os peptídeos antimicrobianos (AMPs) derivados de proteínas contêm o grupamento heme denominados hemocidinas. O carrapato bovino Rhipicephalus (Boophilus) microplus é capaz de adquirir e processar grandes quantidades de sangue para seu metabolismo. Neste trabalho, purificamos hemocidinas produzidas no tubo digestório do carrapato. Identificamos 10 fragmentos da hemoglobina bovina que possuem atividade contra Candida albicans (Hb 1-94, 3-94, 1-87, 93-141, 102-141, 103-141, 107-141, 104-141 e 98-114 e Hb\"beta\" 127-145). Determinamos que todos os peptídeos apresentam um alto conteúdo de aminoácidos básicos, além de uma estrutura secundária em \"alfa\"-hélice, características encontradas em outros AMPs. Verificamos também que as duas principais proteases que atuam na produção das hemocidinas são uma cisteíno- e uma aspártico-proteinase. A identificação de diversas hemocidinas, no tubo digestório de R. (Boophilus) microplus, indica uma possível participação das hemocidinas na proteção contra microorganismos / The hemoglobin posses a great potential as an antimicrobial, being denominated hemocidins the antimicrobial peptides (AMPs) derived from heme containing proteins. The cattle tick Rhipicephalus (Boophilus) microplus is capable of processing large amounts of blood to its own metabolism. In this work we purified the hemocidins that are produced in the tick gut. We identified 10 fragments from bovine hemoglobin that presents activity towards Candida albicans (Hb 1-94, 3-94, 1-87, 93-141, 102-141, 103-141, 107-141, 104-141 e 98-114 e Hb\"beta\" 127-145). We determined that all the peptides presents a high content of basic amino acids, besides a secondary structure in \"alfa\"-helix conformation, characteristics found in other AMPs. We also verified that a cistein- and an aspartic-protease are the two main proteases involved on the production of these hemocids. The identification of several hemocidins in the gut of R. (Boophilus) microplus, suggests that these AMPs may play a role in the defense of the tick against microorganisms

Venenos de aranhas podem ser boas fontes de moléculas com potencial terapêutico e biotecnológico. Sendo assim, esse estudo teve como objetivo analisar moléculas bioativas do veneno de Avicularia juruensis, com foco em PAMs e enzimas. O veneno foi extraído por estimulação elétrica e, através de CLAE-FR, ensaio de inibição do crescimento microbiano e LC-MS/MS, foram identificados e caracterizados 7 PAMs. Todos possuem o motivo nó de cistina do tipo ICK e têm similaridade com neurotoxinas de venenos de outras aranhas. O perfil eletroforético do veneno de A. juruensis indicou que ele possui moléculas com massa entre 130 e abaixo de 10 kDa. Utilizando LC-MS/MS e análise transcriptômica foi possível identificar 6 metaloproteinases. Elas possuem domínios conservados de proteínas do tipo ADAMs, MMPs e metalopeptidases astacina-like. A presença destas enzimas é, provavelmente, importante para a digestão extracorpórea, visto que esta aranha geralmente consome pequenas aves. O estudo de venenos de aranhas terafosídeas é essencial, visto que este é um grupo pouco estudado. / Spider venoms can be good sources of molecules with therapeutic and biotechnological potential. Thus, this study aimed to analyze bioactive molecules from venom of Avicularia juruensis, focusing on AMPs and enzymes. The venom was extracted by electrical stimulation. By RP-HPLC, liquid growth inhibition assay and LC-MS/MS, seven AMPs were identified and characterized. All these peptides have inhibitory cystine knot motif and they showed similarity with venom neurotoxins from others spiders. The electrophoretic profile of A. juruensis venom shows that it has molecules with molecular masses between 130 kDa and below 10 kDa. By LC-MS/MS and transcriptomic analysis yielded the identification of six metalloproteinases, which possess typical conserved domains from ADAMs, MMPs and astacin-like metallopeptidases. These metalloproteinases may be involved in a key role during preoral digestion. The study of Theraphosidae spider venom is essential, since this group is poorly studied.

Estudos atuais revelam que, mesmo na era da terapia antirretroviral (TARV), a infecção pelo HIV-1 é associada com quadros graves e frequentemente refratários de periodontite crônica (PC), despertando para a possibilidade da existência de outros fatores associados ao desenvolvimento da PC nesses pacientes. Acredita-se que fatores relacionados à população microbiana, incluindo as infecções por Candida spp, e a expressão de peptídeos microbianos possam estar envolvidos na patogênese da PC em pacientes infectados pelo HIV-1. O objetivo desse estudo foi determinar a influência do tratamento periodontal não-cirúrgico, na contagem oral de Candida spp, e nos níveis salivares de lactoferrina (Lf) e histatina, por meio de um estudo quase-experimental. Pacientes infectados (Grupo 1) e não infectados pelo HIV-1 (Grupo 2 - controle), todos com PC, foram submetidos à terapia periodontal não cirúrgica. Os pacientes do grupo 1 apresentaram contagem de linfócitos T CD4+ < 200cel/mm3, e estavam em TARV regular. Foi avaliada a contagem de unidades formadoras de colônias (UFC) de Candida spp por meio de enxaguado bucal e níveis salivares de Lf e histatina antes (Tempo 0-1) e após a terapia periodontal (Tempo 2 e 3; 30 e 90 dias após o tratamento, respectivamente). Pacientes do grupo 1 apresentaram contagem de UFC superiores à verificada nos pacientes do grupo 2 (p=0, 268; ANOVAF). Houve tendência a redução de UFC após o tratamento periodontal em ambos os grupos, mas sem diferenças estatisticamente significantes. Os níveis de Lf foram semelhantes entre os grupos, e reduziram 30 dias após o tratamento periodontal (p=0,0111; Mann Whitney). Os níveis salivares de histatina ao tempo 0 foram mais elevados no grupo 1 quando comparado ao grupo 2 (p= 0,6481; Tukey-kramer), mas tiveram comportamento distinto após o tratamento periodontal: foram mais elevados no grupo 1 e reduziram no grupo 2. Estes resultados sugerem a associação entre a presença de Candida spp e a PC, e também a importância da manutenção da higiene oral na prevenção da candidíase. Além disso, Lf salivar pode ser um marcador da PC, tanto em pacientes não-infectados, quanto infectados pelo HIV. / Current studies reveal that, even in the era of antiretroviral therapy (ART), HIV-1 infection is associated with severe and frequently refractory chronic periodontitis (CP), which leads to the possibility of other factors associated with the development of CP in these patients. It is believed that factors related to the microbial population, including Candida spp infections, and the expression of microbial peptides may be involved in the pathogenesis of CP in patients infected with HIV-1. The aim of this study was to determine the influence of non-surgical periodontal treatment on the oral count of Candida spp and on the salivary levels of lactoferrin (Lf) and histatin, by means of a quasi-experimental study. Patients infected (Group 1) and non-HIV-1 infected (Group 2 - control), all with CP, underwent non-surgical periodontal therapy. Patients in group 1 had a CD4 + T-cell count <200cel / mm³, and were on regular ART. The counts of colony forming units (CFU) of Candida spp were evaluated by oral rinsing and salivary levels of Lf and histatin before (Time 0-1) and after periodontal therapy (Time 2 and 3, 30 and 90 days after the treatment, respectively). Patients in group 1 had a higher CFU count than in patients in group 2 (p = 0.268; ANOVAF). There was a tendency to reduce CFU after periodontal treatment in both groups, but without statistically significant differences. Lf levels were similar between groups, and reduced 30 days after periodontal treatment (p = 0.0111; Mann Whitney). Salivary levels of histatin at time 0 were higher in group 1 when compared to group 2 (p = 0.6481; Tukey-Kramer), but had distinct behavior after periodontal treatment: they were higher in group 1 and reduced in group 2. These results suggest the association between the presence of Candida spp and CP, and also the importance of maintaining oral hygiene in the prevention of candidiasis. In addition, Lf salivary can be a marker of CP in both uninfected and HIV infected patients.