Yes / Antimicrobial resistance (AMR) is one of the most alarming global public health challenges of the 21st century. Over 3 million antimicrobial-resistant infections occur in the United States annually, with nearly 50,000 cases being fatal. Innovations in drug discovery methods and platforms are crucial to identify novel antibiotics to combat AMR. We present the isolation and characterization of potentially novel antibiotic lead compounds produced by the cross-feeding of two rhizosphere bacteria, Acinetobacter sp. RIT 592 and Exiguobacterium sp. RIT 594. We used solid-phase extraction (SPE) followed by liquid chromatography (LC) to enrich antibiotic extracts and subsequently mass spectrometry (MS) analysis of collected fractions for compound structure identification and characterization. The MS data were processed through the Global Natural Product Social Molecular Networking (GNPS) database. The supernatant from RIT 592 induced RIT 594 to produce a cocktail of antimicrobial compounds active against Gram-positive and negative bacteria. The GNPS analysis indicated compounds with known antimicrobial activity in the bioactive samples, including oligopeptides and their derivatives. This work emphasizes the utility of microbial community-based platforms to discover novel clinically relevant secondary metabolites. Future work includes further structural characterization and antibiotic activity evaluation of the individual compounds against pathogenic multidrug-resistant (MDR) bacteria. / This work was supported by a National Institutes of Health (NIH) award (R15GM144862) to AH.

Polyethylene glycol-stabilized lipodisks have emerged as a novel type of lipid-based nanoparticles with high potential as both drug carriers and biomimetic membranes. In this thesis we assess both of these applications, and show how the properties of the lipodisks can be further developed and optimized. Initially, we show that the antimicrobial peptides melittin, alamethicin and magainin 2, in spite of their very different physico-chemical properties and suggested modes of action on membranes, all have high affinity to lipodisks. Using melittin as a model peptide, we confirm a maintained antimicrobial effect of disk-formulated peptides. We also show that melittin dissociates slowly from the disks, resulting in extended drug release and prolonged antibacterial effect. Additionally, we present evidence that the peptide is protected against enzymatic degradation when formulated in the disks. Further, we develop a stable HPLC-MS system with immobilized lipodisks as model membranes. The stability of the system is confirmed by drug partitioning analysis using 15 different drug compounds. We also show how the lipodisk column can be supplemented with cyclooxygenase by in situ incorporation of the protein in the lipodisks. The specific binding of the protein to the disks is confirmed using QCM-D. Finally, by changing the polymer length and applying a new preparation protocol, we have optimized the lipodisks for use as drug carriers and biomimetic membranes. Previous lipodisk studies have been conducted on systems containing PEG-lipids with polymer molecular weights of 2000 or 5000 Da. Also, conventional protocols for the preparation of lipodisks typically require a PEG-lipid concentration of 15 mol% or more. Here we show that stable lipodisks can also be produced using PEG-lipids with a 1000 Da molecular weight polymer and that the use of shorter PEG-lipids dramatically improve the amount of lipodisks that can be immobilized on silica surfaces. Moreover, through the development of a method in which lipid mixtures are sonicated at low temperatures, we produce lipodisks containing as little as 2 mol% PEG-lipid. We present data verifying that these disks are superior to disks with higher PEG-lipid content in terms of their ability to incorporate externally added PEG-lipids functionalized with targeting agents.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Biofouling is the attachment and biofilm formation that leads to negative repercussions such as persistent post-harvest infections, infections obtained from medical implants and continual surface contamination of food processing plants. Much of the problem lies with the resistance that develops against conventional treatments due to the formation of mature biofilms. Thus the focus has shifted from the removal of biofilms to the prevention of initial attachment of organisms. This entails the use of antimicrobial surfaces that either have an inherent antimicrobial activity, e.g. certain metals, or surfaces that are modified by the attachment of antimicrobial agents. The attachment of antimicrobial agents can either be through covalent bonding or adsorption, depending on the intended use of the surface as well as the mode of action of the antimicrobial agent. Antimicrobial peptides (AMPs) are ubiquitous in nature, tend to have a broad spectrum of activity, are very stable and have been shown to maintain activity when covalently bound to solid surfaces. Tyrocidines (Trcs), antimicrobial peptides produced by Bacillus aneurinolyticus, are cyclodecapeptides with a broad spectrum of activity against Grampositive bacteria, fungi, yeasts and the human malaria parasite, Plasmodium falciparum. The aim of this study was to determine the antimicrobial activity of surfaces treated with a tyrocidine extract, under which conditions the activity remained stable and to look into possible applications of these peptide-treated surfaces. The study focussed on different solid surfaces namely mixed cellulose, polyvinylidene fluoride, polycarbonate, cellulose acetate, cellulose (paper)(CL) and high density cellulose packing material (HDC), as a pilot study to assess the antimicrobial activity of Trc and gramicidin S (GS) treated solid surfaces. Peptide desorption and subsequent analysis by mass spectrometry was used to confirm the presence and integrity of the Trcs adsorbed. Scanning electron microscopy was utilised to show that the adsorbed peptides did not affect the structural integrity of the treated filters. However, it was shown that the adsorbed peptides changed the hydrophobic/hydrophilic character by means of a wettability assay. A cell viability assay and erythrocyte assay were developed from existing methodologies to determine the biological activity of the AMP-functionalised polymeric material. Seven of the AMP treated solid surfaces showed antimicrobial activity when challenged with >105 Micrococcus luteus cells/cm2. Although the polycarbonate filter lost antimicrobial activity at the high cell concentrations, it was shown to have potent antimicrobial activity at lower cell concentrations. Complete inhibition of M. luteus growth was observed for both the gramicidin S and tyrocidine extract treated high density cellulose and cellulose filters. Stability tests showed that the tyrocidines remained adsorbed to cellulose filters and biologically active when exposed multiple water washes, water washes at different temperatures (25°C - 100°C) and pH changes (pH 1-12). The antimicrobial activity was only affected after exposure to the water wash of pH 13 which is possible due to susceptibility of the CL filters to high pH solvents. A preliminary study on the effect of Trcs treated CL filters on the sterilization, germination and effect on tomato seedlings was conducted. It was found that Trcs had no effect on the germination and did not fully sterilise the seeds or environment against fungi. However, it was observed that 5 μg/mL Trcs treated filters promoted root length opposed to the toxic effect seen with filters treated with higher Trc concentrations. It is hypothesised that Trcs prefer to bind to hydrophilic surfaces exposing the hydrophobic residues and the cationic residue of the peptide to interact with the bacterial membrane to elicit its antimicrobial response. The exposed residues contain some of the hydrophobic residues and the cationic Orn9/Lys9, which are crucial to the antimicrobial activity of the peptides. Hydrophobic interaction is particularly important for the haemolytic activity which is currently the only viable method of detection of the adsorbed Trcs. Trcs also have a preference for adsorption onto cellulose and cellulose analogues which points to possible application in protective food wrapping and wood surface protection. Trcs maintains its antimicrobial activity regardless of adsorption to solid surfaces. It can therefore be concluded that Trcs treated solid surfaces hold great potential in preventing the initial bacterial colonization and subsequent biofilm formation. Antimicrobial peptide enriched solid surfaces can thus be developed and tailored to a specific application such as filters, catheters and packaging materials. / AFRIKAANSE OPSOMMING: Biovervuiling is die aanhegting en vorming van biofilms met negatiewe gevolge soos aanhoudende na-oes infeksies, infeksies op mediese inplantings en voortdurende oppervlak besoedeling van voedselverwerkings fabrieke. Die probleem lê grotendeels by die weerstand wat ontwikkel word teen konvensionele behandelings as gevolg van die vorming van volwasse biofilms. Die fokus het gevolglik verskuif vanaf die verwydering van biofilms na die voorkoming van aanvanklike aanhegting van organismes aan oppervlaktes. Dit behels die gebruik van antimikrobiese oppervlaktes wat of 'n inherente antimikrobiese aktiwiteit het, bv. sekere metale óf oppervlaktes wat aangepas is deur die aanhegting van antimikrobiese middels. Die aanhegting van antimikrobiese agente kan of deur kovalente binding óf adsorpsie plaasvind, afhangende van die beoogde gebruik van die oppervlak, sowel as die metode van werking van die antimikrobiese agent. Antimikrobiese peptiede (AMPe) is alomteenwoordig in die natuur, is geneig om 'n breë spektrum van aktiwiteit te hê, is baie stabiel en het getoon dat aktiwiteit in stand gehou word wanneer dit kovalent gebind word op soliede oppervlaktes. Tirosidiene (Trcs), antimikrobiese peptiede wat deur Bacillus aneurinolyticus geproduseer word, is siklodekapeptiede met 'n breë spektrum van aktiwiteit teen Gram-positiewe bakterieë, swamme, giste en die menslike malaria parasiet Plasmodium falciparum. Die doel van hierdie studie was om die antimikrobiese aktiwiteit te bepaal van oppervlaktes wat met 'n tirosidien ekstrak behandel is, te bepaal onder watter omstandighede die aktiwiteit stabiel bly en om te soek na moontlike toepassings van hierdie peptied-behandelde oppervlaktes. Die studie het gefokus op verskillende soliede oppervlaktes naamlik gemengde sellulose, polyvinylidene fluoried, polikarbonaat, sellulose asetaat, sellulose (papier)(CL) en 'n hoë digtheid sellulose verpakkings materiaal (HDC), as 'n loodsstudie om die antimikrobiese aktiwiteit van die Trcs en gramisidien S (GS) behandelde soliede oppervlaktes te ondersoek. Peptied-desorpsie en daaropvolgende ontleding deur massaspektroskopie is gebruik om die teenwoordigheid en integriteit van die geadsorbeerde Trcs te bevestig. Skandering elektronmikroskopie is gebruik om aan te toon dat die geadsorbeerde peptiede geen invloed op die strukturele integriteit van die behandelde filters het nie. Daar is egter getoon dat die geadsorbeerde peptiede die hidrofobiese / hidrofiliese karakter verander. „n Lewensvatbaarheid selgebaseerde toets en eritrosiet toets is ontwikkel uit bestaande metodes om die biologiese aktiwiteit van die AMP-gefunktionaliseerde polimeriese materiaal te bepaal. Sewe van die AMP behandel soliede oppervlaktes het antimikrobiese aktiwiteit getoon wanneer dit met > 105 Micrococcus luteus selle/cm2 gedaag is. Hoewel die polikarbonaat filter antimikrobiese aktiwiteit met hoë sel konsentrasies verloor het, is dit getoon dat dit wel uitgeproke antimikrobiese aktiwiteit het teen laer konsentrasies selle. Volledige inhibisie van M. luteus groei is waargeneem vir beide die hoë digtheid sellulose en sellulose filters wat met GS en tirosidien ekstrak behandel is. Stabiliteit toetse het getoon dat die tirosidiene geadsorbeer en biologies aktief op sellulose filters bly nadat dit blootgestel is aan verskeie water was-stappe, waterwasse by verskillende temperature (25 °C -100 °C) en pH veranderinge (pH 1-12). Die antimikrobiese aktiwiteit was net beïnvloed ná blootstelling aan die water met 'n pH 13, wat moontlik is te danke aan die vatbaarheid van die CL filters by hoë pH oplosmiddels is. 'n Voorlopige studie is gedoen om die uitwerking van Trcs behandelde CL filters op die sterilisasie, ontkieming en tamatiesaailinge te bepaal. Daar is gevind dat Trcs geen effek op die ontkieming het nie, maar dat dit nie volledig die sade en omgewing steriliseer vir fungiese groei nie. Daar is egter waargeneem dat 5 μg/mL Trcs behandelde filters wortel lengte van die saailinge bevorder teenoor die giftige uitwerking soos waargeneem vir die filters wat met hoër konsentrasies Trcs behandel is. Dit word gepostuleer dat Trcs verkies om aan hidrofiliese oppervlaktes te bind wat die van die hidrofobiese aminosure en die kationiese residu van die peptied blootstel om aan die bakteriële membraan te bind om gevolglik antimikrobiese reaksie te ontlok. Die blootgestelde deel bevat sommige van die hidrofobiese residue en positiewe Orn9/Lys9 wat noodsaaklik vir die antimikrobiese aktiwiteit van die peptiede. Die hidrofobiese interaksies is veral belangrik vir die hemolitiese aktiwiteit wat tans die enigste bruikbare metode van opsporing van die geadsorbeerde Trcs is. Trcs het ook 'n tendens vir adsorpsie op sellulose en sellulose analoë wat dui op die moontlike toepassing in beskermende voedselverpakking en die beskerming van houtoppervlaktes. Trcs handhaaf hul antimikrobiese aktiwiteit, ongeag van adsorpsie aan soliede oppervlaktes. Dit kan dus afgelei word dat Trcs-behandelde soliede oppervlaktes die potensiaal het om die aanvanklike kolonisasie van bakterië te voorkom en die daaropvolgende biofilm vorming. Antimikrobiese peptied verrykde soliede oppervlaktes kan dus ontwikkel en aangepas word vir gebruik in spesifieke toepassing soos in filters, kateters en verpakkingsmateriaal.

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Skin is the largest organ in the human body and serves as a barrier that protects the underlying tissue of the host from infection. Injury, however, destroys this protective barrier and provides a perfect opportunity for microorganisms to invade the host and cause infection, thereby affecting the normal wound healing processes. Furthermore, the ability of microbial pathogens to rapidly develop resistance towards a variety of antimicrobial compounds hampers the effective treatment and control of infections. Antimicrobial-resistant pathogens are increasingly being isolated from patients, placing a huge burden on the health care sector. The search for new and novel antimicrobial agents and treatments is thus of utmost importance and will continue to play an integral role in medical research. Antimicrobial peptides (AMPs) may serve as possible alternatives to antibiotics, or may be used in combination with antibiotics to reduce the risk of antimicrobial resistance. AMPs play a role in innate defence and are produced by a variety of mammals, plants, reptiles, amphibians, birds, fish and insects. The AMPs of bacteria (bacteriocins), especially those of lactic acid bacteria (LAB), are receiving increased attention as antimicrobial agents to treat bacterial infections. Electrospun nanofibers have characteristics that make them suitable as wound dressings, i.e. high oxygen permeability, variable pore size, high surface area to volume ratio and nanofibers are morphologically similar to the extracellular matrix. The ability to incorporate of a variety of biologically active compounds into nanofibers increases their potential as wound dressings. A novel approach would be to incorporate bacteriocins from LAB into nanofiber scaffolds to generate antimicrobial wound dressings. In this study, the feasibility of co-electrospinning bacteriocins from LAB into nanofibers was investigated. Plantaricin 423, produced by Lactobacillus plantarum 423, was successfully co-electrospun into poly(ethylene oxide) (PEO) nanofibers. Plantaricin 423 retained activity after the electrospinning process and continued to inhibit the growth of Lactobacillus sakei DSM 20017T and Enterococcus faecium HKLHS. Viable cells of L. plantarum 423 were also successfully co-electrospun into PEO nanofibers, albeit with a slight reduction in viability. A nanofiber drug delivery system was developed for plantaricin 423 and bacteriocin ST4SA, produced by Enterococcus mundtii ST4SA, by blending PEO and poly(D,L-lactide) (PDLLA) in a suitable solvent before electrospinning. Nanofibers were produced that released the bacteriocins over an extended time period. The PEO:PDLLA (50:50) nanofiber scaffold retained its structure the best upon incubation at 37 °C and released active plantaricin 423 and bacteriocin ST4SA. Nisin A was also successfully co-electrospun into a PEO:PDLLA (50:50) nanofiber scaffold and nisin A, released from the nanofibers, inhibited the growth of Staphylococcus aureus in vitro. Nisin A-containing nanofiber scaffolds significantly reduced viable S. aureus cells in infected skin wounds and promoted wound healing in non-infected wounds. As far as we could determine we are the first to show that bacteriocin-eluting nanofiber scaffolds can be used to treat skin infections and influence wound healing. / AFRIKAANSE OPSOMMING: Vel is die grootse orgaan in die menslike liggaam en dien as buitelaag wat die gasheer se onderliggende weefsel teen infeksie beskerm. Beskadigde vel verloor egter hierdie beskermende eienskap en gee mikroörganismes die geleentheid om die liggaam binne te dring, infeksie te veroorsaak en die normale prosesse geassosieer met wondgenesing te beïnvloed. Die suksesvolle behandeling en beheer van infeksies word gedemp deur die vermoë van mikroörganismes om vinnig weerstand teen antimikrobiese middels te ontwikkel. Mikroörganismes met antimikrobiese weerstand word geredelik van pasiënte geïsoleer en dit plaas enorme druk op die gesondheidssektor. Die soeke na nuwe antimikrobiese middels en behandelings is dus van uiterste belang en sal altyd ‘n integrale rol in geneeskunde navorsing speel. Antimikrobiese peptiede (AMPe) kan moontlik as alternatief tot antibiotika dien, of kan in kombinasie daarmee gebruik word om die ontwikkeling van antimikrobiese- weerstandbiedenheid te verhoed. AMPe speel ‘n rol in ingebore beskerming en word deur soogdiere, plante, reptiele, voëls, visse en insekte geproduseer. AMPe van bakterieë (bakteriosiene), veral die van melksuurbakterieë (MSB), wek toenemende belangstelling as antimikrobiese middels vir die behandeling van bakteriële infeksies. Nanovesels, wat deur middel van ‘n elektrospin proses geproduseer word, het eienskappe wat hul aanloklik maak as wondbedekking, naamlik hoë suurstof deurlaatbaarheid, verskeie porie grottes, ‘n hoë oppervlakte tot volume verhouding, sowel as ‘n morfologiese struktuur wat die ekstrasellulêre matriks naboots. Die vermoë om ‘n verskeidenheid biologies aktiewe komponente in nanovesels te inkorporeer verhoog hul potensiaal as wondbedekkingsmateriaal. ‘n Unieke benadering is die inkorporasie van bakteriosiene van MSB in nanovesels om ‘n antimikrobiese wondbedekking te ontwikkel. In hierdie studie is die vermoë om bakteriosiene van MSB in nanovesels te inkorporeer, deur middel van ‘n mede-elektrospin proses, ondersoek. Plantarisien 423, geproduseer deur Lactobacillus plantarum 423, was suksesvol deur die mede-elektrospin proses in poliëtileen oksied (PEO) nanovesels geinkorporeer. Plantarisien 423 het na die elektrospin proses steeds sy antimikrobiese aktiwiteit behou en het die groei van Lactobacillus sakei DSM 20017T en Enterococcus faecium HKLHS geïnhibeer. Lewende selle van L. plantarum 423 was ook suksesvol deur die mede-elektrospin proses in PEO nanovesels geinkorporeer, alhoewel die lewensvatbaarheid van die selle effens afgeneem het. ‘n Nanovesel matriks is ontwikkel om die vrystelling van plantarisien 423 en bakteriosien ST4SA, geproduseer deur Enterococcus mundtii ST4SA, te beheer deur PEO en poli(D,L-melksuur) (PDLMS) in ‘n geskikte oplosmiddel te vermeng voor die elektrospin proses. Nanovesels is geproduseer wat die bakteriosiene oor ‘n verlengde tydperk kon vrystel. ‘n PEO:PDLMS (50:50) nanovesel matriks het sy stuktuur die beste behou tydens inkubasie by 37 °C en het aktiewe plantarisien 423 en bakteriosien ST4SA vrygestel. Nisien A was met dieselfde tegniek in PEO:PDLMS (50:50) geinkorporeer en nisien A, wat deur die nanovesels vrygestel was, het die groei van Staphylococcus aureus in vitro geïnhibeer. Die nisien A-bevattende nanovesel matriks het die aantal lewende selle van S. aureus noemenswaardig verminder in geïnfekteerde wonde en kon die genesing van wonde, wat nie geïnfekteer was, stimuleer. Sover ons kon vastel is hierdie die eerste gepubliseerde navorsing wat toon dat bakteriosiene, geinkorporeer in nanovesels, gebruik kan word om vel infeksies te beheer en wondgenesing te stimuleer.

Parabutoporin (PP) and opistoporin 1 (OP1) are cation, a-helical antimicrobial peptides isolated from the southern African scorpion species, Parabuthus schlechteri and Opistophthalmus carinatus, respectively. Along with their antimicrobial action against bacteria and fungi, these peptides show pore-forming properties in the membranes of mammalian cells. Pore-formation and ion selectivity in cardiac myocytes were investigated by measuring the whole cell leak current by means of the patch clamp technique. Pore-formation was observed as the induction of leak currents. Ion selectivity of the pores was indicated by the shift of the reversal potential (E,,,) upon substitution of intra (K' with CS' and CI- with aspartate) and extracellular (Na' with NMDG') ions. Results were compared with the effect of gramicidin A used as a positive control for monovalent cation selective pores. PP and OP I induced a fluctuating leak current and indicate non-selectivity of PP and OP1-induced pores. An osmotic protection assay to determine estimated pore size was performed on the cardiac myocytes. PP and OP1-induced pores had an estimate pore size of 1.38-1.78 nm in diameter. The effect of PP and OP1 on the membrane potential (MP) of a neuroblastoma cell line and cardiac myocytes was investigated. TMRM was used to mark the MP fluorescently and a confocal microscope used to record the data digitally. The resting membrane potential (RMP) of the neuroblastoma cells was calculated at -38.3 f 1.9 mV. PP (0.5 uM) and OP1 (0.5-1 uM) depolarized the entire cell uniformly to a MP of -1 1.9 k 3.9 mV and -9.4 k 1.9 mV, respectively. This occurred after 20-30 min of peptide exposure. In the case of the cardiac myocytes depolarization was induced to -39.7 f 8.4 mV and -32.6 f 5.2 mV by 0.5-1 uM PP and 1.5-2.5 uM OPl, respectively. / Thesis (M.Sc. (Physiology))--North-West University, Potchefstroom Campus, 2006.

EN The growing emergence of bacteria resistant to conventional antibiotics is very alarming. This has prompted an intensive search for alternative antimicrobial agents which kill bacteria with different modes of action than do traditional antibiotics and do not develop drug resistance. Among these, antimicrobial peptides (AMPs) are considered as promising compounds against resistant pathogens. These positively charged peptides permeabilize or disrupt bacterial cell envelope which leads to leakage of cytoplasmic components and cell death. The aim of my dissertation thesis was the study of the action mechanism of novel antimicrobial peptides which I have isolated from the venom of different wild bees. I identified six novel AMPs which were named panurgines (PNG), codesane (COD) and antapines (ANTPs). These peptides were isolated from the venom of three different bee species (Panurgus calcaratus, Collete daviesanus and Anthophora plumipes). I was also involved in the structural studies of lasiocepsin (Las), the antimicrobial peptide identified in the venom earlier in our laboratory. All studied peptides possess activity against various strains of bacteria and low or moderate hemolytic activity. We prepared series of PNG, COD and ANTP analogs in order to study the effect of physicochemical properties...

Os peptídeos antimicrobianos tem sido alvo de cada vez mais estudos como candidatos a antibióticos naturais devido a seu amplo espectro de ação e baixa suceptibilidade a induzir resistência microbiana. Carentes de sistema imune específico, os invertebrados contam com um eficiente sistema imune inato para se defenderem de microrganismos patogênicos, incluindo uma coleção de peptídeos antimicrobianos. Através de cromatografia, ensaios antimicrobianos, espectrometria de massas e análise em banco de dados, isolou-se frações do muco de lesmas da espécie P boraceiensis com efeito antimicrobiano e obteve-se sequencias mais prováveis de fragmentos de algumas das frações ativas encontradas, relacionando-os as proteínas e peptídeos conhecidos mais similares e sugerindo relação entre as sequencias encontradas, as proteínas e peptídeos similares, bem como possível relação com atividade antimicrobiana. Obteve-se uma gama de frações ativas contra bactérias e/ou leveduras e sequências e informações relacionadas que podem ser úteis para futuros estudos de isolamento e caracterização de fatores antimicrobianos no muco de P boraceiensis. / Antimicrobial peptides are becoming increasingly more important as natural antibiotics candidates since they show a broad spectrum of action and low suceptibility to induce microbial resistance. Lacking specific imunne system, invertebrates count with an efficient innate imunne system to deffend theirselves from pathogenic microrganisms, including a collection of antimicrobial peptides. Through chromatografy, antimicrobial tests, mass spectometry and database analysis, we isolated fractions from the mucucs of P boraceiensis slugs showing antimicrobial effect. We obtained most propable sequences from several active fractions, relating them with most similar known peptides and proteins. There were sugesteg some possible connections between the found sequences and related peptides and proteins as well as possible antimicrobial activity relations. We found several active fractions against bacteria and/or yeast as well as sequences and related information that may be usefull in further isolation and caracterization studies of antimicrobial factors in P boraceiensis mucus.

Interações de peptídeos antimicrobianos com modelos de membranas biológicas têm sido extensivamente estudadas para entender as funções destes peptídeos e para elucidar seus mecanismos de ação. Muitos esforços têm sido realizados para aumentar a potência e a especificidade desses peptídeos com o propósito de serem mais seletivos aos organismos patogênicos do que às células dos hospedeiros, como também para um melhor entendimento desta classe de processo biológico. Os mecanismos geralmente propostos para a atividade antimicrobiana envolvem a permeabilização das membranas celulares pela formação de poros ou por outras mudanças nas membranas. Um ponto fundamental no entendimento da atividade é que a composição de lipídeos das membranas dos patógenos e dos hospedeiros é diferente, observação que é entendida como a chave principal da seletividade dos peptídeos antimicrobianos. O objetivo do presente trabalho é contribuir para o entendimento da ação de peptídeos antimicrobianos pelo estudo, por simulação molecular, do peptídeo antimicrobiano indolicidina e de alguns de seus mutantes. A indolicidina é um peptídeo constituído por 13 resíduos de aminoácidos que foi isolada dos neutrófilos de bovinos cuja função é ingerir e matar bactérias. Apesar de numerosos estudos experimentais, não se sabe ainda como a indolicidina atua. Este conhecimento é importante tanto no entendimento dos processos de defesa dos organismos multicelulares como no desenvolvimento de novos antibióticos. Estas questões foram abordadas através do estudo do comportamento da indolicidina e de alguns dos seus mutantes em solução aquosa e em interação com modelos de membranas celulares. / Interactions of the antimicrobial peptides with biological membrane models have been broadly studied to understand the function and the action mechanism of this class of peptides. Many efforts have been realized to increase the potency and the specificity of these peptides with the purpose of obtain more selective pathogen antimicrobials with decrease of the toxic effects and for a better explanation of the biological process concerned in the peptide action. The action mechanism approached to antimicrobial peptides concern the cellular membrane permeation by pore formation or other type of membrane disruption. A fundamental point in the knowledge of the activity is the distinct lipid composition of pathogen and host cells that is conceived as the principal key point in the selectivity of the antimicrobial peptides. The aim of the present work is to contribute for the knowledge of the action of the antimicrobial peptide indolicidin and some of its mutants by molecular dynamics simulation. The indolicidin is a short 13 amino acid residues antimicrobial peptide that was isolated from bovine neutrofils that have the function of ingest and kill pathogens. The action mechanism of the indolicidin is not yet known despite of numerous experimental studies realized with this peptide. The interaction of the indolicidin with the membrane models is important both for the knowledge of the defense machinery of the live organisms and for the development of new antimicrobials. These questions were approached by the study of the indolicidin and some of its mutants in solution and in interaction with cell membrane models.

Os invertebrados impedem o estabelecimento de uma infecção através de uma resposta imune eficiente. Esta resposta envolve reações celulares e humorais. Existem poucos trabalhos sobre a imunidade das aranhas. O principal objetivo deste estudo foi verificar o papel dos hemócitos e dos peptídeos antimicrobianos na resposta imune da aranha Acanthoscurria gomesiana. Inicialmente, a localização relativa da gomesina e da acanthoscurrina foi determinada, mostrando que 58% dos hemócitos armazenam os dois peptídeos antimicrobianos. Além disso, foi verificado que a gomesina é direcionada aos grânulos dos hemócitos da forma de pró-peptídeo. Observou-se ainda, que após um desafio, os hemócitos migram para o sítio de infecção, onde deve secretar componentes da cascata de coagulação e peptídeos antimicrobianos. Além disso, os resultados mostraram que a fagocitose não é o principal mecanismo ativado após uma infecção. Assim sendo, as principais respostas imunes da aranha são através da coagulação e secreção dos peptídeos antimicrobianos. / Invertebrates avoid the infection establishment through an efficient immune response. This response consists in cellular and humoral reactions. Few data are available concerning the spider\'s immunity. The main aim of this study was to determine the role of hemocytes and antimicrobial peptides on the immunity of the spider Acanthoscurria gomesiana. Initially, the localization of gomesin and acanthoscurrin was determined, showing that 58% of hemocytes store both antimicrobial peptides. Moreover, our results show that gomesin is addressed to the hemocyte granules as a pro-peptide. We also demonstrate, by in vivo and in vitro experiments, that hemocytes migrate to the site of microbial infection. Once at the site of infection, hemocytes might secrete components of coagulation cascade and antimicrobial peptides. Besides, our results suggest that phagocytosis is not the major defense mechanism activated after microbial challenge. Therefore the main reactions involved in the spider defense might be through the coagulation and antimicrobial peptides secretion.

A saliva é uma mistura de água, eletrólitos, proteínas e enzimas. A secreção diária normal é de 800-1500ml em adultos. A chamada saliva total, o fluído que realmente está presente na cavidade bucal, é produzida por diferentes glândulas salivares e contém ainda fluído crevicular e elementos transudados do plasma, e derivados da rede capilar da mucosa bucal. É importante entender o papel da saliva na proteção dos tecidos bucais, principalmente porque a co-infecção pode ser um fator importante na ativação e supressão do sistema imune, com papel importante no desenvolvimento e severidade das doenças bucais. Além disso, a cavidade bucal tem um vasto número de microrganismos e antígenos presentes, o que faz com que seja considerada em permanente estado de inflamação, em muitos casos, subclínica. Nosso estudo se propõe a observar a variação da composição salivar frente a presença de alterações locais - gengivites e periodontites. O estudo compara as citocinas salivares TNF-α, IL-1β e IL-6, e os fatores de defesa, beta defensinas 1 e 2, catelicidina e mucina 2, em três diferentes grupos de pacientes: Grupo 1 (controle) - 40 Pacientes, total ou parcialmente dentados, sem inflamação/infecção bucal; Grupo 2 - 40 Pacientes total ou parcialmente dentados, com sinais clínicos de gengivite; e Grupo 3 - 40 Pacientes total ou parcialmente dentados, com sinais clínicos de periodontite. A presença das citocinas e fatores salivares foram avaliadas por testes ELISA. Foram significativas as alterações encontradas entre os grupos para os diferentes fatores: TNF-α, e IL-6, beta defensinas 1 e 2, catelicidina e mucina 2. Apenas IL-1β não teve resultados significantes. Assim, indica-se que os componentes salivares possuem importante papel salivar frente à alterações locais. / Saliva is a mixture of water, electrolytes, proteins and enzymes. The daily secretion ranges between 800-1500mL in adults. The called whole saliva is composed by the production of different salivary glands, gingival crevicular fluid, and contain elements transudate from plasma derived from the capillary bed beneath the oral mucosa. It is important to consider the evident and important role of saliva in defense and protection of oral tissues. The effects of co-infecting pathogens have been postulated as an important factor in the activation and/or suppression of immune system, important in many situations, including the severity and rate of disease progression. The oral cavity is continually confronted with a vast number of pathogens and antigens, so, in some way, may be considered an inflammatory environment, although the level of inflammation may be sub-clinical. This study proposed to observe how the presence of local inflammation - gingivitis or periodontitis, may influence the presence of salivary cytokines or defense factors in saliva. The study compared saliva molecular components in three different groups of patients: Group 1 (as control group) - 40 Patients, total or partially dentate, without oral infectious; Group 2 - 40 Patients total or partially dentate, with clinical signs of gingivitis; Group 3 - 40 patients, total or partially dentate, with clinical signs of periodontitis. It checked the presence of TNF-α, IL-1β and IL-6 cytokines, and defense factors, 1 and 2 beta defensins, cathelicidin and mucin 2. ELISA kits determined the levels of these proteins. Found alterations were significant between groups to TNF-α, IL-6, 1 and 2 beta defensins, cathelicidin and mucin 2. Only IL-1β had not significant results. Therefore, it indicated that salivary components have important hole related to local situations.