Peptidomimetics are synthetic foldamers that expected more resistant to proteolytic degradation and enormous chemodiversity when compared with peptides. To date, the functional peptidomimetics such as β-peptides, peptoids, oligoureas, etc have been developed in many science fields. In order to explore the unnatural foldameric architectures, it’s necessary to discover the novel frameworks and molecular scaffolds. γ-AApeptides were reported to be a new class of peptidomimetics that showed its potential applications in drug discovery and chemical biology. However, a wide function and property of γ-AApeptides need to be further explored. To expand the potential application of γ-AApeptides in biochemistry, I have been focusing on the development of bioactive peptidomimetics, such as exploring the antibacterial activity of helical 1:1 α-sulfono-γ-AA heterogeneous peptides, developing the helical peptidomimetic as the inhibitor of the protein Ras_Raf interaction, identifying the protein/peptide ligands by the novel one-bead-two compound macrocyclic γ-AApeptide screening library, and elucidating the de novo dragon-boat-shaped synthetic foldamers.

Peptidomimetics are synthetic oligomers that resemble the activities of peptides. Their advantages over peptides include high stability towards proteolysis and enormous chemical diversity. Over the past two decades, there have been extensive efforts to develop peptide mimics, such as beta-peptides, peptoids, D-peptides, etc. The research on peptidomimetics have led to many important applications in both medicinal and material science. In order to explore new functions, the discovery of peptidomimetics with novel frameworks is essential. We reported the synthesis and evaluation of a new class of peptidomimetics, termed as gamma-AApeptides. Previous studies of gamma-AApeptides have revealed that gamma-AApeptides are highly resistant to proteolysis, and are highly amendable to chemical diversification. However, new biological activities and folding properties of gamma-AApeptides still need to be explored. In order to expand the potential of gamma-AApeptides in chemical biology and medicinal chemistry, I have been focusing on the development of new methods to synthesize linear and cyclic gamma-AApeptides, development of one-bead-one-compound (OBOC) gamma-AApeptide libraries for the discovery of inhibitors against beta-amyloid aggregation, exploring new helical foldamers for the rational design of protein-protein interaction (PPI) inhibitors, and studying cyclic gamma-AApeptides for antimicrobial development.

Η παρούσα εργασία μελετά την ανάπτυξη ενός ευέλικτου συστήματος αντιμικροβιακής προστασίας για εφαρμογή σε είδη ρουχισμού και υφάσματα οικιακής χρήσης. Πιο συγκεκριμένα αναπτύχθηκαν διασυνδεδεμένα πολυμερικά νανοσφαιρίδια πολυστυρολίου-διβινυλοβενζολίου στα οποία ενσωματώθηκε Triclosan, μία ευρέος φάσματος εμπορική αντιμικροβιακή ουσία. Σημειώνεται ότι στο πλαίσιο ανάσχεσης των ενδονοσοκομειακών λοιμώξεων η ανάπτυξη αντιμικροβιακών νοσοκομειακών στολών, σεντονιών και άλλων σχετικών κλωστοϋφαντουργικών προϊόντων αποτελούν τις τελευταίες δεκαετίες αντικείμενο έντονου επιστημονικού ενδιαφέροντος. Το μέγεθος των νανοσωματιδίων βρέθηκε μετά από εξέταση με ηλεκτρονική μικροσκοπία σάρωσης (SEM) και δυναμική σκέδαση φωτός (DLS) να κυμαίνεται μεταξύ 35-350 nm ανάλογα την σύσταση. H θερμική συμπεριφορά τους μελετήθηκε μέσω διαφορικής θερμιδομετρίας σάρωσης (DSC) και διαπιστώθηκε σημείο τήξεως στους ~425 οC. Χρησιμοποιώντας την φασματοσκοπία UVVis προσδιορίστηκε ο πραγματικός εγκλωβισμός του αντιμικροβιακού στο σύστημα κατά μέσο όρο σε ποσοστό ~72% του ονομαστικού και παρακολουθήθηκε ο ρυθμός αποδέσμευσης του σε διαλύματα αιθανόλης-νερού. Επιπλέον, τα σφαιρίδια που όπως διαπιστώθηκε παρουσιάζουν χαρακτηριστικά ελεγχόμενης αποδέσμευσης ενσωματώθηκαν σε μήτρες πολυπροπυλενίου οι οποίες υπό την μορφή φιλμ εφελκύστηκαν μονοαξονικά. Τέλος κάνοντας χρήση της δονητικής φασματοσκοπίας Raman εκτιμήθηκε ο μοριακός προσανατολισμός που επιβλήθηκε στα εφελκυσμένα φιλμ και συσχετίσθηκε με την παρατηρούμενη μείωση που επιτεύχθηκε στην κινητική αποδέσμευσης της εγκλωβισμένης δραστικής ουσίας. Το πρώτο κεφάλαιο της παρούσας εργασίας ασχολείται με το πρόβλημα της μικροβιακής επιμόλυνσης υφασμάτων, τις διάφορες λύσεις που έχουν προταθεί κατά καιρούς και τέλος αναλύει τον στόχο της παρούσας εργασίας. Στο δεύτερο κεφάλαιο επεξηγείται η έννοια της ελεγχόμενης αποδέσμευσης και περιγράφονται οι διάφορες κατηγορίες συστημάτων ελεγχόμενης χορήγησης μαζί με χαρακτηριστικά παραδείγματα. Τα νανοσωματίδια, η σύστασή, οι μοναδικές ιδιότητες, οι εφαρμογές και οι διάφοροι τρόποι σύνθεσης και χαρακτηρισμού τους συζητούνται στο τρίτο κεφάλαιο. Το τέταρτο κεφάλαιο αναφέρει πληροφορίες για τα υλικά και επεξηγεί τις τεχνικές που χρησιμοποιήθηκαν στην σύνθεση, την επεξεργασία και τον χαρακτηρισμό των νανοσωματιδίων και των μιγμάτων τους. Τέλος στο πέμπτο κεφάλαιο παρουσιάζονται τα αποτελέσματα των πειραμάτων που πραγματοποιήθηκαν και ακολουθεί ο σχολιασμός τους. / The present thesis studies the development of a versatile system of antimicrobial protection for use in clothing and household products. In particular Triclosan incorporated crosslinked polystyrenedinylbenzene nanobeads were developed; triclosan is a widely used antimicrobial agent. It is noted that the health hazards arising during nosocomial treatment due to infections caused by microbial pathogens and the means to protect oneself against such threats have become the subject of many research activities during the last few decades. The size of the nanoparticles after examination with scanning electron microcopy (SEM) and dynamic light scattering (DLS) was found to vary between 35-350 nm depending on the system formulation. Their thermal behavior was studied with differential scanning calorimetry (DSC) and their melting point was measured at ~425 oC. Using UV-Vis spectroscopy the real encapsulation efficiency of the antimicrobial in the system was determined at ~72% and its release kinetics were studied in a water-ethanol solution. The nanobeads possess controlled release properties; they were furthermore incorporated into polypropylene matrixes which were uniaxially drawn in film form. Finally utilizing polarized Raman spectra, the draw induced molecular orientation of the films was correlated to the relevant variation of the related antimicrobial release kinetics. The first chapter of the present thesis reviews the textile microbial infections and the various solutions that have been proposed showing up the specific research goal targeted. In the second chapter the meaning of controlled release is explained and the basic system categories involved are presented along with characteristic examples. The nanoparticles, their composition, special attributes, applications, synthesis and characterization techniques are the subject of the third chapter. The fourth chapter reports information about the materials and the methods used in the synthesis, postprocessing and characterization of the nanoparticles and their blends. Finally the last chapter presents the experimental results and relevant comments.

The Min protein system regulates cell division in the bacterium Escherichia coli. The protein MinD undergoes a pole-to-pole oscillation, antagonizing formation of the division septum at the cell poles, thereby confining the septum formation to the mid-cell. The MinD oscillation period is 40 s at room temperature in healthy cells, but has been shown to be sensitive to stress on the cell. By fluorescently labeling MinD with green fluorescent protein (GFP), we are able to measure the MinD oscillation period as an in situ metric of cell viability using high resolution total internal reflection fluorescence (TIRF) microscopy. We have made several improvements to the method by which we measure and analyse the MinD oscillation period. A microscopy flow cell was designed and constructed and it provides temperature control and stability to a precision of 0.05 °C in addition to allowing controlled addition of bacterial cells and reagents of interest to the imaging region of the flow cell. This flow cell enabled us to make a precise measurement of the temperature dependence of the MinD oscillation period, for which we observed an Arrhenius dependence with an activation energy of 11.8 kcal/mol. We developed a centroid-tracking method, performed in a custom MATLAB program, to extract the values of the MinD oscillation periods from our time series of TIRF microscopy images. We measured the effect on the MinD oscillation period of exposure to the cationic antimicrobial peptide polymyxin B (PMB) and the related compound polymyxin B nonapeptide (PMBN), which does not have antimicrobial activity. Exposure to PMB resulted in a 60% increase in the average MinD oscillation period tau, whereas exposure to PMBN resulted in an 20% decrease in tau. After exposure to PMB and PMBN, we measured the Arrhenius temperature dependence of the MinD temperature dependence and calculated the associated activation energy Ea. We found that exposure to PMB resulted in a 40% increase in Ea, whereas exposure to PMBN did not significantly change the value of Ea. These results indicate that careful measurements of the MinD oscillation can yield information that can be helpful in evaluating the mechanism of action of antimicrobial compounds. / Natural Sciences and Engineering Research Council

Made available in DSpace on 2014-06-11T19:27:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-05-28Bitstream added on 2014-06-13T19:35:18Z : No. of bitstreams: 1 rangel_pm_me_jabo.pdf: 202160 bytes, checksum: 7b5c8c75d2c9df10071428271f91da55 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A um longo tempo a mastite tem sido reconhecida como a doença que provoca as maiores perdas econômicas nos rebanhos leiteiros. De fevereiro a novembro de 2004, 670 amostras de leite mastítico bovino, provenientes de dois estados brasileiros foram coletadas, das quais foram isoladas 231 cepas de Escherichia coli. Estas cepas foram analisadas para a detecção dos genes de produção de Shiga toxina (stx 1 e stx 2) e do gene da intimina (eae). Vinte cepas (8,6%) foram detectadas através de PCR contendo os genes da Shiga toxina (8 stx 1, 12 stx 2 e nenhuma delas ambos os genes). Duas cepas (0,8%) de E. coli eram eae positivo não produtoras de Shiga toxina. As cepas de E. coli foram também examinadas para detectar a resistência a 12 agentes antimicrobianos. As resistências mais comuns foram para tetraciclina (92,2%), estreptomicina (90,4%), ácido nalidíxico (88,3%), amicacina (86,5%) e cefalotina (84,8%). A resistência a múltiplas drogas foi encontrada em 152 cepas (65,8%). . Entre os sorogrupos determinados, O111, O26, O158 e O125 foram os mais comuns, todos sorogrupos EPEC clássicos. / Mastitis has been recognized for some time as the most costly disease in dairy herds. From February to November 2004, 670 samples of bovine mastitic milk were collected from two Brazilian states, from which 231 Escherichia coli strains were isolated. These strains were screened for the presence of Shiga toxin-producing (stx 1 and stx 2) and intimin (eae) genes. Twenty (8.6%) strains were detected by PCR to harbor the Shiga toxin genes (8 the stx 1 gene, 12 the stx 2 gene and none both of them) Two (0.8%) of the E. coli strains studied were eae positive non Shiga toxin-producing. The E coli strains were also examined for resistance to 12 antimicrobial agents. The most commonly observed resistance was to tetracycline (92.2%), streptomycin (90.4%), nalidixic acid (88.3%), amikacin (86.5%) and cephalothin (84.8%). Multidrug resistance was found among 152 isolates (65.8%). Among the serogroups determined O111, O26, O158 and O125 were the most commonly, all of them classic EPEC serogroups.

Made available in DSpace on 2016-06-02T19:02:42Z (GMT). No. of bitstreams: 1 5562.pdf: 2010923 bytes, checksum: 99b0ace4e740c99eb2fc0708b19a70fd (MD5) Previous issue date: 2013-05-10 / Financiadora de Estudos e Projetos / The staphylococci belong a diverse group of bacteria that cause diseases ranging from minor skin infections to death-threatening bacteraemia. The two major opportunistic pathogens of this genus, Staphylococcus epidermidis and S. aureus are the most frequent causes of nosocomial infections and infections associated with the use of medical devices. Staphylococci, is the leading cause of infections associated with biofilm formation. Biofilmrelated infections are challenging to treat with conventional antimicrobial agents, limiting the efficacy of antibiotic therapy and becoming a crucial problem for treatment of chronic infections. Therefore, the main aim of this thesis was to study the susceptibility of staphylococcal biofilm cells against antimicrobial agents. For that, it was evaluated in vitro activities of N-acetylcysteine (NAC), rifampicin, linezolid, daptomycin and vancomycin alone and in combination on biofilm cells of Staphylococcus epidermidis and S. aureus. The activities of antimicrobial agents were evaluated in concentrations CIM, 10xCIM and peak serum. The biofilms susceptibility to agents studied was assessed through, colony-forming units (CFU/ml), staining with crystal violet (CV) that is the measure total biofilm biomass and cellular activity using XTT reduction assay. The results of viable cells (expressed as log10 CFU/ml) to N-acetylcysteine alone, in the concentration 10xCIM on both the biofilms of staphylococcal evaluated showed a greater effect compared with other antimicrobial agents evaluated, with reductions of approximately 4-5 log10. The combination NAC (10xCIM) - vancomycin (independent of concentration evaluated) showed a greater reduction (p<0.05) on viable cells of S. epidermidis and S. aureus, compared with other combinations evaluated. This combination presented a reduction about of 5-6 log10 CFU/ml. The results of CV showed loss the total biofilm biomass and were observed decrease in the metabolic activity measured by the XTT, these results are in very good agreement with those obtained in terms of cell viability. In conclusion, the results obtained in the studies of this thesis constitutes a promising therapeutic strategy in the treatment of infections associated with biofilm formation by S. epidermidis and S. aureus. In addition, the use of antimicrobial agents in combinations may be an alternative for monotherapy, thus avoiding the development of resistance. / Os estafilococos pertencem a um grupo diversificado de bactérias que causam doenças que vão desde infecções de pele, a risco de morte como bacteriemia. Os dois principais patógenos oportunistas deste gênero, Staphylococcus epidermidis e S. aureus são as causas mais frequentes de infecções nosocomiais e infecções associadas ao uso de dispositivos médicos. Estafilococos é a principal causa de infecções associadas com a formação de biofilme. As infecções relacionadas à formação de biofilme são difíceis de tratar com agentes antimicrobianos convencionais, limitando a eficácia da terapia com antibióticos e tornando um problema crucial para o tratamento de infecções crônicas. Portanto, o objetivo principal desta tese foi estudar a susceptibilidade das células de estafilococos em biofilme contra agentes antimicrobianos. Para isso, foram avaliadas as atividades in vitro de N-acetilcisteína (NAC), rifampicina, linezolida, daptomicina e vancomicina isoladamente e em combinação contra as células em biofilme de Staphylococcus epidermidis e S. aureus. As atividades dos agentes antimicrobianos foram avaliadas nas concentrações CIM, 10xCIM e a concentração máxima no soro. A susceptibilidade dos biofilmes para os agentes estudados foi avaliada através de unidades formadoras de colônia (UFC/ml), a coloração com cristal violeta (CV), que é a medida da biomassa total do biofilme e, a atividade celular usando ensaio de redução de XTT. Os resultados das células viáveis (expressos em log10 UFC/ml) para N-acetilcisteína sozinho, na concentração 10xCIM para ambos os biofilmes de estafilococos avaliados mostraram um maior efeito em comparação com outros agentes antimicrobianos avaliados, com redução de cerca de 4-5 log10. A combinação de NAC (10xCIM) - vancomicina (independente da concentração avaliada) mostrou uma maior redução (p<0,05) em células viáveis de S. epidermidis e S. aureus, em comparação com as outras combinações avaliadas. Esta combinação apresentou uma redução de cerca de 5-6 log10 CFU/ml. Os resultados de CV mostraram perda da biomassa total do biofilme e, foram observados diminuição da atividade metabólica, medida pelo ensaio de XTT, este resultados estão em boa concordância com os resultados obtidos em termos de viabilidade celular. Em conclusão, os resultados obtidos nos estudos desta tese constituem uma estratégia terapêutica promissora para o tratamento de infecções associadas a formação de biofilme por S. epidermidis e S. aureus. Além disso, a utilização de agentes antimicrobianos em combinação pode ser uma alternativa para a monoterapia, evitando assim o desenvolvimento de resistência.

Made available in DSpace on 2014-10-09T12:27:45Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:05:04Z (GMT). No. of bitstreams: 0 / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Made available in DSpace on 2014-10-09T12:32:58Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:06:11Z (GMT). No. of bitstreams: 0 / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Made available in DSpace on 2014-10-09T12:54:11Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:08:59Z (GMT). No. of bitstreams: 1 12698.pdf: 772894 bytes, checksum: be69d4e1e623ce5d0dc821671a2724dc (MD5) / Dissertacao (Mestrado Profissionalizante em Lasers em Odontologia) / IPEN/D-MPLO / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP; Faculdade de Odontologia, Universidade de Sao Paulo , Sao Paulo

Made available in DSpace on 2014-10-09T12:53:35Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:09:32Z (GMT). No. of bitstreams: 0 / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energéticas e Nucleares - IPEN-CNEN/SP