Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2010.

Orientador: Marcos Antonio Machado / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Civil, Arquitetura e Urbanismo / Made available in DSpace on 2018-08-26T04:12:54Z (GMT). No. of bitstreams: 1 InuiKishi_RosangelaNaomi_D.pdf: 6372359 bytes, checksum: 51a90b9672af7cb590a1b270d3bc5d18 (MD5) Previous issue date: 2014 / Resumo: A cultura do citros tem sido afetada por diversos patógenos como Xylella fastidiosa (Xf), Xanthomonas citri subsp. citri (Xcc) e Candidatus Liberibacter asiaticus (CaLas). Para o controle desses são necessárias grandes quantidades de defensivos agrícolas. Neste contexto, são importantes novas estratégias de manejo sustentável, como o uso de peptídeos antimicrobianos (PAM), que podem controlar o crescimento desses patógenos. Assim, foi realizada uma prospecção de sequências codificantes de peptídeos no banco de sequências expressas de citros (CitEST), e além disso, peptídeos descritos na literatura com atividade antimicrobiana foram utilizados. Para verificar a ação dos PAMs sobre os micro-organismos deste estudo, testes in vitro contendo os PAMs e patógenos foram realizados. Entre os PAMs avaliados in vitro CJ0714, EC0604, SC0902 e o PAM de citros PE1105 apresentaram atividade bactericida sobre Xcc e S. meliloti (bactéria utilizada por ser filogenéticamente próxima a CaLas) e bacterioestática para Rhizobium radiobacter. Todos os peptídeos avaliados in vitro para Xcc, tiveram suas atividades avaliadas em folha destacada, sob condições controladas, e em plantas acondicionadas em casa de vegetação. Os resultados em folha destacada corroboram com os dados obtidos in vitro. Em casa de vegetação os resultados com os PAMs CJ0714, AR0610 e PE1105 não foram semelhantes com os obtidos em testes in vitro e em folhas destacadas. Apesar disso, houve redução de sintomas em comparação com o controle positivo (somente a bactéria). Isso pode ter ocorrido devido a fatores e compostos presentes na planta e a instabilidade dos PAMs em condições mais próximas às naturais. Os PAMs que apresentaram resultados promissores in vitro e em folha destacada ou em trabalhos prévios foram clonados no vetor do CTV (Vírus da tristeza do citros) para avaliação da expressão transiente e sua ação sobre CaLas. Apesar dos resultados satisfatórios nos experimentos anteriores, não foram verificados efeitos destes PAMs sobre a população de CaLas quando expressos através do vetor viral do CTV. No entanto, pode-se observar redução da frequência de CaLas no inseto vetor Diaphorina citri alimentado em plantas com o PAM PE1106, já que o patógeno pode ser transmitido de modo transovariano de uma geração à outra do inseto. Nenhum peptídeo avaliado foi eficiente sobre o crescimento de Xf. Para avaliar o efeito dos PAMs sobre micro-organismos endofíticos foi utilizado Methylobacterium sp., sendo observado que os PAMs apresentaram atividade sobre esta bactéria. Testes para verificar a citotoxicidade dos PAMs para humanos foram realizados. O teste de hemólise permite verificar a atividade do peptídeo sobre eritrócito humano. Os PAMs que apresentaram as menores atividades hemolíticas foram SC0902, PE1105 e PE1106. Além disso, foi verificada a proliferação de linfócitos e desgranulação de mastócitos. Os PAMs CJ0714 e EC0604 apresentaram linfoproliferação e somente os PAMs PE1105 e PE1106, obtidos de citros, não induziram desgranulação de mastócitos. O PAM SC0902 apresentou bons resultados para a atividade antimicrobiana, mas induziu a desgranulação de mastócitos. Dessa forma, este trabalho apresenta o PAM, PE1105 como potencial para o controle desses patógenos bacterianos de citros, pois este apresentou atividade antimicrobiana e nenhum efeito citotóxico sobre células humanas / Abstract: The citrus culture has been affected by various pathogens such as, Xylella fastidiosa (Xf), Xanthomonas citri subsp. citri (Xcc) and Candidatus Liberibacter asiaticus (CaLas).To control these pathogens, large quantities of pesticides are needed. In this context, are important new strategies for sustainable management, such as the use of antimicrobial peptides (PAM), which can control the growth of these pathogens. Here we have prospected at coding peptide sequences in the citrus database of expressed sequence (CitEST) and used other peptides previously tested. To verify the AMPs action on the microorganisms, in vitro tests were performed. Among the peptides evaluated in vitro, CJ0714, EC0604, SC0902 and PE1105 - citrus peptide showing bactericidal activity against Xcc and S. meliloti (phylogenetically close to CaLas) and bacteriostatic effect on Rhizobium radiobacter. All peptides evaluated in vitro for Xcc, had their activities evaluated in a detached leafs, under controlled conditions, and plants placed in the greenhouse. The results in detached leaf results corroborated with the in vitro data. In the greenhouse, results for CJ0714, AR0610 and PE1105 peptides were not similar with those obtained in vitro and in detached leaf. Nevertheless, the presence of symptoms was reduced as compared with the positive control (bacteria only). This may be due to factors and compounds present in the plant and to the instability of the peptide at conditions close to the natural ones. Peptides that showed promising results in vitro and in detached leaf or in previous works were cloned into the CTV (Citrus Tristeza Virus) vector to evaluate the transient expression and its action on CaLas. Despite the satisfactory results in previous experiments, it was not possible to observe the PAM effect on CaLas population when expressed through CTV vector. Nevertheless, reduction of the frequency of the bacteria in the insect vector was observed when Diaphorina citri fed on plants with AMP PE1106, since the pathogen can be transmitted from one generation to another by transovarial mode. None peptides evaluated was efficiently on the growth of Xf. To evaluate the effects of the AMPs on endophytic microorganisms, Methylobacterium sp. was used and it was observed that peptides exhibit activity against this bacterium. Tests to check the cytotoxicity of these AMPs for humans were performed. The hemolysis test allows checking the activity of the peptide on human erythrocyte. The AMPs that showed the lowest hemolytic activities were SC0902, PE1105 and PE1106. Furthermore, we noticed the proliferation of lymphocytes and mast cell desgranulation. Thus, this work presents PAM, PE1105 as potential for the control of these bacterial pathogens of citrus because it showed antimicrobial activity and no cytotoxic effect on human cells / Doutorado / Bioquimica / Doutora em Biologia Funcional e Molecular

Orientador: Daniela Leal Zandim-Barcelos / Resumo: Peptídeos antimicrobianos (PAMs) são importantes componentes da resposta do hospedeiro contra patógenos invasores. Além da ação antimicrobiana direta, podem também participar na modulação do sistema imune. No entanto, o papel dos PAMs na etiopatogênese da doença periodontal e os fatores de risco que podem influenciar sua expressão na cavidade bucal ainda não se encontram totalmente elucidados. Dessa forma, os objetivos do presente estudo foram: (1) avaliar a influência do tabagismo nos níveis de LL-37 e HNP 1-3 no fluido crevicular gengival (FCG) de pacientes com periodontite e investigar a associação entre os níveis destes PAMs e de mediadores inflamatórios nos sítios com ausência e presença de doença periodontal; (2) investigar o impacto do tabagismo nos níveis de hBD1 e 2 em pacientes com periodontite e avaliar os efeitos da nicotina e cotinina na expressão destes PAMs por queratinócitos; (3) avaliar a atividade metabólica e enzimática durante a recolonização de biofilmes multi-espécies periodontopatogênicos após tratamento com peptídeos sintéticos derivados da catelicidina humana (LL31 e D-LL31). A quantificação dos PAMs e mediadores inflamatórios no FCG foi realizada por meio de ensaio ELISA sanduiche e Multiplex, repectivamente, enquanto a RT-qPCR em tempo real detectou a expressão gênica dos PAMs por queratinócitos. A produção de ácido láctico foi mensurada para determinar a atividade metabólica nos biofilmes pós-tratamento, enquanto a atividade enzimática foi analisad... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Antimicrobial peptides (AMPs) are important components of the host response against invading pathogens. In addition to the direct antimicrobial activity, they can also participate in the immune system modulation. However, the role of AMPs in the etiopathogenesis of periodontal disease and the risk factors that may influence their expression in the oral cavity are not yet fully understood. Thus, the purposes of the present study were: (1) to evaluate the influence of smoking on the levels of LL-37 and HNP 1-3 in the gingival crevicular fluid (GCF) of patients with periodontitis and to investigate the association between the levels of these AMPs and inflammatory mediators at sites with absence and presence of periodontal disease; (2) to investigate the impact of smoking on hBD1 and 2 levels in patients with periodontitis and to evaluate the effects of nicotine and cotinine on the expression of these AMPs by keratinocytes; (3) evaluate the metabolic and enzymatic activity during the recolonization of multi-species periodontopathogenic biofilms after treatment with synthetic peptides derived from human cathelicidin (LL31 and D-LL31). The quantification of AMPs and inflammatory mediators in the GCF was performed by sandwich ELISAs and Multiplex assay, respectively, while RT-qPCR in real time detected the gene expression of the AMPs by keratinocytes. The production of lactic acid was measured to determine the metabolic activity in post-treatment biofilms, while the enzymatic activi... (Complete abstract click electronic access below) / Doutor

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Hepcidin is a well-known iron-regulatory hormone that plays a key role in maintaining peripheral iron homeostasis. The presence and wide-spread distribution of hepcidin in the brain suggests that this peptide may also be an important player in brain iron homeostasis. In this study, we tested the hypothesis that hepcidin exerts an important role in the regulation of brain iron content, which might benefit iron-associated NDs such as PD. We also examined the hypothesis that hepcidin could control iron transport processes via regulating iron transport proteins in the brain cells, thus maintaining brain iron homeostasis. / In conclusion, the results of the present study implied that hepcidin plays an important role in maintaining brain iron homeostasis. Hepcidin is beneficial for PD and this effect is related to its iron-regulatory effect via inhibiting iron accumulation in the substantia nigra. Hepcidin effectively controls iron uptake and release through regulating iron transport proteins expressions in the brain, which would contribute to brain iron homeostasis. Therefore, manipulation of hepcidin level in the brain has a potential to be developed into a novel preventive approach for the iron-associated NDs such as PD. / In the second part, we investigated the effect of hepcidin on the processes of iron uptake and release in the cultured brain cells including neurons, astrocytes and brain vascular endothelial cells (BVECs). The expressions of iron uptake proteins such as transferrin receptor 1 (TfR1) and divalent metal transporter 1 (DMT1) as well as the iron exporter ferroportin 1 (Fpn1) were also observed. We found that hepcidin reduced both iron uptake and release via decreasing iron transport proteins expressions in these brain cells, which would contribute to its iron regulatory effect. Finally, we further explored the mechanisms underlying the regulatory effect of hepcidin on the iron transporters in the last part, and found that the action of hepcidin in reducing TfR1 expression is a direct and cAMP-PKA (Cyclic Adenosine 3', 5'-monophosphate/ Protein Kinase-A) pathway-dependent event. / Iron is a transition trace metal essential for mammalian cellular and tissue viability. It also plays important roles in the central nervous system (CNS), including embryonic brain development, myelination, and neurotransmitters synthesis. However, abnormal iron accumulation has been demonstrated in a number of neurodegenerative diseases (NDs) such as Parkinson's (PD), Alzheimer's (AD) and Huntington's diseases (HD). Currently very little is known about the mechanisms involved in brain iron homeostasis and therefore it is not known why and how iron is abnormally increased in the brain. However, given the essential role that excess iron plays in the pathological processes in the NDs, to suppress the accumulated iron is expected to be an effective strategy to prevent and treat these NDs. / To investigate whether hepcidin could benefit iron-associated NDs including PD and whether this beneficial role is related to its iron-regulatory function in the brain, in the first part of study, we investigated the effects of hepcidin on the 6-hydroxydopamine (6-OHDA) in vitro and in vivo PD models. We found that in primary cultured mesencephalic (MES) neurons, hepcidin overexpression via adenovirus-hepcidin (Ad-hepcidin) infection prevented 6-OHDA-induced increase in cellular iron content and protected the MES neurons. In the 6-OHDA model of PD in vivo, overexpression of hepcidin in the substantia nigra via Ad-hepcidin intranigral injection significantly prevented iron accumulation and dopaminergic neurons loss in the pars compacta of substantia nigra (SNc). These effects were accompanied by a marked improvement in motor performance of the PD animals. These findings indicate that hepcidin could benefit iron-associated NDs such as PD and via its iron-regulatory role in the brain. / Du, Fang. / Adviser: Ya Ke. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 152-173). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Lightly cross-linked polyelectrolyte microgels are materials with interesting properties for a range of applications. For instance, the volume of these particles can be drastically changed in response to pH, ionic strength, temperature, or the concentration of specific ions and metabolites. In addition, microgel particles can bind substantial amounts of oppositely charged substances, such as proteins and peptides, and release them upon changes in the external environment. Consequently, microgels have potential in catalysis, photonics, biomaterials, and not at least, as protective and stimuli-sensitive carriers for protein and peptide drugs. In this thesis, the interaction between anionic microgels and cationic peptides was investigated by monitoring microgel deswelling and reswelling in response to peptide binding and release using micromanipulator-assisted light microscopy. In addition, peptide distribution in microgels was analyzed with confocal laser scanning microscopy and peptide uptake determined with solution depletion measurements. The aim of the thesis was to clarify how parameters such as peptide size, charge density, pH, ionic strength and hydrophobicity influences the peptide binding to, distribution in and release from, polyelectrolyte microgels. Results obtained in this thesis show that electrostatic attraction is a prerequisite for interaction to occur although non-electrostatic contributions are responsible the finer details of the interactions. The size and charge density of the interacting peptides play a major role, as large and highly charged peptides are restricted to enter and interact with the microgel core, thus displaying a surface-confined distribution. The peptide-microgel interaction strength is highly reflected in the probability of peptides to be detached from the gel network. For instance, reducing the electrostatic interactions by adding salt induces significant peptide release of sufficiently small and moderately charged peptides, whereas longer and more highly charged peptides is retained in the microgel network due to the strong interaction, insufficient salt screening, and gel network pore size restriction. Decreasing the charge density of microgel network and/or peptides increases the probability for peptide detachment tremendously. To summarize, interactions occurring in oppositely charged microgel-peptide systems can be tuned by varying parameters such as charge density and peptide size and through this, the peptide uptake, distribution and release can be controlled to alter the performance of microgels in peptide drug delivery.

O microbioma intestinal tem sido associado à sepse, causada por infecção, apresentando respostas imunológicas exacerbadas. O peptídeo antimicrobiano CRAMP atua na imunidade inata com característica ambígua, ora pró-inflamatória ora anti-inflamatória. Nesse estudo, a sepse foi induzida por modelo de ligadura e punção cecal (CLP) e, através de RT-qPCR, o DNA de amostras fecais de camundongos selvagens e deficientes em CRAMP foi analisado. A expressão de Alfa-defensina 5 e Beta-defensina 1 foi investigada em RT-qPCR. Técnicas de imunofluorescência, Elisa e Milliplex, foram utilizadas na quantificação de citocinas - IL-1Beta, IL-6, IL-10, MCP-1 e TNF-Alfa -, e Alfa-defensina 7 e Beta-defensina 1. Os resultados mostraram a intensificação da resposta imune, diante da sepse, com alterações pró-inflamatórias de IL-1Beta, IL-6, MCP-1 e TNF-Alfa e anti-inflamatória de IL-10. A Beta-defensina 1 teve expressão aumentada, enquanto a produção foi mantida ou reduzida nos tecidos, após CLP. A liberação de Alfa-defensina 7 foi ampliada no pulmão de animais selvagens durante a sepse, enquanto a expressão de Alfa-defensina 5 foi reduzida no íleo e cólon. A relação entre o microbioma intestinal e a sepse evidenciou-se com o crescimento da espécie Escherichia coli, enquanto o CRAMP apresentou associação com a espécie Bacteroides vulgatus. Novos estudos permitiram mais conhecimento sobre a interação entre sepse e microbioma intestinal, potencializando o uso de biomarcadores e nova terapêutica na recuperação intestinal, tal como transplante microbiológico fecal / The intestinal microbiome has been associated with sepsis, caused by infection, presenting exacerbated immune responses. The antimicrobial peptide CRAMP acts on innate immunity with ambiguous features, pro-inflammatory and anti-inflammatory. In this study, sepsis was induced by cecal ligation and puncture (CLP) model and, through RT-qPCR, the DNA of fecal samples from wild type and CRAMP-deficient mice was analyzed. Expression of Alpha-defensin 5 and Beta-defensin 1 was investigated in RT-qPCR. Immunofluorescence techniques, Elisa and Milliplex, were used to quantify cytokines - IL-1Beta, IL-6, IL-10, MCP-1 and TNF-Alpha -, and Alpha-defensin 7 and Beta-defensin 1. The results showed the enhancement of the immune response to sepsis with proinflammatory alterations of IL-1Beta, IL-6, MCP-1 and TNF-Alpha and anti-inflammatory IL-10. CRAMP and Beta-defensin 1 peptides had increased expression, while production was maintained or reduced in tissues after CLP. The release of Alpha-defensin 7 was amplified in the lungs of wild type animals during sepsis, while expression of Alpha-defensin 5 was reduced in the ileum and colon. The relationship between the intestinal microbiome and sepsis was evidenced by the growth of Escherichia coli, while CRAMP was associated with Bacteroides vulgatus. New studies have allowed more knowledge about the interaction between sepsis and intestinal microbiome, potentializing the use of biomarkers and new therapy in intestinal recovery, such as fecal microbiological transplantation

Mannheimia haemolytica (M. haemolytica)-induced bovine respiratory disease causes millions of dollars in economic losses to Canadian cattle industry. Contemporary management strategies built around the use of antimicrobials are proving to be increasingly unavailing and lead to drug residues in meat which may contribute to the development of multi drug resistant bacteria. Many M. haemolytica vaccines are effective in stimulating antibody responses but studies of vaccina-tion in young calves and the cattle exposed to M. haemolytica (high-risk cattle) have shown poor vaccine efficacy. Antimicrobial peptides (AMPs) may help in the management of respiratory disease caused by M. haemolytica while minimizing the risk of drug residues in animal-derived food products. AMPs are positively charged molecules that can kill bacteria primarily through the electrostatic interactions with the anionic bacterial lipid bilayer. Since the primary target of AMPs is the bac-terial surface charge, which is evolutionarily conserved, the development of resistance towards AMPs seems less likely. These peptides hold potential to replace or reduce the use of antibiotics. Human β-Defensin 3 (HBD3) and Microcin J25 (MccJ25) are cationic peptides that have shown good activity against many Gram-negative bacteria. Five peptides, namely native HBD3, three synthetic HBD3 analogues (28 amino acid, 20AA, and 10AA), and MccJ25 were selected for microbicidal activity against M. haemolytica. Three C-terminal analogues of HBD3 with all cysteines replaced with valines were manually synthesized using solid phase peptide synthesis (SPPS). In all the three analogue, replacement of cysteine with valine rendered them linear and increased their antibacterial activity. Minimum Bactericidal concentration (MBC) assays were performed with the final inoculum size of 1-5x105 cells/ml, with the exception of the 10AA analogue which was incubated with 104 cells/ml final inoculum size. The antimicrobial assay showed that M. haemolytica was intermediately sensitive to HBD3, 28AA and 20AA analogue with an MBC of 50 µg/ml. MccJ25 had limited effect with an MBC greater than 100 µg/ml. The MBC value of 6.3 µg/ml achieved with the 10AA analogue is likely a result of lower final inoculum size. AMPs have several immunomodulatory functions, and these peptides can act as chemoattractant, induce cytokine release that in turn leads to chemotaxis of monocytes and neutrophils. Since neutrophils play an important role in the pathogenesis of BRD, the chemotactic effect of HBD3, 20AA and 28AA peptides on bovine neutrophils was studied using Boyden chamber. Peripheral blood neutrophils isolated from normal healthy cattle showed chemotaxis towards HBD3 and 20AA peptides (P<0.05) but not towards 28AA analogue. Co-incubation of neutrophils with any of the peptides did not affect their chemotaxis towards N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP). Based on these data, it can be concluded that HBD3 and its analogues showed antimicrobial ef-fects against M. haemolytica but MccJ25 had limited microbicidal activity against M. haemolytica. While HBD3 and 20AA analogue were chemotactic for bovine peripheral blood neutrophils, none of the peptides inhibited fMLP-induced migration of neutrophils. These peptides hold potential for further in vivo testing to develop them for use to manage M. haemolytica-induced respiratory disease in cattle.

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