Potential Application of Phage Therapy Against Pseudomonas aeruginosa Biofilm Infection in Cystic Fibrosis Patients

Majority of the microbial activity in humans is in the form of biofilms i.e. an Exopolysaccharide-enclosed bacterial mass. Unlike planktonic cells and the cells on the surface of the biofilm, the biofilm-embedded cells are more resistant to the effects of the antibiotics and the host cellular defense mechanisms. A combination of biofilm growth and inherent resistance prevents effective antibiotics treatment of Pseudomonas aeruginosa infections including those in patients with cystic fibrosis. This has lead to an increasing interest in alternative modalities of treatment. Thus, phages that multiply in situ, only in the presence of susceptible hosts can be used as natural, self-limiting, and deeply penetrating antibacterial agents. The objective of this study is to identify effective phages against a collection of P. aeruginosa isolates (PCOR strains) including the prototype PAOl and the isogenic constitutively alginate-producing PD0300 strains.These PCOR strains were tested against six phages (P105, P134, P140, P168, P175B and P182). Analysis shows 69 % of the PCOR isolates are sensitive and the rest are resistant to all six phages. These phages were then tested for their ability to inhibit biofilm formation using a modified biofilm assay. The analysis demonstrated that the sensitive strains showed increased resistance but none of the sensitive strains from the initial screening were resistant. Using the minimum biofilm eradication concentration (MBEC) assay for biofilm formation, the biofilm eradication ability of the phages was tested. The data showed that a higher volume of phage was required to eradicate preformed biofilms than the volume required to prevent colonization of planktonic cells. This data supports the idea of phage therapy more as a prophylactic treatment.

Molecular Mechanisms Involved in Regulating the Mucoid-to-Nonmucoid Conversion of Pseudomonas aeruginosa Associated with Cystic Fibrosis Patients

Pseudomonas aeruginosa is an opportunistic pathogen that has received attention because of its close association with cystic fibrosis (CF). Chronic pulmonary infection with the mucoid P. aeruginosa is the leading cause of mortality in CF patients. This bacterium has the ability to sense and adapt to the harsh environment in the CF lung by converting from a nonmucoid to a mucoid state. The mucoid phenotype is caused by overproduction of a polysaccharide called alginate. Alginate production is regulated by the algT/U operon containing five genes, algT/U-mucA-mucB-mucC-mucD. Alginate overproduction in CF isolates has been partially attributed to a loss-of-function mutation in mucA that results in the overexpression of algT. This mucoid phenotype is unstable, reverting to the nonmucoid form when the isolates are cultured outside of the CF lung. This study was undertaken to determine the mechanisms involved in the conversion from the mucoid to the nonmucoid form. Thirty-six spontaneous nonmucoid variants of a known mucoid isolate with a mucA mutation were analyzed. Ten of these isolates were complemented in trans by plasmids containing the algT operon and the algT gene. Chromosomal DNA was extracted and the mucA and algT genes were amplified by the polymerase chain reaction. Sequence analysis of the genes showed that these mutants retained the original mucA mutation but acquired secondary mutations in the algT gene.

Comparative transcriptome analyses of Pseudomonas aeruginosa

One of the hallmarks of bacterial survival is their ability to adapt rapidly to changing environmental conditions. Niche adaptation is a response to the signals received that are relayed, often to regulators that modulate gene expression. In the post-genomic era, DNA microarrays are used to study the dynamics of gene expression on a global scale. Numerous studies have used Pseudomonas aeruginosa--a Gram-negative environmental and opportunistic human pathogenic bacterium--as the model organism in whole-genome transcriptome analysis. This paper reviews the transcriptome studies that have led to immense advances in our understanding of the biology of this intractable human pathogen. Comparative analysis of 23 P. aeruginosa transcriptome studies has led to the identification of a unique set of genes that are signal specific and a core set that is differentially regulated. The 303 genes in the core set are involved in bacterial homeostasis, making them attractive therapeutic targets.

Caracterização estrutural e atividades farmacológicas do alginato obtido da alga Dictyopteris delicatula (J.V. Lamouroux., 1809) e seu derivado sulfatado

Marine algae are rich sources of various structural compounds which recently has been increasingly studied as a new source of bioactive substances. The alginate, as come as fucans, are considered the main acidic polysaccharides found in brown seaweed. This molecule consists a linear natural polysaccharide, non-sulfated, and presents monosaccharides: acid β-D-mannuronic (M) and α-L-guluronic acid (G); in a vast amount compositions and threads. Alginate has been widely applied in food and pharmaceutical industries because of its ability to retain water, forming films and gels as well as thickening, stabilizing and form emulsions. In this work we aimed to extract, structurally characterize, compare and analyze the possible pharmacological activities of native alginate molecule obtained from brown seaweed Dyctiopteris delicatula (DYN), and its chemically sulfated derivative (DYS). The alginate structure and composition molecule can be proven through chemical dosing, that showed low protein contamination and high sugar level, existence and separation of M and G blocks in the descending paper chromatography, infrared spectroscopy and nuclear magnetic resonance. Molecule sulfation was proven with sulphate dosage, resulting in 28.56% sulphate in molecule; electrophoresis, verify metachromasia with toluidine blue; and infrared spectroscopy, that showed a characteristic band at 1221cm-1 corresponding a sulfate group vibration. For the pharmacological activities the tests was: antioxidant activity, changes in cell function (MTT test) and anticoagulant test. In the antioxidant activity we observed that DYN showed better results in the kidnapping of hydroxyl radicals and ferric chelation compared to DYS, this had the best result in the total antioxidant capacity. Both showed similar activity in reducing power and the kidnapping radicals DPPH. In MTT test DYN and DYS had not proliferative and cytotoxic activity in fibroblast cells (3T3) and showed antiproliferative and cytotoxic activity in cancer cell lines HeLa and B16 melanoma. In anticoagulant assay DYN showed good activity in the intrinsic pathway of blood coagulation, and a small activity in the extrinsic pathway, in the other hand DYS showed only a very small activity in the extrinsic pathway, but cannot come to be regarded as an anticoagulant agent. From these results it can be concluded that the alginate was extracted and sulfated, revealing a potential compound to be used in the pharmaceutical industry as an anticoagulant agent, antioxidant and antitumor and the sulfation has not been conclusively important to performance in the tested pharmacological activities

Metal transfer mapping for FCAW process by using near-infrared filming

In the Flux Cored Arc Welding (FCAW) process, the transfer of filler metal (metal transfer modes) to the base material to accomplish the weld bead determines the weld quality and therefore studies of such phenomena is demanded. Thus, in this work, the metal transfer through the FCAW process is investigated by filming the phenomena with the assist of near infrared visualization. During the literature survey, it was found that this technic has not been used so far for analyzing the FCAW process. It must be pointed out that the radiation emitted from the weld arc, fumes and particles (spattering) in this process represent a barrier for these studies based in the process visualization. The monitoring of metal transfer for FCAW process was carried out within the operational envelope of voltage and wire feed speed with the electrode E71T-1 (1.2 mm diameter) and Ar+25%CO2 as a shielding gas. A local developed near infrared filming with frame rate of 300 Hz was employed for metal transfer visualization in order to contribute to a better understanding of this process and evaluating characteristics of metal transfer, unlike previous studies, which used shadowgraph technique. It can clearly be seen how the droplet is created and transferred in this process and also identify the different modes of metal transfer by changing the parameters of voltage and wire feed speed in metal transfer maps. The final result of this study is the metal transfer mode maps, which establish suitable conditions and provide the basis for developing arc control strategies for the FCAW process.

Transplantation d'îlots de Langerhans microencapsulés : biocompatibilité, survie et fonction

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Transplantation d'îlots de Langerhans microencapsulés : biocompatibilité, survie et fonction

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Tissue-specific bioscaffolds for adipose-derived stem/stromal cell expansion and delivery

Decellularized adipose tissue (DAT) is a promising biomaterial for soft tissue regeneration, and it provides a highly conducive microenvironment for human adipose-derived stem/stromal cell (ASC) attachment, proliferation, and adipogenesis. This thesis focused on developing techniques to fabricate 3-D bioscaffolds from enzymatically-digested DAT as platforms for ASC culture and delivery in adipose tissue engineering and large-scale ASC expansion. Initial work investigated chemically crosslinked microcarriers fabricated from pepsin-digested DAT as injectable adipo-inductive substrates for ASCs. DAT microcarriers highly supported ASC adipogenesis compared to gelatin microcarriers in a CELLSPIN system, as confirmed by glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity, lipid accumulation, and endpoint RT-PCR. ASCs cultured on DAT microcarriers in proliferation medium also had elevated PPARγ, C/EBPα, and LPL expression which suggested adipo-inductive properties. In vivo testing of the DAT microcarriers exhibited stable volume retention and enhanced cellular infiltration, tissue remodeling, and angiogenesis. Building from this work, non-chemically crosslinked porous foams and bead foams were fabricated from α-amylase-digested DAT for soft tissue regeneration. Foams were stable and strongly supported ASC adipogenesis based on GPDH activity and endpoint RT-PCR. PPARγ, C/EBPα, and LPL expression in ASCs cultured on the foams in proliferation media indicated adipo-inductive properties. Foams with Young’s moduli similar to human fat also influenced ASC adipogenesis by enhanced GPDH activity. In vivo adipogenesis accompanied by a potent angiogenic response and rapid resorption showed their potential use in wound healing applications. Finally, non-chemically crosslinked porous microcarriers synthesized from α-amylase-digested DAT were investigated for ASC expansion. DAT microcarriers remained stable in culture and supported significantly higher ASC proliferation compared to Cultispher-S microcarriers in a CELLSPIN system. ASC immunophenotype was preserved for all expanded groups, with reduced adhesion marker expression under dynamic conditions. DAT microcarrier expansion upregulated ASC expression of early adipogenic (PPARγ, LPL) and chondrogenic (COMP) markers without inducing a mature phenotype. DAT microcarrier expanded ASCs also showed similar levels of adipogenesis and osteogenesis compared to Cultispher-S despite a significantly higher population fold-change, and had the highest level of chondrogenesis among all groups. This study demonstrates the promising use of DAT microcarriers as a clinically relevant strategy for ASC expansion while maintaining multilineage differentiation capacity.

An Ex Vivo Model for Studying Hepatic Schistosomiasis and the Effect of Released Protein from Dying Eggs

BACKGROUND: We report the use of an ex vivo precision cut liver slice (PCLS) mouse model for studying hepatic schistosomiasis. In this system, liver tissue is unfixed, unfrozen, and alive for maintenance in culture and subsequent molecular analysis.

METHODS AND FINDINGS: Using thick naive mouse liver tissue and sterile culture conditions, the addition of soluble egg antigen (SEA) derived from Schistosoma japonicum eggs, followed 4, 24 and 48 hrs time points. Tissue was collected for transcriptional analysis and supernatants collected to quantitate liver enzymes, cytokines and chemokines. No significant hepatotoxicity was demonstrated by supernatant liver enzymes due to the presence of SEA. A proinflammatory response was observed both at the transcriptional level and at the protein level by cytokine and chemokine bead assay. Key genes observed elevated transcription in response to the addition of SEA included: IL1-α and IL1-β, IL6, all associated with inflammation. The recruitment of antigen presenting cells was reflected in increases in transcription of CD40, CCL4 and CSF1. Indications of tissue remodeling were seen in elevated gene expression of various Matrix MetalloProteinases (MMP3, 9, 10, 13) and delayed increases in TIMP1. Collagen deposition was significantly reduced in the presence of SEA as shown in COL1A1 expression by qPCR after 24 hrs culture. Cytokine and chemokine analysis of the culture supernatants confirmed the elevation of proteins including IL6, CCL3, CCL4 and CXCL5.

CONCLUSIONS: This ex vivo model system for the synchronised delivery of parasite antigen to liver tissue provides an insight into the early phase of hepatic schistosomiasis, corresponding with the release of soluble proteins from dying schistosome eggs.

Isolation and structural characterization of fucoidans from the brown algae Fucus vesiculosus

agricultural, pharmaceutical, cosmetic or bioenergy applications. They contain bioactive compounds, namely, polysaccharides Fucoidan. These polysaccharides are mainly constituted by fucose residues and sulfate esters, and have been reported to possess a broad variety of bioactivities, such as anticoagulant, anti-thrombotic, anti-inflammatory, anti-tumor, antiviral and antioxidant. In this work, the fucoidans from brown seaweed Fucus vesiculosus from “Ria de Aveiro” were isolated and characterized in order to add value to this natural resource of the region. The polysaccharides from the algae were extracted with hot water and fractioned by ethanol precipitation and calcium chloride salts. They were further purified by using anion-exchange chromatography, allowing to separate the neutral polysaccharides (laminaranas) from those negatively charged (sulfated fucoidans and alginate). The purified polysaccharides showed high content of fucose (41 mol%) and sulfates (50 mol%), having also galactose residues (6 mol%), which confirm the presence of only sulfated fucoidans. Glycosidic linkages analysis show the presence of high amounts of terminal fucose (25%) and (1→3,4)-Fuc (26%), allowing to infer that the fucoidans were highly branched. These fucoidans are composed also by (1→2)-Fuc (14%) and (1→3)-Fuc linkages (10-16%). In this work it was also tested an alternative extraction technology, the microwave hydrodiffusion and gravity system, where it was possible to extract sugars, although in low yields. However, this methodology allowed to extract polysaccharides, constituted mainly by fucose and uronic acids, as well as mannitol, without the need to add any solvent, obtaining at the end the dry alga. The current work allowed to characterize the structure of the fucoidans isolated from “Ria de Aveiro” F. vesiculosus. The presence of high content of sulfate residues and the high branch degree of the purified fucoidans allow to infer that these polysaccharides could have potential to be studied for biomedical applications, according to their biological activities.

Biomimetic Scaffold with Aligned Microporosity Designed for Dentin Regeneration

Tooth loss is a common result of a variety of oral diseases due to physiological causes, trauma, genetic disorders, and aging and can lead to physical and mental suffering that markedly lowers the individual’s quality of life. Tooth is a complex organ that is composed of mineralized tissues and soft connective tissues. Dentin is the most voluminous tissue of the tooth and its formation (dentinogenesis) is a highly regulated process displaying several similarities with osteogenesis. In this study, gelatin, thermally denatured collagen, was used as a promising low-cost material to develop scaffolds for hard tissue engineering. We synthetized dentin-like scaffolds using gelatin biomineralized with magnesium-doped hydroxyapatite and blended it with alginate. With a controlled freeze-drying process and alginate cross-linking, it is possible to obtain scaffolds with microscopic aligned channels suitable for tissue engineering. 3D cell culture with mesenchymal stem cells showed the promising properties of the new scaffolds for tooth regeneration. In detail, the chemical–physical features of the scaffolds, mimicking those of natural tissue, facilitate the cell adhesion, and the porosity is suitable for long-term cell colonization and fine cell–material interactions.

Investigations on growth ecology and reproduction of Sargassum sp. and Gracilaria on intertidal region in Bandar Lenge

Investigations on growth and quantity of phycocolloids of Sargassum sp. and Gracilaria corticata was done on field and laboratorial works over one year from January 2003 to May 2004. Sargassum thalli began growth from January. The highest biomass value recorded 1611.04 gm^-2 was obtained in November. The receptacles appeared on November and released eggs. The senescence of Sargassum thalli was in December and the new thallus began to grow from January, The highest relative growth rate (6.74 percent) was in February. The relative growth rate showed significant correlation (p<0,05) with temperature. The highest value of alginate was in November (10.02 percent). Alginat content showed significant correlations (p<0.05) with Sargassum biomass. There was no significant effect of environmental factors on alginate content. The highest biomass of Graciiaria was in Match (49.88 gm^-2). Maximum relative growth rate of Gracilara (2 percent) was in December. Relative growth rate of Gracilaria (2.8 percent) was in December. Relative growth rate of Gracilaria showed significant correlations (p<0.05) with temperature. There was significant effect of ammonium (p<0.05) on growth factors of Gracilaria, maximum agar content was in August {10.005 percent). The yield of agar showed significant correlation (p<0.05) with Gracilaria biomass and ammonium in field and laboratory.

Desempenho de sementes de soja revestidas com biopolímeros

The use of biopolymers that help to fix pesticides efficiently and degrade easily without harming the environment, and still improve the physiological performance of field soybean seed may bring contributions to the soybean yield. This study aimed to evaluate the effect of cassava starch polymers (AM), sodium alginate (ALG) and polyvinyl alcohol (PVOH), in the concentrations 2, 4 and 6 g / 100 ml of solution, in the physiological attributes of seeds soy, seed speed soaking and performance of soybean seeds after three months of storage. The soybean variety used was the NK 7059 RR. The experimental design used for the three studies was a factorial with 48 experimental units: 3 polymers (AM, ALG and PVOH), 4 different concentrations (0%, 2%, 4% and 6%), with four replications, in a completely randomized design. It was observed the level of significance of the factors and their interactions, applying the test F. The polymers were evaluated by the Tukey test at 5% probability, and the concentrations were evaluated by polynomial regression. The witness obtained better results for most variables studied. Among the polymers, the best coating was observed PVOH because it was the less viscous polymer and visually not served as a substrate for microorganisms. However, also, satisfactory results were obtained for the AM and ALG polymers at a concentration of 2%. There was not interference of the polymers studied with regard to reduction of imbibition rate of soybean seeds. The hydrophilicity of polymers, mainly the AM and ALG accelerated soaking seeds harming germination at concentrations 4% and 6%. In general, the higher the concentration of polymers tended to worse results.

Produção enzimática de biodiesel a partir de resíduos agro-industriais usando a lipase imobilizada de origem microbiana

Trabalho Final de Mestrado para obtenção do grau de Mestre em Engenharia Química e Biológica

Immunologic Storm Simulating Systemic Lupus Erythematosus Following Parvovirus B19 Infection

Background: The appearance of symptoms compatible with systemic autoimmune diseases has been described in relation to several viral infections like HIV, cytomegalovirus and especially PVB19, depending on the evolution of the immunological condition of the host and their age. We present a young immunocompetent male patient, with clinical manifestations simulating systemic lupus erythematosus (SLE) with important activation of cytokines. Methods: For quantification of the different cytokines in plasma, a commercially available multiplex bead immunoassay, based on the Luminex platform (Cat # HSCYTO-60SK-08, Milliplex® MAP High Sensitivity, Millipore), was used according to the manufacturer’s instructions. All samples were run in duplicate and the data (mean fluorescence intensity) were analyzed using a Luminex reader. The mean concentration was calculated using a standard curve. Results: The clinical evolution was favourable without the need for any specific treatment, showing complete recovery after two months. Whilst the symptoms and viral charge were disappearing, the anti-DNA continued to increase and we demonstrate important activation of IL-10, IL-6 and TNFα cytokines as a result of a hyperstimulating response by an immunocompetent hyperfunctional system, which persists after clinical improvement. We should emphasize the behaviour of two cytokines: IL-12p70 and IL-2, which showed opposite tendencies. Conclusions: Viral infections, especially PVB19, can produce or simulate several autoimmune diseases as a hyperstimulation response from an immunocompetent hyperfunctional system. Consequently, a persistent increase of autoantobodies and important activation of cytokines, even after clinical improvement and seroconversion, can be demonstrated.