Background: Alginate overproduction in P. aeruginosa, also referred to as mucoidy, is a poor prognostic marker for patients with cystic fibrosis (CF). We previously reported the construction of a unique mucoid strain which overexpresses a small envelope protein MucE leading to activation of the protease AlgW. AlgW then degrades the anti-sigma factor MucA thus releasing the alternative sigma factor AlgU/T(σ22)to initiate transcription of the alginate biosynthetic operon. Results: In the current study, we mapped the mucE transcriptional start site, and determined that P mucEactivity was dependent on AlgU. Additionally, the presence of triclosan and sodium dodecyl sulfate was shown to cause an increase in P mucEactivity. It was observed that mucE-mediated mucoidy in CF isolates was dependent on both the size of MucA and the genotype of algU. We also performed shotgun proteomic analysis with cell lysates from the strains PAO1, VE2 (PAO1 with constitutive expression of mucE) and VE2ΔalgU (VE2 with in-frame deletion of algU). As a result, we identified nine algU-dependent and two algU-independent proteins that were affected by overexpression of MucE. Conclusions: Our data indicates there is a positive feedback regulation between MucE and AlgU. Furthermore, it seems likely that MucE may be part of the signal transduction system that senses certain types of cell wall stress to P. aeruginosa.

Gels and hydrogels have attracted a great attention for potential applications in tissue engineering, drug delivery, actuators, and soft robots. There has been a significant progress to engineer hydrogels from both synthetic and natural precursors to be as tough as a solid and as stretchable as a rubbery material while maintaining high water/solvent content. Despite considerable advances in rationally designing hydrogels, our understanding of their complex nonlinear mechanical deformation behavior is incomplete. This is partially due to the difficulty in conducting mechanical characterization on slippery, soft and swollen gels. Thus, it is required to develop new experimental techniques in order to better characterize them. Further, analyzing the experimental observations and link it with the molecular networks is an important factor. With this perspective, in this dissertation, nonlinear mechanical properties of different gel like materials have been investigated. We chose different gels with varied molecular structure, from molecular gel to self-assembled copolymer gels with flexible chains, to semiflexible polysaccharide based polymers. By developing suitable experimental protocols, strain-stiffening behavior of these materials, similar to that observed in biological materials, have been captured. Chain flexibility is a dominant factor in mechanical behavior of gels. For example, gels with flexible chains dilate orthogonal to an external shear load, whereas gels with semilexible chains contract similar to biological gel-like materials. In order to investigate the failure mechanism in our gels, cavitation rheology technique was also applied. We found that cavitation phenomenon in gels is related to the molecular architecture of the gels. The present work provides a better understanding of the deformation behavior of soft gels when subjected to a large load.

Calcification is linked to a high prevalence of cardiovascular events and mortality due to arterial stiffness. Stiffening of the arteries in the case of medial calcification is due to hydroxyapatite mineral deposited in the artery thus leading to the loss of elastin. A possibility of removing this rogue mineral along the vessel walls could be the use of osteoclasts. Osteoclasts, a type of osteocyte, have the unique ability to absorb bone in the bone turnover process. It is proposed that in the future, osteoclasts be delivered to the site of mineralization through oxidized alginate-fibrin microbeads. Alginate hydrogels have proven great in drug delivery and could be a revolutionary cell delivery device to provide care for multitudes of people suffering from adjacent cardiovascular health problems such as arterial stiffness.

No description available.

No description available.

The neuronal differentiation of neural stem cells (NSCs) has received much attention due to its potential for the treatment of neurodegenerative diseases (i.e., Parkinson’s and Alzheimer’s diseases). In this regard, discovering compounds that direct differentiation of NSCs is highly required to facilitate therapeutic applications. In this study, we examined various bioactive compounds (SA1, SA2, LiCl, compound B, and DHED) to induce the neuronal differentiation of human neural stem cells (hNSCs). The study was conducted on the cells grown in three dimensional (3D) hydrogel or two dimensional (2D) environment since 3D hydrogel mimics the extracellular matrix and provides physiologically more relevant environment than 2D cell culture system. Three-dimensional (3D) hydrogel systems in this study involve polysaccharides such as alginate and hyaluronic acid. Neuronal differentiation of hNSCs was monitored in genetic level and protein level by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunocytochemistry (ICC), respectively. This study will show the effect of bioactive compounds on hNSCs differentiation in 2D and 3D culture systems. / Bioengineering

The type IV pilus is a motility organelle found in a range of bacteria, including the opportunistic pathogen Pseudomonas aeruginosa. These flexible fibres mediate twitching motility, biofilm maturation, surface adhesion, and virulence. The principle structural protein of the pilus is the major pilin, PilA, while a set of low abundance “minor pilins” are proposed to constitute the pilus tip. The minor pilins, FimU and PilVWXE, along with the non-pilin protein PilY1, prime assembly of surface-exposed pili. The fimU-pilVWXY1E operon is positively regulated by the FimS-AlgR two-component system. Independent of pilus assembly, PilY1 is an adhesin and mechanosensor that, along with PilW and PilX, triggers virulence upon surface attachment. Here, we aimed to uncover the mechanism for PilWXY1-mediated virulence. We hypothesized that loss of PilWXY1 would relieve feedback inhibition on FimS-AlgR, resulting in increased transcription of the minor pilin operon and dysregulation of virulence factors in the AlgR regulon. Caenorhabditis elegans slow killing assays revealed that pilW, pilX, and pilY1 mutants had reduced virulence relative to a pilA mutant, implying a role in virulence independent of pilus assembly. FimS-AlgR were required for the increased promoter activity of the minor pilin operon upon loss of pilV, pilW, pilX, or pilY1. Overexpression or hyperactivation of AlgR by point mutation led to reduced virulence, and the virulence defects of pilW, pilX, and pilY1 mutants were dependent on FimS-AlgR expression. We propose that PilWXY1 inhibit their own expression at the level of FimS-AlgR, such that loss of pilW, pilX, or pilY1 leads to FimS-mediated activation of AlgR, and reduced expression of acute-phase virulence factors. Accumulation of mutations in the minor pilin operon may represent an evolutionary strategy for P. aeruginosa populations in chronic lung infections, as loss of PilWXY1 would upregulate the expression of AlgR-dependent virulence factors – such as alginate – characteristic of such infections. / Thesis / Master of Science (MSc) / Pseudomonas aeruginosa is a bacterium that causes dangerous infections, including lung infections in cystic fibrosis patients. The bacteria use many strategies to infect their hosts, one of which involves a grappling hook-like fibre called the type IV pilus. There are many components involved in assembly and function of the pilus, including five proteins called “minor pilins” and a larger protein called PilY1 that may help the pilus detect surface attachment. We used a roundworm infection model to show that loss of PilY1 and specific minor pilins leads to delayed killing, while loss of other pilus proteins has no effect on worm survival. This effect was due to increased activation of a regulatory system called FimS-AlgR that inhibits expression of other factors used by this bacterium to infect its hosts. By studying how P. aeruginosa causes infection, we can design better strategies to disarm it and reduce the severity of infections.

Yes / Cells encapsulation is a micro-technology widely applied in cell and tissue research, tissue transplantation, and regenerative medicine. In this paper, we proposed a growth of microtissue model for the human keratinocytes (HaCaT) cell line and an oral squamous cell carcinoma (OSCC) cell line (ORL-48) based on a simple aerosol microencapsulation technique. At an extrusion rate of 20 μL/min and air flow rate of 0.3 L/min programmed in the aerosol system, HaCaT and ORL-48 cells in alginate microcapsules were encapsulated in microcapsules with a diameter ranging from 200 to 300 μm. Both cell lines were successfully grown into microtissues in the microcapsules of alginate within 16 days of culture. The microtissues were characterized by using a live/dead cell viability assay, field emission-scanning electron microscopy (FE-SEM), fluorescence staining, and cell re-plating experiments. The microtissues of both cell types were viable after being extracted from the alginate membrane using alginate lyase. However, the microtissues of HaCaT and ORL-48 demonstrated differences in both nucleus size and morphology. The microtissues with re-associated cells in spheroids are potentially useful as a cell model for pharmacological studies. / Malaysia Ministry of Education (Fundamental Research Grant Scheme, FRGS Vot. 1482 and IGSP Vot. 679).

Oxidative stress-inducing chemical contamination in the environment is a significant concern for public health. The depletion of antioxidants by these chemicals results in oxidative stress which may cause detrimental effects in many cell types. For example, multiple stress responses may be activated in bacteria and several disorders including neurodegenerative disorders may occur in mammalian organisms. Oxidative chemicals also have negative effects on engineered water systems as an oxidative stress response in bacteria has been implicated to cause process failure in wastewater treatment facilities. Therefore, it is essential to monitor oxidative chemical contamination in water environments to provide early warning of potential negative effects. Whole-cell biosensors that indicate bacterial stress responses to oxidative toxic agents can be powerful tools in environmental monitoring. An oxidative stress response found in many Gram-negative heterotrophic bacteria called the glutathione-gated potassium efflux (GGKE) mechanism is a good biological indicator to be used in a biosensor designed to detect the presence of oxidative chemicals in water. The authors of this study propose the development of a GGKE biosensor using an environmental strain of Pseudomonas aeruginosa. The abundance of the global antioxidant glutathione, the gating compound in GGKE, in various cell types suggests that there may be connections between the responses of the different cell types to oxidative stress. In this study, specific oxidative stress responses in two distantly related cell types were studied: the GGKE mechanism in Gram-negative heterotrophic bacteria, and mitochondrial dysfunction in rat brain cells. Furthermore, the use of an octanol-based emulsification method for the immobilization of P. aeruginosa in calcium alginate microbeads was evaluated for long-term mechanical stability, viability, and GGKE response of the immobilized cells. The immobilization of cells is an important factor in the design of a whole-cell biosensor, and must yield viable and active cells over time. This study showed that the dose-dependent responses of GGKE in Pseudomonas aeruginosa cells and of mitochondrial dysfunction in a mixed culture of rat brain cells to a model oxidative electrophilic chemical, N-ethylmaleimide, correspond well to each other. We also showed that both responses are accompanied by the depletion of intracellular glutathione, which precedes the GGKE response in P. aeruginosa as well as mitochondrial damage in rat brain cells. Thus, this study suggests that bacterial responses to oxidative stress involving glutathione, such as GGKE, could potentially be used as an early warning to predict the presence of bioavailable oxidative chemicals that can induce oxidative stress in eukaryotic systems. Although further research is needed, this suggests that bacterial stress response biosensors may be used to predict oxidative stress responses in mammalian brain cells. The octanol-based emulsification method produced P. aeruginosa encapsulated alginate microbeads with an average diameter of 200 μm. The microbeads were mechanically stable in solutions containing up to 20 mg/L K+ for 15 days. LIVE/DEAD® and specific oxygen uptake rate (SOUR) analyses showed that the microbead-immobilized cells recovered their membrane integrity within 5 days but not their net respiration potential. The microbead immobilized cells had no net GGKE potential in response to 50 mg/L N-ethylmaleimide after 14 days whereas water-based alginate bead (2mm) immobilized cells did, albeit at a reduced level to planktonic cells. Confirmation experiments revealed that octanol impeded cellular activities of the immobilized cells. Overall, this study showed that the octanol-based emulsification method is not suitable for the immobilization of P. aeruginosa for use in the GGKE biosensor and other microscale immobilization methods should be evaluated. / Master of Science

Francisella tularensis is a Gram-negative, zoonotic bacterium that causes tularemia in animals and humans. The two subspecies tularensis (Type A) and holarctica (Type B) are considered Tier I Select Agents due to the bioweapon potential of these subspecies. Type A strains, considered the more virulent of the subspecies, are highly infective producing respiratory tularemia with inhalation of as few as 10 cells. Due to classification as a Select Agent, a vast amount of F. tularensis research has occurred in the last two decades after the September 11th terrorism attack and the use of Bacillus anthracis spores in a biological attack on the United States Postal Services in 2001. This research has uncovered many of the various virulence factors of F. tularensis including an intracellular nature, the unique lipopolysaccharide produced, and a genetic pathogenicity island. This dissertation aims to further characterize outer surface antigens of F. tularensis subspecies in regards to virulence, biofilm formation, and role in vaccine development. In addition, this dissertation will also investigate the use of a novel vaccine delivery vehicle, alginate microencapsulation, in increasing the efficacy of these mutant strains. F. novicida is a subspecies of F. tularensis and usually classified as being non-encapsulated. However, F. novicida has a similar capsule glycosylation locus as F. tularensis and could produce a similar capsule-like complex that has previously been described for the F. tularensis LVS strain. I was able to isolate and characterize this CLC of F. novicida, which contained a heterogenous mixture of proteins and possible glycosylated proteins. A mutant with a multi-gene interruption within the glycosylation locus (F. novicidaΔ1212-1218) produced significantly less carbohydrate than the parent strain, was attenuated in the mouse model, and was partially protective when used to immunize mice against a virulent challenge. Biofilms of F. novicida were also characterized in regards to biofilm formation in various growth media and biofilm formation of strains lacking the O-antigen of the lipopolysaccharide (LPS). In general, F. novicida produced the greatest amount of biofilm in a brain heart infusion (BHI) broth, compared to other media. Loss of the O-antigen led to increased biofilm production when grown in BHI and decreased or similar biofilm production as the wildtype when grown in other media. This highlights the need to carefully select the growth medium when assessing biofilm formation of Francisella strains in the future. A final study of this dissertation characterized the use of alginate microspheres as a vaccine vehicle for an attenuated F. tularensis type A O-antigen deficient strain. O-antigen deficient strains of F. tularensis are highly attenuated in vivo and would be a safe choice for a vaccine candidate. However, these strains produce less than ideal protection against virulent challenge when used to immunize mice, possibly due to a lack of persistence in the host. In an attempt to increase persistence, we encapsulated an O-antigen deficient strain within sodium alginate microspheres and used those microspheres to immunize mice. The immunized mice produced a higher level of antibody response than mice immunized with a non-encapsulated version. However, this immunization only partially protected mice from a virulent challenge and did not match the protection afforded by the former Live Vaccine Strain (LVS). In part the deficiency in protection appears to be due to a lack of a robust cellular immune response in mice immunized with the alginate microspheres. In summary, this dissertation focuses on the various extracellular polysaccharides of F. tularensis: the glycosylation of CLC, the O-antigen, and the biofilm. Each polysaccharide plays a role in the virulence and pathogenesis of F. tularensis. Glycosylation of the CLC and the O-antigen are important virulence factors in mammalian disease, and mutants lacking either (not type A strains) are attenuated in the mouse model. Both also appear to play a role in the formation of the F. tularensis biofilm in a manner dependent on the environment or culture medium used. Each of these extracellular polysaccharides contribute to the lifecycle of Francisella. / Ph.D. / Francisella tularensis is a highly infectious bacterial pathogen that can cause disease in a wide array of animals and in humans. F. tularensis is also considered a potential weapon of bioterrorism and the development of an effective vaccine is a critical area of research. One strategy of developing a tularemia vaccine includes mutating a strain of F. tularensis to reduce expression of extracellular components that include polysaccharides. Strains that cannot express these components are usually unable to produce clinical signs in the host and may provide protection against fully virulent F. tularensis strains. The work presented in this dissertation will focus on characterizing the polysaccharide extracellular components of F. tularensis and developing a novel vaccine vehicle to increase protection from strains that do not cause disease.