Bacteria have been confirmed as the main etiological factor for root canal infection as well as for root canal treatment failure. Thus the success of endodontic treatment depends on the complete elimination of bacteria and prevention of bacterial recolonization in the root canal system. The major challenge for conventional root canal disinfection strategies is the ability of bacteria to persist as biofilms within the anatomical complexities of the root canal system. In addition, the alterations in the ultrastructure of dentin tissue results in compromised structural integrity of root dentin leading to higher risk of fracture in root-filled teeth. The objectives of this study are twofold: 1) develop and test functionalized nanoparticles to eliminate biofilm bacteria and, 2) to stabilize and strengthen the dentin organic matrix by crosslinking collagen fibrils in the presence of biopolymeric nanoparticles. A bioactive polymeric nanoparticle functionalized with a photosensitizer may present as a single step treatment to achieve both the objectives. Chitosan a bioactive polymer was used owing to their inherent antibacterial and biocompatible characteristics. Chitosan micro-/nanoparticles were synthesized as well as functionalized with photosensitizer (rose bengal) for photodynamic activation. Bioactive chitosan nanoparticle functionalized with a rose bengal is expected to combine the properties of chitosan i.e., polycationic with higher affinity to bacterial cell wall and alter membrane integrity; that of a photosensitizer i.e., to generate singlet oxygen when photoactivated; and the nano-form further potentiate these specific properties. These photodynamically activable chitosan nanoparticles showed the distinct characteristics of chitosan and rose bengal. The synergistic effect of the chitosan conjugated nanoparticles was able to eliminate monospecies and multi-species bacterial biofilms with complete disruption of the biofilm structure. The singlet oxygen generated during photoactivation produced photochemical crosslinking of dentin collagen and infiltration of chitosan nanoparticles. Following crosslinking the dentin collagen showed significantly improved mechanical properties (ultimate tensile strength and toughness) and improved resistance to degradation by bacterial collagenase. In conclusion, this study presents a potential photosensitizer functionalized chitosan nanoparticles based treatment strategy to improve the success of endodontic treatment to achieve complete disinfection of the root canal system and enhanced the mechanical/ structural integrity of the root-filled teeth.

No description available.

No description available.

Cardiac fibroblasts are capable of a phenotype shift to myofibroblasts and the latter contribute to wound healing and interstitial fibrosis. TGF-β1 signals through R-Smads and Co-Smad proteins and modulates fibrillar collagen deposition. It also influences myofibroblast cells contractility, which they confer torsional forces on the surrounding matrix. c-Ski plays an inhibitory role in TGF-β1 signaling. C184M is a 27 kDa protein that is a novel cytosolic partner of c-Ski. c-Ski-C184M complexes may negatively regulate TGF-β1 signaling via sequestering R-Smad in the cytosol, however, the role of C184M in cardiac fibrosis is unknown. Herein we characterize the expression of C184M and explore its role in TGF-β1 signaling. We found that C184M is expressed in P0 primary fibroblasts, P1 and P2 cardiac myofibroblasts and as well in NIH 3T3 cells. Western blot analysis revealed that the C184M is not responsive to TGF-β1 treatment (10ng/ml, 12, 24 and 48hr treatment) and that Smad3 overexpression does not influence expression of C184M protein in P1 cardiac myofibroblasts. In the presence of overexpressed C184M, immunofluorescence studies indicated a shift in localization of Smad3 from a diffuse cytosolic pattern to a distinctly punctuate cytosolic pattern. C184M overexpression abrogates the effects of TGF-β1 mediated increased collagen synthesis in NIH 3T3 cells. Further, C184M is involved in reduction of contractility of NIH 3T3 cells.

Intramuscular connective tissue is critical in maintaining muscle structure and the transfer of force from contractile elements to the bone. We examined intramuscular connective tissue characteristics in young and old men and women, as well as men and women subjected to simulated microgravity. We hypothesized that intramuscular collagen content, collagen cross-linking and formation of advanced glycation endproducts of old individuals would be greater than young, and that intramuscular collagen content would be elevated following prolonged periods of unloading spanning 35, 60 and 90 days. Vastus lateralis muscle biopsies revealed that intramuscular collagen (Young: 9.6±1.1, Old: 10.2±1.2 ug•mg muscle wet wf-') and collagen cross-links (hydroxylysylpyridinoline, HP) (Young: 395±65, Old: 351±45 mmol HP•mol collagen-1) were unchanged (p>0.05) with aging. The advanced glycation endproduct, pentosidine, was increased (p<0.05) by 203% (Young: 5.2±1.3, Old: 15.9±4.5 mmol pentosidine•mol collagen"') with aging. With unloading, collagen content of the vastus lateralis was unchanged (p>0.05) following all time periods but was found to be elevated (p<0.05) in the soleus following 90 days of unloading. Furthermore, baseline collagen content was found to greater (p<0.05) in the soleus compared to the vastus lateralis. These results suggest the age related decline in whole muscle function is not related to increases in intramuscular collagen content or cross-linking but may be related to the accumulation of advanced glycation endproducts. Muscle function following unloading does not appear to be impacted by collagen content in the vastus lateralis but may play a role in the soleus. / School of Physical Education, Sport, and Exercise Science

Cardiac fibroblasts are capable of a phenotype shift to myofibroblasts and the latter contribute to wound healing and interstitial fibrosis. TGF-β1 signals through R-Smads and Co-Smad proteins and modulates fibrillar collagen deposition. It also influences myofibroblast cells contractility, which they confer torsional forces on the surrounding matrix. c-Ski plays an inhibitory role in TGF-β1 signaling. C184M is a 27 kDa protein that is a novel cytosolic partner of c-Ski. c-Ski-C184M complexes may negatively regulate TGF-β1 signaling via sequestering R-Smad in the cytosol, however, the role of C184M in cardiac fibrosis is unknown. Herein we characterize the expression of C184M and explore its role in TGF-β1 signaling. We found that C184M is expressed in P0 primary fibroblasts, P1 and P2 cardiac myofibroblasts and as well in NIH 3T3 cells. Western blot analysis revealed that the C184M is not responsive to TGF-β1 treatment (10ng/ml, 12, 24 and 48hr treatment) and that Smad3 overexpression does not influence expression of C184M protein in P1 cardiac myofibroblasts. In the presence of overexpressed C184M, immunofluorescence studies indicated a shift in localization of Smad3 from a diffuse cytosolic pattern to a distinctly punctuate cytosolic pattern. C184M overexpression abrogates the effects of TGF-β1 mediated increased collagen synthesis in NIH 3T3 cells. Further, C184M is involved in reduction of contractility of NIH 3T3 cells.

No description available.

No description available.

In the cancer research, it is important to understand protein dynamics which are involved in cell signaling. Therefore, particular protein detection and analysis of target protein behavior are indispensable for current basic cancer research. However, it usually performed by conventional biochemical approaches, which require long process time and a large amount of samples. We have been developed the new applications based on microfluidics and Raster image Correlation spectroscopy (RICS) techniques. A simple microfluidic 3D hydrodynamic flow focusing device has been developed for quantitative determinations of target protein concentrations. The analyte stream was pinched not only horizontally, but also vertically by two sheath streams by introducing step depth cross junction structure. As a result, a triangular cross-sectional flow profile was formed and the laser was focused on the top of the triangular shaped analyte stream. Through this approach, the target protein concentration was successfully determined in cell lysate samples. The RICS technique has been applied to characterize the dynamics of protein 53 (p53) in living cells before and after the treatment with DNA damaging agents. P53 tagged with Green Fluores-cent Protein (GFP) were incubated with and without DNA damaging agents, cisplatin or eptoposide. Then, the diffusion coefficient of GFP-p53 was determined by RICS and it was significantly reduced after the drug treatment while that of the one without drug treatment was not. It is suggested that the drugs induced the interaction of p53 with either other proteins or DNA. This result demonstrates that RICS is able to detect protein-protein or protein-DNA interactions in living cells and it may be useful for the drug screening. As another application of microfluidics, an integrated microfluidic platform was developed for generating collagen microspheres with encapsulation of viable cells. The platform integrated four automated functions on a microfluidic chip, (1) collagen solution cooling system, (2) cell-in-collagen microdroplet generation, (3) collagen microdroplet polymerization, and (4) incubation and extraction of the microspheres. This platform provided a high throughput and easy way to generate uniform dimensions of collagen microspheres encapsulating viable cells that were able to proliferate for more than 1 week.

It is well established that failure of the lamellar basement membrane (BM) occurs during the development of equine laminitis. This is due to loss of the crucial BM components; laminins and collagens along with loss of attachment complex, the hemidesmosome, of the basal cell to the underlying BM. Previous studies have suggested that Ln-332 may be the primary protein involved in lamellar failure. However, the details of the progression and mechanism involved in this pathology are not currently fully known. This thesis aimed to refine the proteolytic processes and mechanisms occurring during the development of oligofructose induced laminitis. Through the use of novel temporal lamellar biopsies obtained during the development of laminitis induction, it was determined that loss of both Ln-332 and collagen type IV occurs as early as 12 hours post induction. This loss of reactivity initially occurred in a focal pattern with increasing loss as the disease progressed in severity. At the later stages of laminitis, separation of the basal epithelial cell from the dermal tissue was also observed, however at these points the BM still appeared intact. This suggests that more than one mechanism may be involved in disease pathology; one resulting in fragmentation of the BM while a second results in loss of the cell attachment allowing the intact BM to slip away. Immunohistochemical analysis of lamellar tissue revealed a unique pattern of reactivity for the Ln-332 γ2 antibody D4B5, in which no reactivity was observed in normal lamellar tissue, yet the epitope recognized by this antibody becomes apparent during disease development. This initially led to the hypothesis that cleavage of the γ2 subunit and the release of biologically active fragments may occur. However, at the molecular level, no γ2 fragments were detected by western blotting. In vitro cleavage of partially purified equine Ln-332 revealed that both MMP-2 and MT1-MMP were able to process the molecule to produce fragments corresponding to the biologically active counterparts. This suggests that the change in reactivity with this antibody may be due to other mechanisms such as decreased interaction of Ln-332 with other BM components resulting in loss of structural stability of the BM allowing for a change in the orientation of Ln-332. Increased MMP-2, MMP-9 and MT1-MMP expression has been demonstrated in laminitis and this was assumed to be the causative agent resulting in tissue destruction and failure. However, work in this thesis found no increase in gene expression of MMP- 2 and MT1-MMP, as well as no activation of pro MT1-MMP. Increased pro MMP-9 gene and protein expression was observed early in the disease progression yet no MMP- 9 activation occurred. Additionally, activation of MMP-2 was found to occur late in laminitis progression at least 12 hours following BM degradation, thus MMP-2 activation is a secondary effect of laminitis development. Thus, other proteases are expected to result in BM processing. Gene expression of the metalloprotease ADAMTS-4, was observed to increase early during laminitis development, suggesting this is a putative factor involved in intensifying the degradation of the lamellar BM. Work in this thesis also revealed that both Ln-332 and collagen type IV are widely distributed throughout organs in the equine body and localized primarily to BM structures. A novel finding of this thesis is that not only does BM degradation occur in the lamellar BM, it also occurs in organs remote from the hoof. At both the onset of lameness and the acute phase of laminitis, fragmentation of both Ln-332 and collagen type IV also occurs in both the skin and stomach. Recent studies have indicated that both leukocyte emigration and increased cytokine expression occurs in the lamellar tissue during laminitis. Work in this thesis added to this knowledge as leukocyte infiltration into the lamellar tissue occurs early during oligofructose laminitis induction as does increased IL-6 gene expression. Overall, work conducted in this thesis has added to the knowledge of the events occurring during laminitis development. Even though the complete mechanism of tissue destruction and lamellar failure was not established, the progression of events is now more clear in that BM degradation is one of the first events to occur, while MMP-2 activation occurs secondarily. Thus, other mechanisms must be at work early during laminitis development and discovering what they are must remain a research priority for the realization of effective therapeutic strategies.