Bond strength of adhesives to dentin contaminated with smoker's saliva.

The purpose of this study was to determine the effects of contamination with smoker's and non-smoker's saliva on the bond strength of resin composite to superficial dentin using different adhesive systems. The interfacial structure between the resin and dentin was evaluated for each treatment using environmental scanning electron microscopy (ESEM). Freshly extracted human molars were ground with 600-grit SiC paper to expose the superficial dentin. Adhesives [One-Up-Bond-F-Plus (OUFP) and Adper-Prompt-L-Pop (APLP)] and resin composite (TPHSpectrum) were bonded to the dentin (n = 8/group, 180 total specimens) under five surface conditions: control (adhesive applied following manufacturers' instructions); saliva, then 5-s air dry, then adhesive; adhesive, saliva, 5-s air dry; adhesive, saliva, 5-s water rinse, 5-s air dry (ASW group); and adhesive, saliva, 5-s water rinse, 5-s air dry, reapply adhesive (ASWA group). After storage in water at 37 degrees C for 24 h, the specimens were debonded under tension at a speed of 0.5 mm/min. ESEM photomicrographs of the dentin/adhesive interfaces were taken. Mean bond strength ranged from 8.1 to 24.1 MPa. Fisher's protected least significant difference (P = 0.05) intervals for critical adhesive, saliva, and surface condition differences were 1.3, 1.3, and 2.1 MPa, respectively. There were no significant differences in bond strength to dentin between contamination by smoker's and nonsmoker's saliva, but bond strengths were significantly different between adhesive systems, with OUFP twice as strong as APLP under almost all conditions. After adhesive application and contamination with either smoker's or nonsmoker's saliva followed by washing and reapplication of the adhesive (ASWA group), the bond strength of both adhesive systems was the same as that of the control group.

Identification of the Causative Bacteria in Musculoskeletal Infections Using 16s rDNA - Denaturing Gradient Gel Electrophoresis Analysis

Musculoskeletal infections are infections of the bone and surrounding tissues. They are currently diagnosed based on culture analysis, which is the gold standard for pathogen identification. However, these clinical laboratory methods are frequently inadequate for the identification of the causative agents, because a large percentage (25-50%) of confirmed musculoskeletal infections are false negatives in which no pathogen is identified in culture. My data supports these results. The goal of this project was to use PCR amplification of a portion of the 16S rRNA gene to test an alternative approach for the identification of these pathogens and to assess the diversity of the bacteria involved. The advantages of this alternative method are that it should increase sample sensitivity and the speed of detection. In addition, bacteria that are non-culturable or in low abundance can be detected using this molecular technique. However, a complication of this approach is that the majority of musculoskeletal infections are polymicrobial, which prohibits direct identification from the infected tissue by DNA sequencing of the initial 16S rDNA amplification products. One way to solve this problem is to use denaturing gradient gel electrophoresis (DGGE) to separate the PCR products before DNA sequencing. Denaturing gradient gel electrophoresis (DGGE) separates DNA molecules based on their melting point, which is determined by their DNA sequence. This analytical technique allows a mixture of PCR products of the same length that electrophoreses through agarose gels as one band, to be separated into different bands and then used for DNA sequence analysis. In this way, the DGGE allows for the identification of individual bacterial species in polymicrobial-infected tissue, which is critical for improving clinical outcomes. By combining the 16S rDNA amplification and the DGGE techniques together, an alternative approach for identification has been used. The 16S rRNA gene PCR-DGGE method includes several critical steps: DNA extraction from tissue biopsies, amplification of the bacterial DNA, PCR product separation by DGGE, amplification of the gel-extracted DNA, and DNA sequencing and analysis. Each step of the method was optimized to increase its sensitivity and for rapid detection of the bacteria present in human tissue samples. The limit of detection for the DNA extraction from tissue was at least 20 Staphylococcus aureus cells and the limit of detection for PCR was at least 0.05 pg of template DNA. The conditions for DGGE electrophoreses were optimized by using a double gradient of acrylamide (6 – 10%) and denaturant (30-70%), which increased the separation between distinct PCR products. The use of GelRed (Biotium) improved the DNA visualization in the DGGE gel. To recover the DNA from the DGGE gels the gel slices were excised, shredded in a bead beater, and the DNA was allowed to diffuse into sterile water overnight. The use of primers containing specific linkers allowed the entire amplified PCR product to be sequenced and then analyzed. The optimized 16S rRNA gene PCR-DGGE method was used to analyze 50 tissue biopsy samples chosen randomly from our collection. The results were compared to those of the Memorial Hermann Hospital Clinical Microbiology Laboratory for the same samples. The molecular method was congruent for 10 of the 17 (59%) culture negative tissue samples. In 7 of the 17 (41%) culture negative the molecular method identified a bacterium. The molecular method was congruent with the culture identification for 7 of the 33 (21%) positive cultured tissue samples. However, in 8 of the 33 (24%) the molecular method identified more organisms. In 13 of the 15 (87%) polymicrobial cultured tissue samples the molecular method identified at least one organism that was also identified by culture techniques. Overall, the DGGE analysis of 16S rDNA is an effective method to identify bacteria not identified by culture analysis.

Sibling relationships in the family of a child with a burn injury

Purpose. Understanding siblings' experiences after a major childhood burn injury was the purpose of this mixed method, qualitative dominant study. The following research questions guided this project: How do siblings describe the impact of a major childhood burn injury experience? How do sibling relationship factors of warmth/closeness, relative status/power, conflict, and rivalry further clarify their relationship and their experience after a major burn injury? ^ Methods. A mixed method, qualitative dominant, design was implemented to understand the sibling experiences in a family with a child suffering from a major burn injury. Informants were selected from patients with childhood burn injuries attending the reconstructive clinic at a Gulf coast children's specialty hospital. The qualitative portion used the life story method, a narrative process, to portray the long-term impact on sibling relationships. A "case" represents a family unit and could be composed of one or multiple family members. Participants from 22 cases (N = 40 participants) were interviewed. Interviews were conducted in person and via telephone. The quantitative portion, or the embedded part of this mixed method design, used the Sibling Relationship Questionnaire Revised (SRQ-R) to conduct an additional structured interview and acquire scoring data. It was postulated that the SRQ-R would provide another perspective on the sibling experience and expand the qualitative data analysis. Thematic analysis was implemented on the qualitative interview data including the qualitative data from the interviews structured on the SRQ-R. Additionally, scores on the SRQ-R were tabulated to further describe the cases. ^ Results. The overall thematic pattern for the sibling relationship in families having a child with a major burn injury was that of normalization. Areas of normalization as well as the process of adjustment were the major themes. Areas of normalization were found in play and other activities, in school and work, and in family relations with their siblings and their parents. The process of adjustment in the sibling relationship was described as varied, involved school and work re-entry, and might even change their life perspective. Further analysis included an examination of the cases in which more than one person were interviewed and completed the SRQ-R. Participants from five ( n = 11) of six cases (n = 14), scored above 3.0 on the five-point scale on the Warmth/Closeness construct, indicating they perceived the sibling relationship as close. Five participants scored high on the Conflict construct and four participants scored high on the Rivalry construct. Finally, Relative Status/Power was low or negative in the six cases (n = 13). ^ Conclusions/implications. These findings suggest the importance of returning to normalcy for many of the families and the significance of sibling relationships on the process. Some of these families were able to use this major life event in a positive way to promote normalization. ^

Structural determinants of subunit -subunit interactions and subcellular localization of the calcium /calmodulin -dependent protein kinase II

The multifunctional Ca$\sp{2+}$/calmodulin-dependent protein kinase II (CaM kinase) is a Ser/Thr directed protein kinase that participates in diverse Ca$\sp{2+}$ signaling pathways in neurons. The function of CaM kinase depends upon the ability of subunits to form oligomers and to interact with other proteins. Oligomerization is required for autophosphorylation which produces significant functional changes that include Ca$\sp{2+}$/calmodulin-independent activity and calmodulin trapping. Associations with other proteins localize CaM kinase to specific substrates and effectors which serves to optimize the efficiency and speed of signal transduction. In this thesis, we investigate the interactions that underlie the appropriate positioning of CaM kinase activity in cells. We demonstrate that the subcellular distribution of CaM kinase is dynamic in hippocampal slices exposed to anoxic/aglycemic insults and to high K$\sp{+}$-induced depolarization. We determine the localization of CaM kinase domains expressed in neurons and PC-12 cells and find that the C-terminal domain of the $\alpha$ subunit is necessary for localization to dendrites. Moreover, monomeric forms of the enzyme gain access to the nucleus. Attempts made to identify novel CaM kinase binding proteins using the yeast two-hybrid system resulted in the isolation of hundreds of positive clones. Those that have been sequenced are identical to CaM kinase isoforms. Finally, we report the discovery of specific regions within the C-terminal domain that are necessary and sufficient for subunit-subunit interactions. Differences between the $\alpha$ and $\beta$ isoforms were discovered that indicate unique structural requirements for oligomerization. A model for how CaM kinase subunits interact to form holoenzymes and how structural heterogeneity might influence CaM kinase function is presented. ^