The role of the ClpA chaperone in proteolysis by ClpAP

ClpA, a member of the Clp/Hsp100 family of ATPases, is a molecular chaperone and, in combination with a proteolytic component ClpP, participates in ATP-dependent proteolysis. We investigated the role of ClpA in protein degradation by ClpAP by dissociating the reaction into several discrete steps. In the assembly step, ClpA–ClpP–substrate complexes assemble either by ClpA–substrate complexes interacting with ClpP or by ClpA–ClpP complexes interacting with substrate; ClpP in the absence of ClpA is unable to bind substrates. Assembly requires ATP binding but not hydrolysis. We discovered that ClpA translocates substrates from their binding sites on ClpA to ClpP. The translocation step specifically requires ATP; nonhydrolyzable ATP analogs are ineffective. Only proteins that are degraded by ClpAP are translocated. Characterization of the degradation step showed that substrates can be degraded in a single round of ClpA–ClpP–substrate binding followed by ATP hydrolysis. The products generated are indistinguishable from steady-state products. Taken together, our results suggest that ClpA, through its interaction with both the substrate and ClpP, acts as a gatekeeper, actively translocating specific substrates into the proteolytic chamber of ClpP where degradation occurs. As multicomponent ATP-dependent proteases are widespread in nature and share structural similarities, these findings may provide a general mechanism for regulation of substrate import into the proteolytic chamber.

The quorum-sensing transcriptional regulator TraR requires its cognate signaling ligand for protein folding, protease resistance, and dimerization

Complexes between the quorum-sensing regulator TraR and its inducing ligand autoinducer (AAI) are soluble in Escherichia coli, whereas apo-TraR is almost completely insoluble. Here we show that the lack of soluble TraR is due in large part to rapid proteolysis, inasmuch as apo-TraR accumulated to high levels in an E. coli strain deficient in Clp and Lon proteases. In pulse labeling experiments, AAI protected TraR against proteolysis only when it was added before the radiolabel. This observation indicates that TraR proteins can productively bind AAI only during their own synthesis on polysomes, whereas fully synthesized apo-TraR proteins are not functional AAI receptors. Purified apo-TraR was rapidly degraded by trypsin to oligopeptides, whereas TraR–AAI complexes were more resistant to trypsin and were cleaved at discrete interdomain linkers, indicating that TraR requires AAI to attain its mature tertiary structure. TraR–AAI complexes eluted from a gel filtration column as dimers and bound DNA as dimers. In contrast, apo-TraR was monomeric, and incubation with AAI under a variety of conditions did not cause dimerization. We conclude that AAI is critical for the folding of nascent TraR protein into its mature tertiary structure and that full-length apo-TraR cannot productively bind AAI and is consequently targeted for rapid proteolysis.

Antifreeze Proteins in Winter Rye Leaves Form Oligomeric Complexes1

Antifreeze proteins (AFPs) similar to three pathogenesis-related proteins, a glucanase-like protein (GLP), a chitinase-like protein (CLP), and a thaumatin-like protein (TLP), accumulate during cold acclimation in winter rye (Secale cereale) leaves, where they are thought to modify the growth of intercellular ice during freezing. The objective of this study was to characterize the rye AFPs in their native forms, and our results show that these proteins form oligomeric complexes in vivo. Nine proteins were separated by native-polyacrylamide gel electrophoresis from apoplastic extracts of cold-acclimated winter rye leaves. Seven of these proteins exhibited multiple polypeptides when denatured and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After isolation of the individual proteins, six were shown by immunoblotting to contain various combinations of GLP, CLP, and TLP in addition to other unidentified proteins. Antisera produced against individual cold-induced winter rye GLP, CLP, and TLP all dramatically inhibited glucanase activity in apoplastic extracts from cold-acclimated winter rye leaves, and each antiserum precipitated all three proteins. These results indicate that each of the polypeptides may be exposed on the surface of the protein complexes. By forming oligomeric complexes, AFPs may form larger surfaces to interact with ice, or they may simply increase the mass of the protein bound to ice. In either case, the complexes of AFPs may inhibit ice growth and recrystallization more effectively than the individual polypeptides.

Induction of Acclimative Proteolysis of the Light-Harvesting Chlorophyll a/b Protein of Photosystem II in Response to Elevated Light Intensities1

Most plants have the ability to respond to fluctuations in light to minimize damage to the photosynthetic apparatus. A proteolytic activity has been discovered that is involved in the degradation of the major light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCII) when the antenna size of photosystem II is reduced upon acclimation of plants from low to high light intensities. This ATP-dependent proteolytic activity is of the serine or cysteine type and is associated with the outer membrane surface of the stroma-exposed thylakoid regions. The identity of the protease is not known, but it does not correspond to the recently identified chloroplast ATP-dependent proteases Clp and FtsH, which are homologs to bacterial enzymes. The acclimative response shows a delay of 2 d after transfer of the leaves to high light. This lag period was shown to be attributed to expression or activation of the responsible protease. Furthermore, the LHCII degradation was found to be regulated at the substrate level. The degradation process involves lateral migration of LHCII from the appressed to the nonappressed thylakoid regions, which is the location for the responsible protease. Phosphorylated LHCII was found to be a poor substrate for degradation in comparison with the unphosphorylated form of the protein. The relationship between LHCII degradation and other regulatory proteolytic processes in the thylakoid membrane, such as D1-protein degradation, is discussed.

Habilidades funcionais de autocuidado, mobilidade e função social em crianças com fissura labiopalatina e Espectro Óculo-Aurículo-Vertebral (EOAV)

As anomalias craniofaciais ocasionam comprometimentos estéticos e funcionais com grande impacto na saúde e na integração social da criança, com interferência no desenvolvimento global e social. Das anomalias craniofaciais este estudo abordou as Fissuras Labiopalatinas (FLP) e o Espectro Óculo Aurículo Vertebral (EOAV). As FLP constituem malformações resultantes de falta do fechamento completo dos tecidos que compõe o lábio e o palato. O EOAV, também conhecido como Síndrome de Goldenhar, é uma anomalia congênita de etiologia desconhecida, com manifestação genética variável e de causa bastante heterogênea. Conhecer as habilidades funcionais e o impacto destas no desenvolvimento global de crianças com EOAV e FLP pode otimizar o desenvolvimento de programas de prevenção e intervenção para promover a saúde e a integração social destes indivíduos. Este estudo foi delineado com objetivo de verificar e comparar o desempenho em habilidades funcionais quanto ao desempenho nas áreas de autocuidado, mobilidade, função social e nível de independência entre crianças com EOAV, crianças com FLP e um grupo comparativo, de crianças sem anomalias. O modelo de pesquisa foi observacional descritivo transversal com uma casuística de 39 pais/responsáveis de crianças na faixa etária entre três anos e sete anos e seis meses, de ambos os gêneros. Foram convidados para participar pais/responsáveis de crianças em tratamento no Hospital de Reabilitação de Anomalias Craniofaciais da Universidade e São Paulo (HRAC-USP) os quais foram divididos em três grupos: dois experimentais e um grupo comparativo. O instrumento para coleta dos dados das habilidades funcionais foi o Pediatric Evaluation of Disability Inventory (PEDI), em sua versão adaptada para o português. A avaliação é realizada por meio de entrevista com o cuidador, o qual deve saber informar sobre o desempenho da criança em atividades e tarefas típicas da rotina diária. Os dados foram apresentados por análise descritiva com medidas de tendência central (média aritmética), dispersão (desvio-padrão) e distribuição de frequência, nas variáveis: idades, gênero e nível socioeconômico da família e caracterização da casuística. Para as análises das pontuações bruta e normativa do questionário PEDI no que se refere às habilidades funcionais e a assistência do cuidador nas três áreas de função autocuidado, mobilidade e função social, foi utilizado o teste de variância One Way, e para o teste de normalidade foi utilizado Shapiro Wilk para variável dependente. A análise comparativa foi realizada pelo teste de Kruskal-Wallis, adotando-se o valor de significância de p< 0,05. Os resultados deste estudo na análise comparativa nas habilidades funcionais na mobilidade, houve diferença estatisticamente significante na comparação entre os grupos GC vs GEEOAV, no escore bruto, e entre os grupos GC vs GEEOAV e GC vs GEFLP, no escore normativo.Na assistência do cuidador no autocuidado, houve diferença estatisticamente significante na comparação entre os grupos GC vs GEEOAV, no escore normativo. Na assistência do cuidador na mobilidade, houve diferença estatisticamente significante na comparação entre os grupos GC vs GEEOAV nos escores bruto e normativo.Na assistência do cuidador na função social houve diferença estatisticamente significante na comparação entre os grupos GC vs GEFLP.

Influence of nitrogen deficiency on senescence and the amounts of RNA and proteins in wheat leaves

Senescence-associated coordination in amounts of enzymes localized in different cellular compartments were determined in attached leaves of young wheat (Triticum aestivum L. cv. Arina) plants. Senescence was initiated at the time of full leaf elongation based on declines in total RNA and soluble protein. Removal of N from the growth medium just at the time of full leaf elongation enhanced the rate of senescence. Sustained declines in the amount of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39), and a marked decrease in the rbcS transcripts, just after full leaf elongation indicated that Rubisco synthesis/degradation was very sensitive to the onset of senescence. Rubisco activase amount also declined during senescence but the proportion of rca transcript relative to the total poly A RNA pool increased 3-fold during senescence. Thus, continued synthesis of activase may be required to maintain functional Rubisco throughout senescence. N stress led to declines in the amount of proteins located in the chloroplast, the peroxisome and the cytosol. Transcripts of the Clp protease subunits also declined in response to N stress, indicating that Clp is not a senescence-specific protease. In contrast to the other proteins, mitochondrial NADH-glutamate dehydrogenase (EC 1.4.1.2) was relatively stable during senescence and was not affected by N stress. During natural senescence with adequate plant nitrate supply the amount of nitrite reductase (EC 1.7.7.1) increased, and those of glutamine synthetase (EC 1.4.7.1) and glutamate synthase (EC 6.3.1.2) were stable. These results indicated that N assimilatory capacity can continue or even increase during senescence if the substrate supply is maintained. Differential stabilities of proteins, even within the same cellular compartment, indicate that proteolytic activity during senescence must be highly regulated.

Salivary Gland and Tooth Abnormalities Massively Increase Caries Susceptibility in A Mouse Model of Cleft Lip/Palate

Thesis (Ph.D.)--University of Washington, 2016-04

A decision support system for waste collection management and its potential improvement with Radio-Frequency Identification Technology (RFID)

Due to vigorous globalisation and product proliferation in recent years, more waste has been produced by the soaring manufacturing activities. This has contributed to the significant need for an efficient waste management system to ensure, with all efforts, the waste is properly treated for recycling or disposed. This paper presents a Decision Support System (DSS) framework, based on Constraint Logic Programming (CLP), for the collection management of industrial waste (of all kinds) and discusses the potential employment of Radio-Frequency Identification Technology (RFID) to improve several critical procedures involved in managing waste collection. This paper also demonstrates a widely distributed and semi-structured network of waste producing enterprises (e.g. manufacturers) and waste processing enterprises (i.e. waste recycling/treatment stations) improving their operations planning by means of using the proposed DSS. The potential RFID applications to update and validate information in a continuous manner to bring value-added benefits to the waste collection business are also presented. © 2012 Inderscience Enterprises Ltd.

An investigation of multiple pathways of developmental intervention change

Convergence among treatment, prevention, and developmental intervention approaches has led to the recognition of the need for evaluation models and research designs that employ a full range of evaluation information to provide an empirical basis for enhancing the efficiency, efficacy, and effectiveness of prevention and positive development interventions. This study reports an investigation of a positive youth development program using an Outcome Mediation Cascade (OMC) evaluation model, an integrated model for evaluating the empirical intersection between intervention and developmental processes. The Changing Lives Program (CLP) is a community supported positive youth development intervention implemented in a practice setting as a selective/indicated program for multi-ethnic, multi-problem at risk youth in urban alternative high schools. This study used a Relational Data Analysis integration of quantitative and qualitative data analysis strategies, including the use of both fixed and free response measures and a structural equation modeling approach, to construct and evaluate the hypothesized OMC model. Findings indicated that the hypothesized model fit the data (χ2 (7) = 6.991, p = .43; RMSEA = .00; CFI = 1.00; WRMR = .459). Findings also provided preliminary evidence consistent with the hypothesis that in addition to having effects on targeted positive outcomes, PYD interventions are likely to have progressive cascading effects on untargeted problem outcomes that operate through effects on positive outcomes. Furthermore, the general pattern of findings suggested the need to use methods capable of capturing both quantitative and qualitative change in order to increase the likelihood of identifying more complete theory informed empirically supported models of developmental intervention change processes.

A developmental intervention science outreach research approach to promoting positive youth development

Recent intervention efforts in promoting positive identity in troubled adolescents have begun to draw on the potential for an integration of the self-construction and self-discovery perspectives in conceptualizing identity processes, as well as the integration of quantitative and qualitative data analytic strategies. This study reports an investigation of the Changing Lives Program (CLP), using an Outcome Mediation (OM) evaluation model, an integrated model for evaluating targets of intervention, while theoretically including a Self-Transformative Model of Identity Development (STM), a proposed integration of self-discovery and self-construction identity processes. This study also used a Relational Data Analysis (RDA) integration of quantitative and qualitative analysis strategies and a structural equation modeling approach (SEM), to construct and evaluate the hypothesized OM/STM model. The CLP is a community supported positive youth development intervention, targeting multi-problem youth in alternative high schools in the Miami Dade County Public Schools (M-DCPS). The 259 participants for this study were drawn from the CLP’s archival data file. The model evaluated in this study utilized three indices of core identity processes (1) personal expressiveness, (2) identity conflict resolution, and (3) informational identity style that were conceptualized as mediators of the effects of participation in the CLP on change in two qualitative outcome indices of participants’ sense of self and identity. Findings indicated the model fit the data (χ2 (10) = 3.638, p = .96; RMSEA = .00; CFI = 1.00; WRMR = .299). The pattern of findings supported the utilization of the STM in conceptualizing identity processes and provided support for the OM design. The findings also suggested the need for methods capable of detecting and rendering unique sample specific free response data to increase the likelihood of identifying emergent core developmental research concepts and constructs in studies of intervention/developmental change over time in ways not possible using fixed response methods alone.

Cognitive competence and life course change in multi-problem adolescents

The dissertation reports on two studies. The purpose of Study I was to develop and evaluate a measure of cognitive competence (the Critical Problem Solving Skills Scale – Qualitative Extension) using Relational Data Analysis (RDA) with a multi-ethnic, adolescent sample. My study builds on previous work that has been conducted to provide evidence for the reliability and validity of the RDA framework in evaluating youth development programs (Kurtines et al., 2008). Inter-coder percent agreement among the TOC and TCC coders for each of the category levels was moderate to high, with a range of .76 to .94. The Fleiss' kappa across all category levels was from substantial agreement to almost perfect agreement, with a range of .72 to .91. The correlation between the TOC and the TCC demonstrated medium to high correlation, with a range of r(40)=.68, p<.001 to r(40)=.79, p<.001. Study II reports an investigation of a positive youth development program using an Outcome Mediation Cascade (OMC) evaluation model, an integrated model for evaluating the empirical intersection between intervention and developmental processes. The Changing Lives Program (CLP) is a community supported positive youth development intervention implemented in a practice setting as a selective/indicated program for multi-ethnic, multi-problem at risk youth in urban alternative high schools in the Miami Dade County Public Schools (M-DCPS). The 259 participants for this study were drawn from the CLP's archival data file. The study used a structural equation modeling approach to construct and evaluate the hypothesized model. Findings indicated that the hypothesized model fit the data (χ2 (7) = 5.651, p = .83; RMSEA = .00; CFI = 1.00; WRMR = .319). My study built on previous research using the OMC evaluation model (Eichas, 2010), and the findings are consistent with the hypothesis that in addition to having effects on targeted positive outcomes, PYD interventions are likely to have progressive cascading effects on untargeted problem outcomes that operate through effects on positive outcomes.

The possible selves method in evaluating youth development interventions: The use of Integrated Qualitative /Quantitative Data Analytic Strategies (IQ -DAS)

This study investigated the feasibility of using qualitative methods to provide empirical documentation of the long-term qualitative change in the life course trajectories of “at risk” youth in a school based positive youth development program (the Changing Lives Program—CLP). This work draws from life course theory for a developmental framework and from recent advances in the use of qualitative methods in general and a grounded theory approach in particular. Grounded theory provided a methodological framework for conceptualizing the use of qualitative methods for assessing qualitative life change. The study investigated the feasibility of using the Possible Selves Questionnaire-Qualitative Extension (PSQ-QE) for evaluating the impact of the program on qualitative change in participants' life trajectory relative to a non-intervention control group. Integrated Qualitative/Quantitative Data Analytic Strategies (IQ-DAS) that we have been developing a part of our program of research provided the data analytic framework for the study. ^ Change was evaluated in 85 at risk high school students in CLP high school counseling groups over three assessment periods (pre, post, and follow-up), and a non-intervention control group of 23 students over two assessment periods (pre and post). Intervention gains and maintenance and the extent to which these patterns of change were moderated by gender and ethnicity were evaluated using a mixed design Repeated Measures Multivariate Analysis of Variance (RMANOVA) in which Time (pre, post) was the within (repeated) factor and Condition, Gender, and Ethnicity the between group factors. The trends for the direction of qualitative change were positive from pre to post and maintained at the year-end follow-up. More important, the 3-way interaction for Time x Gender x Ethnicity was significant, Roy's Θ =. 205, F(2, 37) = 3.80, p <.032, indicating that the overall pattern of positive change was significantly moderated by gender and ethnicity. Thus, the findings also provided preliminary evidence for a positive impact of the youth development program on long-term change in life course trajectory, and were suggestive with respect to the issue of amenability to treatment, i.e., the identification of subgroups of individuals in a target population who are likely to be the most amenable or responsive to a treatment. ^

Participation in a positive youth development program and multi -problem alternative high school students' transformative goals: An investigation of the theoretical and methodological utility and validity of the Transformative Goal Attainment (TGAS)

There is a growing body of literature that provides evidence for the efficacy of positive youth development programs in general and preliminary empirical support for the efficacy of the Changing Lives Program (CLP) in particular. This dissertation sought to extend previous efforts to develop and preliminarily examine the Transformative Goal Attainment Scale (TGAS) as a measure of participant empowerment in the promotion of positive development. Consistent with recent advances in the use of qualitative research methods, this dissertation sought to further investigate the utility of Relational Data Analysis (RDA) for providing categorizations of qualitative open-ended response data. In particular, a qualitative index of Transformative Goals, TG, was developed to complement the previously developed quantitative index of Transformative Goal Attainment (TGA), and RDA procedures for calculating reliability and content validity were refined. Second, as a Stage I pilot/feasibility study this study preliminarily examined the potentially mediating role of empowerment, as indexed by the TGAS, in the promotion of positive development. ^ Fifty-seven participants took part in this study, forty CLP intervention participants and seventeen control condition participants. All 57 participants were administered the study's measures just prior to and just following the fall 2003 semester. This study thus used a short-term longitudinal quasi-experimental research design with a comparison control group. ^ RDA procedures were refined and applied to the categorization of open-ended response data regarding participants' transformative goals (TG) and future possible selves (PSQ-QE). These analyses revealed relatively strong, indirect evidence for the construct validity of the categories as well as their theoretically meaningful structural organization, thereby providing sufficient support for the utility of RDA procedures in the categorization of qualitative open-ended response data. ^ In addition, transformative goals (TG) and future possible selves (PSQ-QE), and the quantitative index of perceived goal attainment (TGA) were evaluated as potential mediators of positive development by testing their relationships to other indices of positive intervention outcome within a four-step method involving both analysis of variance (ANOVA and RMANOVAs) and regression analysis. Though more limited in scope than the efforts at the development and refinement of the measures of these mediators, the results were also promising. ^

A model of self -transformative identity development in troubled adolescent youth

Within the Stage II program evaluation of the Miami Youth Development Project's (YDP) Changing Lives Program (CLP), this study evaluated CLP intervention effectiveness in promoting positive change in emotion-focused identity exploration (i.e. feelings of personal expressiveness; PE) and a "negative" symptom of identity development (i.e. identity distress; ID) as a first step toward the investigation of a self-transformative model of identity development in adolescent youth. Using structural equation modeling techniques, this study found that participation in the CLP is associated with positive changes in PE (path = .841, p < .002), but not changes in ID. Increase in ID scores was found to be associated with increases in PE (path = .229, p < .002), as well. Intervention effects were not moderated by age/stage, gender, or ethnicity, though differences were found in the degree to which participating subgroups (African-American/Hispanic, male/female, 14-16 years old/17-19 years old) experience change in PE and ID. Findings also suggest that moderate levels of ID may not be deleterious to identity exploration and may be associated with active exploration. ^

Development of Innovative Load Transfer Mechanism to Reduce Hurricane-Induced Failures in New and Existing Residential Construction

Implicit in current design practice of minimum uplift capacity, is the assumption that the connection's capacity is proportional to the number of fasteners per connection joint. This assumption may overestimate the capacity of joints by a factor of two or more and maybe the cause of connection failures in extreme wind events. The current research serves to modify the current practice by proposing a realistic relationship between the number of fasteners and the capacity of the joint. The research is also aimed at further development of non-intrusive continuous load path (CLP) connection system using Glass Fiber Reinforced Polymer (GFRP) and epoxy. Suitable designs were developed for stud to top plate and gable end connections and tests were performed to evaluate the ultimate load, creep and fatigue behavior. The objective was to determine the performance of the connections under simulated sustained hurricane conditions. The performance of the new connections was satisfactory.