Family and group cognitive behavior therapy: Evaluation of treatment outcome and treatment specificity

This dissertation examined the efficacy of family cognitive behavior treatment (FCBT) and group cognitive behavior treatment (GBCT) for reducing anxiety disorders in children and adolescents using several approaches: clinical significant change, equivalence testing, and analyses of variance. It also examined treatment specificity in terms of targeting family/parents (in FCBT) and peers/group (in GCBT) contextual variables using two main approaches: analyses of variance and structural equation modeling (SEM). The sample consisted of 143 children and their parents who presented to the Child Anxiety and Phobia Program housed within the Child and Family Psychosocial Research Center at Florida International University. Diagnostic interviews and questionnaires were administered to assess youth anxiety. Questionnaires were administered to assess child and parent views of family/parents and peers/group contextual variables. In terms of clinical significant change, results indicated that 84.6% of youth in FCBT and 71.2% of youth in GBCT no longer met diagnostic criteria for their primary/targeted anxiety disorder. In addition, results from analyses of variance indicated that FCBT and GCBT were both efficacious in reducing anxiety disorders in youth across both child and parent ratings. Results using both analyses of variance and structural equation modeling also indicated that there was no meaningful treatment specificity between FCBT and GCBT in terms of either family/parents or peers/group contextual variables. That is, child social skills improved in GCBT in which these skills were targeted and in FCBT in which these skills were not targeted; parenting skills improved in FCBT in which these skills were targeted and in GCBT in which these skills were not targeted. Clinical implications and future research recommendations are discussed.

Specificity, stability and comparative physiology of coral-Symbiodinium mutualisms: Evaluating the potential for acclimation and/or adaptation in reef corals

The relationship between reef corals and endosymbiotic dinoflagellates is fundamental to the existence of coral reefs. To evaluate the fidelity of coral-Symbiodinium mutualisms, corals maintained in aquaria for years were analyzed by denaturant gradient gel electrophoresis (DGGE). Comparing Symbiodinium populations of captive aquarium colonies with known associations in nature is a practical way of examining partner flexibility. The finding of "normal" symbiont populations in corals existing under highly variable conditions supports the premise that most coral colonies possess stable associations. High sensitivity real-time PCR (rtPCR) was used to evaluate background populations of the putatively stress-tolerant Symbiodinium D in reef corals of the Caribbean. Analyses of samples collected during periods of environmental stability indicate the ability of Symbiodinium D to associate with a wider diversity of host taxa than previously recognized. To gain a broader perspective with regard to the ecology of Symbiodinium D1a, rtPCR and DGGE were used to evaluate the symbiont populations of reef corals from Barbados before and after the 2005 mass coral bleaching. Background populations were observed in 56% of the host genera prior to observations of bleaching. These findings indicate that 'stress', not 'bleaching', caused the displacement of 'natural' symbiont population and the opportunistic proliferation of D1a in many host taxa. Of the 12 host taxa monitored before and after the bleaching event, there was a 40% increase in colonies hosting Symbiodinium D1a. Together, these studies elucidate the mechanism responsible for recent observations reporting the emergence of Symbiodinium D following thermal disturbances. These observations are now most easily explained as the disproportionate growth of existing in hospite symbiont populations, rather than novel symbiont acquisition subsequent to bleaching. To evaluate the comparative "fitness" of corals able to host multiple symbiont types, rates of calcification were measured in P. verrucosa hosting either Symbiodinium C1b-c or D1 at elevated temperature. Rates of calcification decreased significantly for both host-symbiont combinations, but differences attributable to symbiont composition were not detected. This research improves our knowledge of the symbiosis biology and ecology of reef corals and contributes information necessary to most accurately predict the response of these ecosystems to global climate changes.

Parental involvement and group cognitive behavioral treatment for anxiety disorders in children and adolescents: Treatment specificity and mediation effects of parent and peer variables

Phobic and anxiety disorders are one of the most common, if not the most common and debilitating psychopathological conditions found among children and adolescents. As a result, a treatment research literature has accumulated showing the efficacy of cognitive behavioral treatment (CBT) for reducing anxiety disorders in youth. This dissertation study compared a CBT with parent and child (i.e., PCBT) and child group CBT (i.e., GCBT). These two treatment approaches were compared due to the recognition that a child’s context has an effect on the development, course, and outcome of childhood psychopathology and functional status. The specific aims of this dissertation were to examine treatment specificity and mediation effects of parent and peer contextual variables. The sample consisted of 183 youth and their mothers. Research questions were analyzed using analysis of variance for treatment outcome, and structural equation modeling, accounting for clustering effects, for treatment specificity and mediation effects. Results indicated that both PCBT and GCBT produced positive treatment outcomes across all indices of change (i.e., clinically significant improvement, anxiety symptom reduction) and across all informants (i.e., youths and parents) with no significant differences between treatment conditions. Results also showed partial treatment specific effects of positive peer relationships in GCBT. PCBT also showed partial treatment specific effects of parental psychological control. Mediation effects were only observed in GCBT; positive peer interactions mediated treatment response. The results support the use CBT with parents and peers for treating childhood anxiety. The findings’ implications are further discussed in terms of the need to conduct further meditational treatment outcome designs in order to continue to advance theory and research in child and anxiety treatment.

Specific binding of the mammalian high mobility group protein AT-hook 2 to the minor groove of AT -rich DNAs: Thermodynamic and specificity studies

The mammalian high mobility group protein AT-hook 2 (HMGA2) is a small transcriptional factor involved in cell development and oncogenesis. It contains three "AT-hook" DNA binding domains, which specifically recognize the minor groove of AT-rich DNA sequences. It also has an acidic C-terminal motif. Previous studies showed that HMGA2 mediates all its biological effects through interactions with AT-rich DNA sequences in the promoter regions. In this dissertation, I used a variety of biochemical and biophysical methods to examine the physical properties of HMGA2 and to further investigate HMGA2's interactions with AT-rich DNA sequences. The following are three avenues perused in this study: (1) due to the asymmetrical charge distribution of HMGA2, I have developed a rapid procedure to purify HMGA2 in the milligram range. Preparation of large amounts of HMGA2 makes biophysical studies possible; (2) Since HMGA2 binds to different AT-rich sequences in the promoter regions, I used a combination of isothermal titration calorimetry (ITC) and DNA UV melting experiment to characterize interactions of HMGA2 with poly(dA-dT) 2 and poly(dA)poly(dT). My results demonstrated that (i) each HMGA2 molecule binds to 15 AT bp; (ii) HMGA2 binds to both AT DNAs with very high affinity. However, the binding reaction of HMGA2 to poly(dA-dT) 2 is enthalpy-driven and the binding reaction of HMGA2 with poly(dA)poly(dT) is entropy-driven; (iii) the binding reactions are strongly depended on salt concentrations; (3) Previous studies showed that HMGA2 may have sequence specificity. In this study, I used a PCR-based SELEX procedure to examine the DNA binding specificity of HMGA2. Two consensus sequences for HMGA2 have been identified: 5'-ATATTCGCGAWWATT-3' and 5'-ATATTGCGCAWWATT-3', where W represents A or T. These consensus sequences have a unique feature: the first five base pairs are AT-rich, the middle four to five base pairs are GC-rich, and the last five to six base pairs are AT-rich. All three segments are critical for high affinity binding. Replacing either one of the AT-rich sequences to a non-AT-rich sequence causes at least 100-fold decrease in the binding affinity. Intriguingly, if the GC-segment is substituted by an AT-rich segment, the binding affinity of HMGA2 is reduced approximately 5-fold. Identification of the consensus sequences for HMGA2 represents an important step towards finding its binding sites within the genome.

Tailoring heme -thiolate proteins into efficient biocatalysts with high specificity and selectivity

Cytochrome P450 monooxygenases, one of the most important classes of heme-thiolate proteins, have attracted considerable interest in the biochemical community because of its catalytic versatility, substrate diversity and great number in the superfamily. Although P450s are capable of catalyzing numerous difficult oxidation reactions, the relatively low stability, low turnover rates and the need of electron-donating cofactors have limited their practical biotechnological and pharmaceutical applications as isolated enzymes. The goal of this study is to tailor such heme-thiolate proteins into efficient biocatalysts with high specificity and selectivity by protein engineering and to better understand the structure-function relationship in cytochromes P450. In the effort to engineer P450cam, the prototype member of the P450 superfamily, into an efficient peroxygenase that utilizes hydrogen peroxide via the “peroxide-shunt” pathway, site-directed mutagenesis has been used to elucidate the critical roles of hydrophobic residues in the active site. Various biophysical, biochemical and spectroscopic techniques have been utilized to investigate the wild-type and mutant proteins. Three important P450cam variants were obtained showing distinct structural and functional features. In P450camV247H mutant, which exhibited almost identical spectral properties with the wild-type, it is demonstrated that a single amino acid switch turned the monooxygenase into an efficient preoxidase by increasing the peroxidase activity nearly one thousand folds. In order to tune the distal pocket of P450cam with polar residues, Leu 246 was replaced with a basic residue, lysine, resulting in a mutant with spectral features identical to P420, the inactive species of P450. But this inactive-species-like mutant showed catalytic activities without the facilitation of any cofactors. By substituting Gly 248 with a histidine, a novel Cys-Fe-His ligation set was obtained in P450cam which represented the very rare case of His ligation in heme-thiolate proteins. In addition to serving as a convenient model for hemoprotein structural studies, the G248H mutant also provided evidence about the nature of the axial ligand in cytochrome P420 and other engineered hemoproteins with thiolate ligations. Furthermore, attempts have been made to replace the proximal ligand in sperm whale myoglobin to construct a heme-thiolate protein model by mimicking the protein environment of cytochrome P450cam and chloroperoxidase.

Tailoring Heme-Thiolate Proteins into Efficient Biocatalysts with High Specificity and Selectivity

Cytochrome P450 monooxygenases, one of the most important classes of heme-thiolate proteins, have attracted considerable interest in the biochemical community because of its catalytic versatility, substrate diversity and great number in the superfamily. Although P450s are capable of catalyzing numerous difficult oxidation reactions, the relatively low stability, low turnover rates and the need of electron-donating cofactors have limited their practical biotechnological and pharmaceutical applications as isolated enzymes. The goal of this study is to tailor such heme-thiolate proteins into efficient biocatalysts with high specificity and selectivity by protein engineering and to better understand the structure-function relationship in cytochromes P450. In the effort to engineer P450cam, the prototype member of the P450 superfamily, into an efficient peroxygenase that utilizes hydrogen peroxide via the “peroxide-shunt” pathway, site-directed mutagenesis has been used to elucidate the critical roles of hydrophobic residues in the active site. Various biophysical, biochemical and spectroscopic techniques have been utilized to investigate the wild-type and mutant proteins. Three important P450cam variants were obtained showing distinct structural and functional features. In P450camV247H mutant, which exhibited almost identical spectral properties with the wild-type, it is demonstrated that a single amino acid switch turned the monooxygenase into an efficient preoxidase by increasing the peroxidase activity nearly one thousand folds. In order to tune the distal pocket of P450cam with polar residues, Leu 246 was replaced with a basic residue, lysine, resulting in a mutant with spectral features identical to P420, the inactive species of P450. But this inactive-species-like mutant showed catalytic activities without the facilitation of any cofactors. By substituting Gly 248 with a histidine, a novel Cys-Fe-His ligation set was obtained in P450cam which represented the very rare case of His ligation in heme-thiolate proteins. In addition to serving as a convenient model for hemoprotein structural studies, the G248H mutant also provided evidence about the nature of the axial ligand in cytochrome P420 and other engineered hemoproteins with thiolate ligations. Furthermore, attempts have been made to replace the proximal ligand in sperm whale myoglobin to construct a heme-thiolate protein model by mimicking the protein environment of cytochrome P450cam and chloroperoxidase.

Cognitive behavior therapy for anxiety disorders in youth: Treatment specificity and mediation effects

The present study investigated the efficacies of Individual CBT (ICBT), Parent Relationship Skill Training (RLST, which targets increasing parental acceptance of youth and increasing autonomy granting) and Parent Reinforcement Skills Training (RLST, which targets increasing parental positive reinforcement and decreasing negative reinforcement). The specific aims were to examine treatment specificity and mediation effects of parenting variables. ICBT was used as a baseline comparison condition. The sample consisted of 253 youth (ages 5-16 years; M = 9.38; SD = 2.42) and their parents. To examine treatment outcome and specificity, the data were analyzed using analysis of variance within a structural equation modeling framework. Mediation was analyzed via structural equation modeling using MPlus. Results indicated that ICBT, RLST, and RFST produced positive treatment outcomes across all indices of change (i.e., clinically significant improvement, anxiety symptom reduction) and across all informants (i.e., youths and parents). RLST was associated with incremental reduction in youth anxiety symptoms beyond ICBT, as per youth report. Treatment specificity effects were found for participants in RFST in terms of parental reinforcement, as per parent report only. Treatment mediation was not found for any of the hypothesized parenting variables (i.e., parental acceptance, parental autonomy granting, parental reinforcement). The results support the use of CBT involving only the youth and the parent and youth together for treating youth anxiety. The findings’ implications are further discussed in terms of the need to conduct further meditational treatment outcome designs in order to continue to advance theory and research in youth anxiety treatment.