PSD-95/Dlg/ZO-1 (PDZ) domain - PDZ binding motif (PBM) interactions have been one of the most well studied protein-protein interaction systems through biochemical, biophysical and high-throughput screening (HTS) strategies. This has allowed us to understand the mechanism of individual PDZ-PBM interactions and the re-engineering of PBMs to bind tighter or to gain or lose certain specificity. However, there are several thousand native PDZ domains whose biological ligands remain unknown. Because of the low sequence identity among PDZ domain homologues, promiscuous binding profiles (defined as a PDZ domain that can accommodate a set of PBMs or a PBM that can be recognized by many PDZ domains), and context-dependent interaction mechanism, we have an inadequate understanding of the general molecular mechanisms that determine the PDZ-PBM specificity. Therefore, predicting PDZ specificity has been elusive. In addition, no de novo PBM ligand or artificial non-native PDZ domain have been successfully designed. This reflects the general challenges in understanding the general principles of PDZ-ligand interactions, namely that they are context-dependent, exhibit weak binding affinity, narrow binding energy range, and larger interaction surface than other protein-ligand interactions. Together, PDZ domains make good model systems to investigate the fundamental principles of protein-protein interactions with a wide spectrum of biomedical implications. My studies suggest that understanding PBM specificity with the set of structural positions forming the binding pocket can connect sequence, structure and function of a PDZ domain in a general context. They also suggest that this way of understanding the specificity will shed light on prediction and engineering of specificity rationally. Structural analysis on most of the available PDZ domain structures was established to support the principle (Chapter I). The principle was tested against two different types of PBM; C-terminal PBM (Chapter II) and internal PBM (Chapter III), and shown to support better understanding and design of PDZ domain specificity. We further applied the principle to design de novo PDZ domains, and the preliminary data hints that it is optimistic to engineer PDZ domain specificity (Appendix A and B).

In this study we investigated whether a musical prime would produce a contextspecificity effect in predictive learning. Participants were divided into six conditions of a spy-radio predictive learning task. The six conditions were comprised of a combination of three primes (i.e. visual, music, or both) and two learning phase groups (i.e. retrieve, default). The primes indicated the type of stimulus used to prime the temporal context for the test cue-outcome association. The learning phase groups indicated which temporal context would be primed. In the retrieve group, learning Phase 1 was primed; in the default group learning Phase 2 was primed. The presence of a temporal contextspecificity effect was indicated by lower test predictive judgments for the test cue X in the retrieve group and higher test predictive judgments for this cue in the default group. We hypothesized that all three types of primes would lead to a significant contextspecificity effect. Furthermore, we hypothesized that the context-specificity effect would be strongest in the both prime condition because, with the presentation of both the visual and musical primes, participants would have more information about the learning phase temporal context to inform their test predictive judgment. The results partially supported the first hypothesis as there was a significant context-specificity effect with the visual prime. However, contrary to our hypotheses, we did not obtain a context-specificity effect with the music prime or both prime. Despite the lack of a context-specificity effect in the music prime condition, a relationship between participant musical expertise and predictive judgment suggested that the music did have an effect on context-specificity in predictive learning.

Depression is a mental illness that requires prompt identification and treatment due to grave consequences if untreated. Depression can affect a person's level of functioning, lead to worsening health conditions, comorbid substance abuse, and suicide. Despite these facts, the current state of nursing practice includes an inadequate diagnosis of patients with depression, lack of guidelines for the use of assessment tools and diagnostic tests to identify depression, and insufficient information concerning the accuracy of depression assessment tools. This systematic literature review examined 6 depression assessment tools in regard to their accuracy as identified by specificity, sensitivity, reliability, and validity. This project also examined the pros and cons, demographics, and healthcare settings that use these depression inventory tools. This project used the Orlando nursing process theory as a theoretical framework. Based on the review of 10 articles selected, evidence showed that the Hamilton depression rating scale has the highest sensitivity (93%) and specificity (97%) rates. The implications for positive social change include the opportunity for clinicians to use the findings of this project in their selection of depression assessment tools in healthcare settings. Other researchers can use this project as a valuable resource for management of major depressive disorders.

Many disorders found in humans are caused by abnormalities in DNA. Genetic testing of DNA provides a way for clinicians to identify disease-causing mutations in patients. Once patients with potentially disease-causing mutations are identified, they can be enrolled in treatment or preventative programs to improve the patients' long term quality of life. Array-based comparative genomic hybridization (aCGH) provides a high- resolution, genome-wide method for detecting chromosomal abnormalities. Using computer software, chromosome abnormalities, or copy number variations (CNVs) can be identified from aCGH data. The development of a software tool to analyze the performance of CGH microarrays is of great benefit to clinical laboratories. Calibration of parameters used in aCGH software tools can maximize the performance of these arrays in a clinical setting. According to the American College of Medical Genetics, the validation of a clinical chromosomal microarray platform should be performed by testing a large number (200-300) of well-characterized cases, each with unique CNVs located throughout the genome. Because of the Clinical Laboratory Improvement Amendment of 1988 and the lack of an FDA approved whole genome chromosomal microarray platform the ultimate responsibility for validating the performance characteristics of this technology falls to the clinical laboratory performing the testing. To facilitate this task, we have established a computational analytical validation procedure for CGH microarrays that is comprehensive, efficient, and low cost. This validation uses a higher resolution microarray to validate a lower resolution microarray with a receiver operating characteristic (ROC)-based analysis. From the results we are able to estimate an optimal log2 threshold range for determining the presence or absence (calling) of CNVs.

This dissertation investigates second language (L2) development in the domains of morphosyntax and semantics. Specifically, it examines the acquisition of definiteness and specificity in Russian within the Feature Re-assembly framework (Lardiere, 2009), according to which the hardest L2 learning task is not to reset parameters but to reconfigure, or remap features from the way they are assembled in the L1 into new formal configurations in the L2. Within the Feature Re-assembly approach, it has been argued that re-assembling features that are represented overtly in the L1 and mapping them onto those that are encoded covertly by context in the L2 will present a greater difficulty than re-assembling features in the opposite direction (Slabakova, 2009). This dissertation examines the acquisition of four linguistic properties (types of modifiers, word order, indefinite determiners and case marking) that encode definiteness and specificity overtly or covertly in L2 Russian by English and Korean speakers. The native languages of the learners were chosen specifically in order to test various overt-covert mappings. The data obtained from two experimental tasks (grammaticality and felicity judgments) completed by 56 Russian native speaker controls, 51 English- and 53 Korean-speaking learners support Slabakova's prediction that overt-to-covert realization of the feature is more challenging than covert-to-overt realization. In addition, the findings uncovered other important factors facilitating or impeding acquisition, such as the nature of the form-to-meaning mapping (one-to-one or one-to-many) and the availability of clear, unambiguous evidence for a certain mapping in the input available to learners. Results also reveal that the presence or absence of the L1 transfer depends on the overt/covert status of the feature in the L2. That is, when the feature is marked overtly in both the L1 and L2, L1 transfer has facilitative effect on the acquisition of the feature. On the contrary, when the feature is marked covertly in both the L1 and L2, L1 transfer has no or negative effects. These findings provide new insights into the effects of the native language on L2 learnability and enable us to come to a more precise and fine-grained understanding of grammatical meaning acquisition in the second language.

Finding what is alike among the personalities of creative people has been a dream of many researchers. No single personality type has been discovered as prototypical, yet the promise of common attributes among creative people remains enticing. This study examines one of these promising characteristics - Openness to Experience, a personality factor from the Five-Factor Model. This factor has been shown to correlate positively with creativity in past studies. In the present study this relationship was partially confirmed in a sample of employees whose jobs require technical problem solving, by correlating the employees self-rated Work-specific Openness to Experience and NEO PI-R Openness with supervisory ratings of their creative work performance. The Work-specific Openness scale demonstrated a significant correlation with supervisory ratings of creativity, whereas the NEO PI-R Openness scale did not. Although none of the NEO PI-R facets were significant predictors of criterion, four Work-specific facets were significant predictors based on zero order correlations. These facets are Openness to Ideas, Fantasy, Values, and Actions. However, although individual facets of Openness were expected to differ in validity, the magnitude of their correlations with creative performance scores did not differ significantly. Convincing results showing incremental validity of the Work-specific scale over the NEO PI-R scale are also discussed.

The majority of genetic differences between species and individuals have been hypothesised to impact on the regulation, rather than the structure, of genes. As the details of genetic variation are uncovered by the various genome sequencing projects, understanding the functional effects on gene regulation will be key to uncovering the molecular mechanisms underying the genesis and inheritance of common phenotypes, such as complex human disease and commercially important traits in plants and animals. Unlike coding sequence polymorphisms, genetic variants affecting gene expression will reside in the transcriptional machinery and its regulatory inputs. As these are largely specific to cell- or tissue-types, we would expect that regulatory variants will also affect final mRNA levels in a tissue specific manner. Genetic variation between individuals may therefore be more complex than the sum total of sequence differences between them. Demonstrating this hypothesis is the main focus of this thesis. We use microarrays to measure mRNA levels of approximately 22,000 transcripts in inbred and recombinant inbred strains of mice, and present compelling evidence that the genetic influences on these levels are tissue-specific in at least 85% of cases. We uncover two loci which apparently influence transcript levels of multiple genes in a tissue-specific manner. We also present evidence that failure of microarray data normalisation may cause spurious linkage of expression phenotypes leading to erroneous biological conclusions, and detail a novel, extensible mathematical framework for performing tailored normalisation which can remove such systematic bias. The wider context of these results is then discussed.

<p>Protein kinases are enzymes that catalyse the phosphoryl transfer from the g-phosphate of ATP to acceptor amino acids in proteins. The specificity of selected model protein kinases was studied at three different levels using a) novel bi-substrate-analogue inhibitors, b) synthetic peptide substrates and c) mutated protein substrate analogues. </p><p>A new class of protein kinase bi-substrate-analogue inhibitors was designed on the basis of adenosine-5’-carboxylic acid derivatives, where a short arginine containing peptide was attached to the 5'-carbon atom of the adenosine sugar moiety via a linker chain. These compounds showed high inhibitory potential against two basophilic protein kinases, the protein kinase A (PKA) and protein kinase C (PKC), with IC50 values in the nanomolar range, but no inhibitory activity towards the acidophilic kinases CK1 and CK2. The inhibitors were efficiently applied for affinity purification of PKA using MgATP as well as L-arginine as eluting agents. </p><p>Ca2+-dependent protein kinase (CDPK-1) was purified from maize seedlings and its substrate specificity was studied using a set of synthetic peptides. These were derived from the phosphorylatable sequence RVLSRLHS(15)VRER of maize sucrose synthase 2 (SuSy2), and a consensus sequence motif A/LXRXXSXRZR (where X denotes a position with no strict amino acid requirements and Z a position strictly not tolerating arginine) was defined from a study using arrays of systematically varied peptides attached to cellulose membrane (SPOTs^TM membranes). The SuSy2 derived peptides were also found to be efficient substrates for mammalian PKC, but showed low reactivity in the case of PKA. On the basis of this peptide motif, a positionally oriented peptide library approach based on ESI-MS detection of phosphopeptides in initial velocity conditions was designed for quantitative kinetic characterization of protein kinase specificity profiles. On the basis of the obtained data an optimal peptide substrate for PKC, FRRRRSFRRR, was designed. </p><p>The specificity of protein kinase A was studied using site-directed mutagenesis in the phosphorylation site of L-type pyruvate kinase (L-PK), and comparison of the obtained data with the data from previous studies on structurally altered peptide substrates revealed that amino acid alterations in short peptide substrates cause stronger effects on the phosphorylation rate than the corresponding alterations in the protein substrate L-PK.</p>

<p>It is well established that mast cells (MC) are key players in airway pathologies such as allergic asthma, but they are also known to contribute to host defense and tissue remodeling. MC serine proteases are the major protein components of mast cell granules and accordingly, are most likely involved in many aspects of MC function. Two major groups of MC serine proteases have been described; chymases, which cleave a target preferentially after aromatic amino acids, and tryptases, which cleave preferentially after positively charged residues. Biochemical characterization of these proteases is a first step towards understanding their contribution to MC function. One of the issues addressed in this thesis is the target specificity of two rodent MC chymases, rat mast cell protease (rMCP)-4 and rMCP-5. The substrate specificity was analyzed using a substrate phage display technique, in which a large library of peptide substrates is screened simultaneously in a single reaction. The substrate analysis revealed that rMCP-4 displays very stringent substrate specificity, with striking preference for two subsequent aromatic amino acids N-terminal of the cleavage site. This chymase therefore holds a substrate recognition profile clearly distinct from other chymases. Database searches using the generated peptide sequence identified several interesting potential targets for rMCP-4, such as the FcγRIII and the TGFβ receptor. The phage display technique was also used to analyze the substrate specificity of rMCP-5. rMCP-5 is the rat chymase most closely related in sequence to human chymase. Interestingly, rMCP-5, unlike human chymase, was shown to hydrolyze substrates after small aliphatic amino acids, but not after aromatic residues. rMCP-5 and human chymase might therefore have different biological functions. Thus, studies of cleavage specificity can be a successful approach both to elucidate subtle differences in specificity of closely related proteases, as well as to identify new biological targets for a protease.</p><p>The MC tryptases contribute to the pro-inflammatory activities of the MC. To assess the requirements for activation and stability of a mouse tryptase, mMCP-6, recombinant mMCP-6 protein was produced in mammalian cells. A low pH (<6.5), as well as a negatively charged proteoglycan, e.g. heparin, were shown to be necessary both to obtain and maintain activity. With this in mind, heparin antagonists were studied for their potential to inhibit mMCP-6 and human tryptase. Indeed, the heparin antagonists were shown to be highly efficient tryptase inhibitors. Thus, heparin antagonists might be promising candidates to attenuate inflammatory disorders, such as allergic asthma. </p>

<p>The aim was to identify diagnostic difficulties for acute abdominal pain at the emergency department and during hospital stay. A total of 3349 patients admitted to Mora Hospital with acute abdominal pain of up to seven days duration, were registered prospectively for history and clinical signs according to a structured schedule. The preliminary diagnosis from the attending physician at the emergency department, any investigations or surgery and final diagnosis were registered at a follow-up after at least one year. </p><p>There were no differences in diagnostic performance between physicians with 0.5 to 5 years of medical experience. The information collected and a careful examination of the patient was more important than formal competence. The main differential diagnostic problem was non-specific abdominal pain; this was the same for diagnoses requiring surgery. Patients originally diagnosed as not needing surgery had a median delay before operation of 22 hours (mean 40 hours, with 95% confidence interval of 30-50 hours), compared to 8 hours (mean 15 hours, 95% confidence interval of 12-28 hours) for patients with the same final follow-up diagnosis as the preliminary diagnosis. Constipation was a diagnostic pitfall, as 9% of the patients considered constipated required surgery for potentially life threatening reasons and 8% were later found to have an abdominal malignancy. Both the preliminary diagnosis and the discharge diagnosis were less reliable for elderly patients than for younger patients. Elderly patients often had specific organ disease and arrived at the emergency department after a longer history of abdominal pain. </p><p>This study confirms that assessment of suspected appendicitis can still be based on clinical judgements combined with laboratory tests. Classical clinical findings indicating localised inflammation, such as isolated pain in the right iliac fossa, rebound tenderness, right-sided rectal tenderness, pain migration to the right iliac fossa, local guarding and aggravation of pain when moving, were reliable for predicting acute appendicitis. A CT scan can be saved for the more equivocal cases of acute abdominal pain. A generous strategy regarding CT scan among elderly patients with acute abdominal pain, even in the absence of pronounced signs of an inflammatory intra-abdominal process, is recommended.</p>