The glutathione transferases (GSTs) represent a superfamily of dimeric proteins involved in cellular detoxication by catalyzing the nucleophilic addition of the reduced glutathione (GSH) to the hydrophobic electrophiles. The present work focuses on the functional role of the conserved structures of GSTP1-1. The lock-and-key motif is a highly conserved hydrophobic interaction in the subunit interface of Pi, Mu, and Alpha class GSTs. The key residue (Tyr50 in hGSTP1-1) of one subunit is wedged into a hydrophobic pocket of the neighboring subunit. The heterodimer GSTP1/Y50A was constructed from the fully active wild-type GSTP1-1 and the nearly inactive Y50A in order to study how an essentially inactive subunit influences the activity of the neighboring subunit. The results illuminate the vital role of the lock-and-key motif in modulating the GSH binding and the rate of catalysis. Additionally, the two active sites of the dimeric enzyme work synergistically. An observed water network, in hGSTP1-1 structures, connects the two active sites, thereby offering a mechanism for communication between the two active sites. Cys48 and Tyr50 were targeted by mutations and chemical modifications for understanding how the α2 loop residues modulate GSH binding and catalysis. The replacement of Tyr50 with different unnatural amino acids showed that the nature of the key residue side-chain influences the interaction with the lock structure and, consequently, the catalytic activity. The KMGSH, GSH affinity and protein stability can be modulated by fitting key residue into the lock cavity of the neighbor subunit and, consequently, restriction of the flexibility of the α2 loop. Optimization of the interaction between the key residue and the lock-cavity increases kcat. Also, the crystal structure of the Cys-free variant was determined. The result indicated that Cys48 restricts the flexibility of the α2 loop by interacting with surrounding residues and, consequently, contributes to GSH binding and protein stability.

Developing radiation delivery systems for optimisation of absorbed dose to the target without normal tissue toxicity requires advanced calculations for transport of radiation. In this thesis absorbed dose and fluence in different radiotherapy applications were calculated by using Monte Carlo (MC) simulations. In paper I-III external neutron activation of gadolinium (Gd) for intravascular brachytherapy (GdNCB) and tumour therapy (GdNCT) was investigated. MC codes MCNP and GEANT4 were compared. MCNP was chosen for neutron capture reaction calculations. Gd neutron capture reaction includes both very short range (Auger electrons) and long range (IC electrons and gamma) products. In GdNCB the high-energetic gamma gives an almost flat absorbed dose delivery pattern, up to 4 mm around the stent. Dose distribution at the edges and inside the stent may prevent stent edge and in-stent restenosis. For GdNCT the absorbed dose from prompt gamma will dominate over the dose from IC and Auger electrons in an in vivo situation. The absorbed dose from IC electrons will enhance the total absorbed dose in the tumours and contribute to the cell killing. In paper IV a model for calculation of inter-cluster cross-fire radiation dose from β-emitting radionuclides in a breast cancer model was developed. GEANT4 was used for obtaining absorbed dose. The dose internally in cells binding the isotope (self-dose) increased with decreasing β-energy except for the radionuclides with substantial amounts of conversion electrons and Auger electrons. An effective therapy approach may be a combination of radionuclides where the high self-dose from nuclides with low β-energy should be combined with the inter-cell cluster cross-fire dose from high energy β-particles. In paper V MC simulations using correlated sampling together with importance sampling were used to calculate spectra perturbations in detector volumes caused by the detector silicon chip and its encapsulation. Penelope and EGSnrc were used and yielded similar results. The low energy part of the electron spectrum increased but to a less extent if the silicon detector was encapsulated in low z-materials.

Protein-protein interactions (PPIs) are attractive targets because of their therapeutic potential. One approach to design small molecules that can disrupt the PPIs is to use structural information of proteins. With this approach, triazole-based peptidomimetics that mimic beta-turn hot-spot regions in neurotrophins were synthesized. The monovalent mimics were assembled into bivalent mimics via a combinatorial method. Three different bivalent mimics were prepared for different studies. Bivalent mimics with long-linkers bound to TrkA or TrkC receptor and showed partial antagonism for the receptors. Other mimics were conjugated with cytotoxic compounds and they were used for TrkC targeted drug delivery. The last group of bivalent mimics previously showed targeted delivery effects for pancreatic cancer cells. In this study, we synthesized Eu-chelated bivalent mimics to perform a competitive binding assay for pancreatic cancer cells. Previous research in our group focused on design of secondary structures' mimics on rigid scaffolds as "minimalist mimics." We sought to establish structural design criteria for the minimalist mimics, and we wanted to propose that sets of such compounds could mimic local pairs of amino acids in any secondary structures as "universal peptidomimetics." Thus, we designed five compounds, such as oxazoline-, pyrrole-, dyine- "kinked" and "linear" bistrizole-based peptidomimetics, and performed molecular modelings, DFT calculations, and QMD for them to validate our hypothesis. On the concepts of "minimalist mimics" and "universal peptidomimetics," we developed the C alpha ? C beta vector matching program to evaluate preferred orientations of C alpha - C beta coordinates for secondary structures. We applied the program to omegatides and pyrrolinone-pyrrolidine oligomers. The compounds matched better with strands than for helices. We expanded the C alpha ? C beta vector matching idea to a method that ranks preferred conformations of small molecules on any combination of three interface side-chains in all structurally characterized PPIs. We developed a PDB mining program (explores key orientation, EKO) to do this, and EKO applied to pyrrolinone-pyrrolidine oligomers to find targets. EKO found several interesting targets, such as AICAR Tfase, GAPDH, and HIV-1 protease. HIV-1 dimerization inhibition and Zhang-Poorman kinetic assays were performed to validate our hypothesis, and the results showed that pyrrolinone-pyrrolidine derivatives inhibited HIV-1 dimerization.

Severe Traumatic Brain Injury (TBI) and aneurysmal Subarachnoid Hemorrhage (SAH) are severe cerebral emergencies. They are common reasons for extensive morbidity and mortality in young people and adults in the western world. This thesis, based on five clinical studies in patients with severe TBI (I-IV) and SAH (V), is concentrated on examination of pathophysiological developments and of evaluation of therapeutic approaches in order to improve outcome after cerebral emergency. The treatment for severe TBI patients at Umeå University Hospital, Sweden is an intracranial pressure (ICP)-targeted therapy according to “the Lund-concept”. This therapy is based on physiological principles for cerebral volume regulation, in order to preserve a normal cerebral microcirculation and a normal ICP. The main goal is to avoid development of secondary brain injuries, thus avoiding brain oedema and worsened microcirculation. Study I is evaluating retrospectively 41 children with severe TBI, from 1993 to 2002. The boundaries of the ICP-targeted protocol were obtained in 90%. Survival rate was 93%, and favourable outcome (Glasgow Outcome Scale, score 4+5) was 80%. Study II is retrospectively analysing fluid administration and fluid balance in 93 adult patients with severe TBI, from 1998 to 2001.The ICP-targeted therapy used, have defined fluid strategies. The total fluid balance was positive day one to three, and negative day four to ten. Colloids constituted 40-60% of total fluids given/day. Severe organ failure was evident for respiratory insufficiency and observed in 29%. Mortality within 28 days was 11%. Study III is a prospective, randomised, double-blind, placebo-controlled clinical trial in 48 patients with severe TBI. In order to improve microcirculation and prevent oedema formation, prostacyclin treatment was added to the ICP-targeted therapy. Prostacyclin is endogenously produced, by the vascular endothelium, and has the ability to decrease capillary permeability and vasodilate cerebral capillaries. Prostacyclin is an inhibitor of leukocyte adhesion and platelet aggregation. There was no significant difference between prostacyclin or placebo groups in clinical outcome or in cerebral microdialysis markers such as lactatepyruvate ratio and brain glucose levels. Study IV is part of the third trial and focus on the systemic release of pro-inflammatory mediators that are rapidly activated by trauma. The systemically released pro-inflammatory mediators, interleukin-6 and CRP were significantly decreased in the prostacyclin group versus the placebo group. Study V is a prospective pilot study which analyses asymmetric dimethylarginine (ADMA) concentrations in serum from SAH patients. Acute SAH patients have cerebral vascular, systemic circulatory and inflammatory complications. ADMA is a marker in vascular diseases which is correlated to endothelial dysfunction. ADMA concentrations in serum were significantly elevated seven days after the SAH compared to admission and were still elevated at the three months follow-up. Our results show overall low mortality and high favourable outcome compared to international reports on outcome in severe TBI patients. Prostacyclin administration does not improve cerebral metabolism or outcome but significantly decreases the levels of pro-inflammatory mediators. SAH seems to induce long-lasting elevations of ADMA in serum, which indicates persistent endothelial dysfunction. Endothelial dysfunction may influence outcome after severe cerebral emergencies.

A multi-tiered system of supports offers a comprehensive model for the prevention of academic and behavior problems in schools. To date, research has emphasized the impact of universal and intensive interventions. However, the need for research on secondary or targeted group interventions (Tier 2) for those students who do not respond to the universal level of support is growing. This study evaluated CICO, a Tier 2 intervention, in improving student behavior when it is used with three elementary students from a high-need population and in conjunction with student accountability tracking, designed to promote parental involvement. Functional assessments indicated that all three students had attention-maintained problem behavior during instruction sessions. The study employed a concurrent multiple baseline design across students to assess the effects of CICO and CICO with accountability tracking on academic engagement and problem behavior. Results indicated that the team members were able to implement CICO with fidelity and their implementation of the intervention was effective in increasing academic engagement and reducing problem behavior. The CICO with accountability tracking implementation with one student contributed to further improvement of his target behaviors. These effects were shown to be maintained moderately well for two students who underwent fading. Results are discussed in terms of the study limitations and implications for practice and future research.

An educational framework known as School Wide Positive Behavior Support being implemented in school systems across the country provides the schools with three tiers of support to address both academic and behavior challenges. The purpose of this study was to evaluate the use of peer tutors when applied to a Tier 2 intervention known as Check-In Check-Out (CICO). Peer tutors performed the morning check-in with the tutees by setting the expectations for the day and giving the tutees their Daily Progress Report (DPR) form. Throughout the day, the tutees took the DPR form to each class where they received a score from the teacher. At the end of the class period, the peer tutors provided the tutees with feedback on the scores received on the DPR form. Once the school day finished, the peer tutees checked-out with the tutors and received a reward if they met their percentage goal. The results of this study showed that CICO implemented by peers improved classroom behavior for all three participants.

Hardware verification currently takes more than 50% of the whole verification time. There is a sustained effort to improve the efficiency of the verification process, which in the past helped deliver a large variety of supporting tools. The past years though did not see any major technology change that would bring the improvements that the process really needs (H. Foster 2013) (Wilson Research Group 2012). The existing approach to verification does not provide that type of qualitative jump anymore. This work is introducing a new tactic, providing a modern alternative to the existing approach to the verification problem. The novel approach I use in this research has the potential of significantly improve the process, way beyond incremental changes. It starts with acknowledging the huge amounts of data that follows the hardware development process from inception to the final product and in considering the data not as a quantitative by-product but as a qualitative supply of information on which we can develop a smarter verification. The approach is based on data already generated throughout the process currently used by verification engineers to zoom into the details of different verification aspects. By using existing machine learning approaches we can zoom out and use the same data to extract information, to gain knowledge that we can use to guide the verification process. This approach allows an apparent lack of accuracy introduced by data discovery, to achieve the overall goal. The latest advancements in machine learning and data mining offer a base of a new understanding and usage of the data that is being passed through the process. This work takes several practical problems for which the classical verification process reached a roadblock, and shows how the new approach can provide a jump in productivity and efficiency of the verification process. It focuses on four different aspects of verification to prove the power of this new approach: reducing effort redundancy, guiding verification to areas that need it first, decreasing time to diagnose, and designing tests for coverage efficiency. / text

Η Σωματοστατίνη (SRIF) είναι μια ορμόνη του υποθαλάμου. Ραδιοεπισημασμένα ανάλογα της χρησιμοποιούνται στη σπινθηρογραφική απεικόνιση όγκων που υπερεκφράζουν υποδοχείς της (στοχευμένη διάγνωση). Μειονεκτήματα της στοχευμένης διάγνωσης είναι η υψηλή και παρατεταμένη εντόπιση της ακτινοβολίας στους νεφρούς και η πρόσληψη των ραδιοπεπτιδίων από όργανα μη στόχους. Στόχος της παρούσας διατριβής είναι η ανάπτυξη νέων αναλόγων της SRIF τα οποία να πληρούν τις απαραίτητες προϋποθέσεις ώστε να καταστούν νέα διαγνωστικά μέσα. Αναπτύχθηκαν 33 νέα συνθετικά παράγωγα της SRIF. Σε πρώτη φάση συνετέθησαν απλά ανάλογα της SRIF. Ακολούθησε αξιολόγηση και σε αυτά τα οποία παρουσίαζαν ικανοποιητική συμπεριφορά σε σχέση με την συγγένεια τους με τον υποδοχέα και την πολικότητα τους συζεύχθηκε τετρααμινικός υποκαταστάτης για σύμπλεξη ραδιομετάλλου. Συνετέθησαν επίσης διμερή ανάλογα της SRIF και γλυκοπεπτιδικά ανάλογα. Τα πεπτίδια συντέθηκαν εφαρμόζοντας τεχνικές της Fmoc/tBu μεθοδολογίας. Ο συνδυασμός των μεθόδων που εφαρμόστηκαν οδήγησε στη λήψη των επιθυμητών δομών σε υψηλές αποδόσεις και καθαρότητα. Από τα αποτελέσματα των μελετών ανταγωνιστικής δέσμευσης τα οποία έγιναν σε μεμβράνες κυττάρων AR4-2J, αποδείχτηκε ότι όλα τα ανάλογα διαθέτουν πολύ υψηλές τιμές IC50. Ικανοποιήθηκε επίσης η απαίτηση για αύξηση της πολικότητας τους. Τα νέα ανάλογα πληρούν καταρχήν τις απαραίτητες προϋποθέσεις ώστε να καταστούν υποψήφια νέα διαγνωστικά μέσα και να βρουν κλινική εφαρμογή. Αναμένεται τα τελικά ραδιοπεπτίδια να οδηγούν σε μειωμένη πρόσληψη της ακτινοβολίας από τα όργανα μη στόχους και να εμφανίζουν αυξημένη απέκκριση από τους νεφρούς. Από προκαταρτικά πειράματα βρέθηκε ότι τα πιο πάνω επετεύχθησαν. / Somatostatin is a hyrothalamic regulatory peptide hormone with a pan-antisecretory profile. A large variety of human tumors are expressing multple somatostatin receptors. Synthetic somatostatin analogues radiolabelled with a variety of metallic radionuclide’s are used for the diagnosis and staging of sstr positive tumors but there is the main problem of high accumulation of the radiopeptides on non target organs and the high renal uptake of the radioactivity which leads to nephrotoxicity. Aim of this study was the development of new SRIF analogues as candidate useful clinical tools for the diagnosis and staging of sstr positive tumors. There have been developed thirty three new SRIF analogues and a glycoaminoacid suitable for use in SPPS. First, new analogues of [Tyr3]Octreotate were developed and the best of them, these that satisfied the relation between polarity and binding affinity to the sstr2, were coupled with a tetraaminic chelator ligand for the future complexation of a radionuclide. In other approach peptide dimers were developed. New glycopeptides were also developed. All analogues were synthesized on the solid phase applying several techniques of Fmoc/tBu strategy in high yield and purity. The binding affinity of the new SRIF analogs was checked with experiments of competitive binding with membranic preparations of AR42J cells, positive on SRIF receptors. All the new analogues are able to bind the sstr2 with high affinities. The new analogues accomplish the pre-condition to become new and useful clinical tools after their radiolabelling in the field of peptide-receptor imaging. It is expected that the new molecules will be able to have increased renal excretion via the kidneys and low non target accumulation. From preliminary experiments was shown that the above were succeeded.

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