Obwohl fetale Mißbildungen seit langem bekannt sind, wird eine intensive Prüfung von Arzneimitteln und anderen Substanzen erst seit den 1960er Jahren durchgeführt. Dem Tierschutzbericht von 2001 ist zu entnehmen, daß im Jahr 1999 insgesamt etwa 1,6 Millionen Wirbeltiere zu Versuchszwecken benötigt wurden. Als mögliche Alternative zu Untersuchungen mit Säugetieren wurde ein Testmodell mit Embryonen des Zebrabärblings (D. rerio) entwickelt. Ziel der vorliegenden Arbeit ist es zu klären, ob sich mit den Embryonen von Danio rerio ein teratogenes Potential von Substanzen erkennen und quantifizieren läßt. Dazu wurde DarT (?Danio rerio Teratogenicity Assay?) als Teratogenitäts-Screening Test entwickelt. Es können anhand von toxikologischen Endpunkten sowohl die letalen als auch die subletalen Wirkungen von Substanzen bestimmt werden. Darüber hinaus werden anhand von teratogenen Endpunkten speziell Malformationen erfaßt. Der Vergleich der beobachteten Effekte und der daraus berechneten Wirkkonzentrationen gestattet eine Einschätzung des teratogenen Potentials von Substanzen. Die im DarT erzielten Ergebnisse werden mit der bekannten Zuordnungen des ?säugerteratogenen? Potentials verglichen. Für 88 % der getesteten Substanzen gibt DarT die aus säugertoxikologischen Untersuchungen bekannten Einordnungen hinsichtlich des teratogenen Potentials wieder. Für 10 % der Testsubstanzen wurde das teratogene Potential zu hoch, für 2 % zu niedrig eingeschätzt. Mit dem Testsystem ?Danio rerio Teratogenicity Assay ? DarT? ist ein Vergleich von Substanzen hinsichtlich ihres teratogenen und allgemein toxischen Potentials möglich. In einem Modell können Wirkkonzentrationen und Konzentrations-Wirkungs-Beziehung ermittelt und direkt verglichen werden. Mit DarT kann eine große Anzahl von Substanzen zeit- und kostengünstig untersucht werden. Die aus Untersuchungen mit Säugetieren bekannten Zuordnungen der teratogenen Potentiale von Substanzen wird gut wiedergegeben.

Gegenwärtig ist das Interesse und der Bedarf von Proteinbindern insbesondere in der Biotechnik und Pharmaforschung sehr groß. Kombinatorische, Partikel-basierte (One-Bead-One-Compound) Peptidbibliotheken sind eine Technik, um selektiv bindende Proteine zu identifizieren. Allerdings beinhaltet das Screening dieser Peptidbibliotheken aufwendige Schritte, wie die Separation, Sequenzierung und Charakterisierung von identifizierten Bindern. In dieser Arbeit wurde ein Chip-System entwickelt, auf dem alle Schritte eines Screenings durchgeführt werden können. Dafür wurde ein Glasobjektträger mit einem magnetisch leitenden, doppelseitigen Klebeband versehen. Die Partikel der Bibliothek wurden durch ein Sieb aufgetragen. Dies führte zu einer geordneten Immobilisierung der Partikel auf dem Chip. Über 30.000 Partikel konnten so auf einem Chip immobilisiert werden. Für die Identifizierung von selektiven Protein-bindenden Peptiden wird die immobilisierte Peptidbibliothek mit einem Fluorophor-markierten Protein inkubiert, bindende Partikel mittels Fluoreszenzscan identifiziert und die Peptidsequenz direkt auf dem Chip mittels Matrix-Assisted-Laser-Desorption/Ionization-(MALDI)-Flugzeit-(TOF)-Massenspektroskopie (MS) bestimmt. Die Durchführung einer Abbruchsequenz-Methode erlaubt die eindeutige Bestimmung der Peptidsequenzen mit einer nahezu 100 % Genauigkeit. Die entwickelte Technologie wurde in einem FLAG-Peptid-Modell validiert. Bei dem Screening wurden neue anti-FLAG-Antikörper-bindende Peptide identifiziert. Anschließend wurden in einem Screening von ca. 30.000 Partikeln IgG-bindende Peptide mit mittleren mikromolaren Dissoziationskonstanten identifiziert. Für die Identifizierung stärkerer Binder wurde eine magnetische Anreicherung entwickelt, die dem Chip-Screening vorgeschaltet werden kann. Hiermit wurden aus ca. 1 Million gescreenter Partikel, Peptide mit Dissoziationskonstanten im niedrigen mikromolaren Bereich identifiziert. / The screening of one-bead-one-compound (OBOC) libraries is a well-established technique for the identification of protein-binding ligands. The demand for binders with high affinity and specificity towards various targets has surged in the biomedical and pharmaceutical field in recent years. The combinatoric peptide screening traditionally involves tedious steps such as affinity selection, bead picking, sequencing and characterization. In this thesis, a high-throughput “all-on-one chip” system is presented to avoid slow and technically complex bead picking steps. Beads of a combinatorial peptide library are immobilized on a conventional glass slide equipped with an electrically conductive tape. The beads are applied by using a precision sieve, which allows the spatially ordered immobilization of more than 30,000 beads on one slide. For the target screening, the immobilized library is subsequently incubated with a fluorophore-labeled target protein. In a fluorescence scan followed by matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry (MS), high-affinity binders are directly and unambiguously sequenced directly from the bead. The use of an optimized ladder sequencing approach improved the accuracy of the de-novo sequencing step to 100 %. This new technique was validated by employing a FLAG-based model system. In a first step, new peptide binders for the M2 anti-FLAG monoclonal antibody were identified. Finally, this system was utilized to screen for IgG-binding peptides. The screening of about 30.000 peptides on one chip led to the identification of peptide binders in the mid micromolar range. A magnetic enrichment technique was developed to increase the number of screened beads. By combining the magnetic enrichment strategy with the chip system, 1 million beads were screened and IgG-binders in the low micromolar range were identified.

No description available.

Master of Science / Department of Biochemistry and Molecular Biophysics / Phillip Klebba / Iron acquisition is a component of Gram-negative bacteria pathogenesis, therefore as a form of 'nutritional immunity' host organisms sequester iron. To obtain iron bacteria secrete siderophores that scavenge iron. The E. coli outer membrane protein FepA actively transports the siderophore ferric enterobactin into the periplasm. We observe this uptake reaction by fluorescently labeling FepA in live bacteria, monitoring quenching that occurs upon binding of FeEnt, and then fluorescence recovery during transport. Energy poisons azide, arsenate, and 2,4-dinitrophenol were evaluated to determine sensitivity to known transport inhibitors. We developed and optimized methods to screen for iron transport inhibitors using a cell-based high-throughput screening platform. These inhibitors may have broad spectrum bacteriostatic antibiotic properties.

Colorectal cancer is second only to lung cancer as a leading cause of internal cancer death. Individuals over 65 years of age are most at risk yet least likely to engage in screening for colorectal cancer. The purpose of this descriptive-correlational study using a modified Pender Health Promotion Model was to identify motivations of elderly individuals to engage in health protective behavior. A convenience sample of 90 subjects answered a four-part motivations questionnaire in which three subscales--early detection, powerful others, and chance--met reliability standards (alpha >.70). Chance was significantly related to compliance (r = -.28; p =.003); Hemoccult compliers believed less in chance and powerful others than did non-compliers (p =.005;.002). The 88 percent who performed a Hemoccult stool test as a screening method for early detection of colorectal cancer demonstrated that these elders willingly engaged in health protective behavior and supported the nurses' role in promoting primary prevention in elderly clients.

Drug discovery has witnessed an increase in the application of in silico methods to complement existing in vitro and in vivo experiments, in an attempt to 'fail fast' and reduce the high attrition rates of clinical phases. Computer algorithms have been successfully employed for many tasks including biological target selection, hit identification, lead optimization, binding affinity determination, ADME and toxicity prediction, side-effect prediction, drug repurposing, and, in general, to direct experimental work. This thesis describes a multifaceted approach to virtual screening, to computationally identify small-molecule inhibitors against a biological target of interest. Conformer generation is a critical step in all virtual screening methods that make use of atomic 3D data. We therefore analysed the ability of computational tools to reproduce high quality, experimentally resolved conformations of organic small-molecules. We selected the best performing method (RDKit), and developed a protocol that generates a non-redundant conformer ensemble which tends to contain low-energy structures close to those experimentally observed. We then outline the steps we took to build a multi-million, small-molecule database (including molecule standardization and efficient exact, substructure and similarity searching capabilities), for use in our virtual screening experiments. We generated conformers and descriptors for the molecules in the database. We tagged a subset of the database as `drug-like' and clustered this to provide a reduced, diverse set of molecules for use in more computationally-intensive virtual screening protocols. We next describe a novel virtual screening method we developed, called Ligity, that makes use of known protein-ligand holo structures as queries to search the small-molecule database for putative actives. Ligity has been validated against targets from the DUD-E dataset, and has shown, on average, better performance than other 3D methods. We also show that performance improved when we fused the results from multiple input structures. This bodes well for Ligity's future use, especially when considering that protein structure databases such as the Protein Data Bank are growing exponentially every year. Lastly, we describe the fruitful application of structure-based and ligand-based virtual screening methods to Plasmodium falciparum Subtilisin-like Protease 1 (PfSUB1), an important drug target in the human stages of the life-cycle of the malaria parasite. Our ligand-based virtual screening study resulted in the discovery of novel PfSUB1 inhibitors. Further lead optimization of these compounds, to improve binding affinity in the nanomolar range, may promote them as drug candidates. In this thesis we postulate that the accuracy of computational tools in drug discovery may be enhanced to take advantage of the exponential increase of experimental data and the availability of cheaper computational power such as cloud computing.

The overall aim of this thesis was to describe pregnant women's and partners' views and experiences on early prenatal screening with the combined test, with special focus on the two-step information model. Interviews were performed with 15 couples who had taken part in the extended information visit about prenatal screening, describing their perceptions of the information model (I) and ten couples or women of those, for a follow-up interview exploring their decision-making process (II). Seven couples, who had not taken part in the extended information visit, were interviewed describing their views and experiences about prenatal screening (III). A questionnaire was answered by 295 women and by 223 partners about their satisfaction about the decision whether or not to participate in the combined test, and their assessment of whether or not this choice had been difficult (IV). The results showed that different opinions were expressed about the offer of the extended information visit. The separate visit was welcomed by most couples (I). The decision-making process regarding whether to take part in the test or not was described by most couples as a fairly straightforward decision, while for others it was a more complex process that required a great deal of consideration (II). An apprehension of the test, by some of those who had refrained the extended information visit, was that it was an expression of society's involvement in decisions that belong to the expectant parents (III). Ninety-three percent of both women and partners considered the decision about participating in the combined tests as uncomplicated, and well over 90%, of both women and partners were satisfied with their decision (IV). The conclusions in this thesis, are that the decision whether or not to participate in the combined test is multidimensional and influenced by different views. The two-step information model helped the pregnant woman and the partner to make a decision in a fairly straightforward process or a more complex process with mixed feelings.

In all kinds of disease models, many proteins involved in protein-protein interactions (PPIs) are mutated and do not function properly. The important role of PPIs in disease makes the design of small molecule inhibition an interesting proposition. This project looks at mouse double minute 2 (MDM2) and mouse double minute X (MDMX) which binds and inhibits the tumour suppressor protein p53. MDM2 and MDMX are therefore attractive therapeutic targets due to their role in tumour progression. The aim is to identify small molecule dual inhibitors that are able to disrupt MDM2 and MDMX from binding to p53. Both N-terminal MDM2 and MDMX were successfully expressed and purified with high purity and decent yield. These proteins were used to develop Fluoresence Polarization (FP) and Capillary Electrophoresis (CE) assays for small molecule inhibitors screening. This work has successfully developed FP and CE assays for detecting weakly interacting fragments. The CE assay is a novel method for detecting weak fragments for protein-protein interactions, which are a challenging target. Two approaches were employed to identify small molecule inhibitors for MDM2- N/p53 interaction. At first, small molecules were identified using in silico screening and these hits were verified using FP and CE assays. Second, analogue exploration was applied to identify fragments from the small molecule inhibitors discovered from the in silico screening. Diphenylamine and oxindole fragments were identified as the most potent. However, diphenylamine fragment was discovered to aggregate MDM2-N and was ranked as a false positive hit. No protein aggregation was found when incubated with the oxindole fragment. Therefore oxindole can provide a good starting point for the design of higher affinity analogues. Studying the interaction of MDMX has only recently been undertaken. MDMX contains a high homology binding site with MDM2. Hence, developing a dual MDM2/MDMX inhibitor has become an attractive target to focus on. FP and CE assays were developed to screen compounds against MDMX-N. In silico screening against MDM2-N and MDMX-N found several hits. One compound was discovered as a dual binder to MDM2-N and MDMX-N with low μM affinity. This novel hit is potentially a good starting point for the design of higher affinity analogues.

MDM2, murine double minute 2, is a RING type-E3 ligase protein and also an oncogene. MDM2 plays a critical role in determining the steady levels and activity of p53 in cells using two mechanisms. The N-terminal domain of MDM2 binds to the transactivation domain of p53 and inhibits its transcriptional activity. The RING domain of MDM2 plays a role in the ubiquitination (and degradation) of p53. Several proteins are responsible for the ubiquitination mechanism including the ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin ligase (E3). Since the E2-E3 interaction is essential for ubiquitination, the protein-protein recognition site is a potential drug target. Two different MDM2 RING constructs were expressed and purified: MDM2RING (residues 386-491) and MDM2RING△C (residues 386-478). Both constructs were characterised using dynamic light scattering, size exclusion chromatography, mass spectrometry, NMR and electron microscopy. E3 ligase activity in vitro was also studied. Taken together these results showed that the MDM2RING construct formed a concentration-dependent oligomeric structure. In contrast, the MDM2RING△C construct formed a dimer at all concentrations. Both MDM2RING and MDM2RING △ C retain E3 ligase activity. However, the MDM2RING△C construct is less active. Full length E2 enzyme UbcH5a was also purified. Various biophysical techniques were used to study its interaction with MDM2 as well as with potential small molecule inhibitors as in principle, small molecules which disrupt the interaction between MDM2 and UbcH5a, could prevent/promote ubiquitination of p53. The dimerisation of MDM2 is important for its E3 activity and the C8-binding site potentially provides a second druggable site. In this work, peptide 9, which has the same sequence as the C-terminus of MDMX (an MDM2 homologue) was found to inhibit MDM2 E3 activity. Various biological techniques including NMR, fluorescence anisotropy, and electrospray mass spectrometry were used to investigate the interaction between two inhibitory peptides and MDM2. A major part of project involved virtual screening (VS) to search for small molecules which can affect MDM2-dependent ubiquitination. Three potential targets were considered: (1) the C8-binding site of MDM2; (2) the UbcH5a-binding site of MDM2; and (3) the MDM2-binding site of UbcH5a. Several small molecules were identified using our virtual screening database-mining and docking programs that were shown to affect MDM2-dependent ubiquitination of p53. In terms of understanding the complex biochemical mechanism of MDM2 this work provides two interesting and functionally relevant observations: (i) the MDM2 RING△C construct is a dimer as this would not be expected form the existing studies, and has less E3 ligase activity than MDM2RING; (ii) small molecules that bind MDM2 on the E2 binding site enhanced E3 ligase activity. One model to explain these observations is that binding of small molecule activators family to the RING induces a change in the conformation of the Cterminal tail residues which may enhance E2 binding.

Despite there being pragmatic national guidelines for assigning risk to women with a family history of breast cancer, the evidence base is still sparse. There are three major questions: First, how can an assignment of "low" risk be made most efficiently? Second, what are the actual outcomes for higher-risk women enrolled in special surveillance programmes? Third, what are the costs and benefits of current management of members of breast cancer families? My thesis reviews the evolution of clinical services for familial breast cancer and the existing literature in the field. I describe the gathering of information from the service records of the Tayside Breast Cancer Family History Clinic and from specific research exercises that involved collaboration with other centres in the UK and abroad. My findings are as follows: 1. Histories provided by the families are not sufficient to assign risk accurately. They must be extended and verified from other records by clinical geneticists. Women assigned a low risk can be informed by post, but some may require further support. The 2004 NICE guidelines for assigning risk are fairly accurate, but may under-estimate it for some women aged 45--55 years. 2. Annual screening of young women at increased risk results in detection of most cancers at a curable stage. Women who carry BRCA1 mutations fare less well, even when tumours are detected at an apparently early stage. 3. Costs of accurate risk assessment are outweighed by savings from the better targeting of surveillance programmes. Early cancer detection in young women enrolled in these programmes achieves a substantial gain in life expectancy at a cost of £3,700 per quality adjusted life year (QALY). Prophylactic surgery for carriers of BRCA1 mutations is highly cost-effective. The thesis concludes with a discussion as to how these findings might be extended and clinical practice improved in the future.