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.

published_or_final_version / Nursing Studies / Doctoral / Doctor of Philosophy

The purpose of this study was to determine the factors related to young black men’s intention to screen for prostate cancer as well as their engagement in prostate cancer risk-reduction behaviors. The study tested the significance of the constructs – age, attitude (direct and indirect), social influence, comfortability, cues to action, health screening experiences and knowledge – in predicting young black men’s intention to screen for prostate cancer; as well as the significance of the constructs – age, cues to action, exercise and knowledge – in predicting engagement in prostate cancer risk-reduction behaviors. Demographic/personal factors were also explored in related to the model predictors. Web-based and paper-pencil surveys were administered to 279 black men aged between 18 – 40 years from the Austin area. Three focus groups were conducted to collect information regarding young black men’s behavioral beliefs toward prostate cancer screening as well as their comfortability with prostate examinations. The number of usable surveys was 267. Using direct and indirect measures, the combination of attitude, social influence, comfortability (indirect model), and knowledge explained 41.0 and 43.0 percent of the variance in intention to screen for prostate cancer, respectively; with social influence being the strongest predictor ([Beta]=0.41; p <0.01 for the direct model and [Beta]=0.47 for the indirect model). For the model with prostate cancer risk-reduction as the outcome variable, the model accounted for 10.0 percent of the variance in behavior with only knowledge ([Beta]=0.19; p=0.03) as significant predictor. Interventions that address young black men’s attitude, social influence, comfortability, and knowledge may be necessary to increase young men’s intention to screen for prostate cancer when it is recommended by a physician. Additionally, factors surrounding exercise and knowledge may be important in increasing young men’s engagement in prostate cancer risk-reduction behaviors. Future studies using intention as a predictor of young men’s behavior are needed to assess the influence of intention on prostate cancer screening. / text

Die Identifikation von Personen mit hoher Expertise in einem spezifischen Gebiet ist in vielen Bereichen des Wirtschaftslebens von zentraler Bedeutung. Um beispielsweise Marktinformationen einzuholen, wird in Unternehmen normalerweise die Marktforschung bemüht. Deren Instrumentarium ist aus guten Gründen stark am Ideal der Repräsentativität ausgerichtet. Was aber, wenn das Ziel nicht ist, allgemeingültige Aussagen zu gewinnen, sondern seltene, besonders wertvolle Informationen gesucht werden, wie etwa Ideen für neue Produkte? Methoden wird hierzu in der Marktforschungsliteratur kaum Aufmerksamkeit gewidmet. Wissen dazu ist jedoch von hoher Bedeutung. Unternehmen sind einem zunehmenden Innovationsdruck ausgesetzt und daher wird auch die Frage immer wichtiger, wie man zu Ideen für viel versprechende Innovationen gelangen kann. Eine besondere Bedeutung als Träger solcher Ideen haben die so genannten "Lead User" - Produktnutzer, die ihrer Zeit voraus sind. Wie findet man sie? Diese Frage ist keineswegs trivial. Ziel der vorliegenden Arbeit ist es daher, zentrale Fragen bei der Vewendung der Pyramiding-Methode für die Suche nach Personen mit besonderen Merkmalen theoretisch und empirisch zu beantworten. Die beiden Forschungsfragen dieser Dissertation lauten:(1)Wie effizient ist der Suchprozess im Rahmen der Pyramiding-Methode (im Vergleich zur Screening-Methode)? (2)Welche Faktoren beeinflussen die Effizienz eines Verweises der Auskunftsperson bei der Suche mit der Pyramiding-Methode? Ein aufwändiges Feldexperiment ermöglicht die Analyse von 292 Pyramiding-Verweisketten. Das Ergebnis: Im Rahmen der vorliegenden empirischen Untersuchung sind nur zwischen rund 8% und 70% des Aufwands (Anzahl der befragten Personen) für die Suche mittels der Screening-Methode notwendig, um die gesuchte Person zu identifizieren. Als zentrale Einflussfaktoren auf die Effizienz eines Pyramiding-Verweises einer Auskunftsperson konnten in dieser empirischen Untersuchung unterschiedliche Variablen identifiziert und empirisch bestätigt werden: sowohl personenbezogene als auch populationsbezogene Variablen zeigen einen signifikanten Einfluss. (Autorenref.)

Problem: There is a lack of knowledge about women who are screened for breast cancer, have an abnormal finding on mammogram, and then do not return in a timely manner for diagnostics and treatment. Lack of follow- up likely constitutes delayed treatment and poorer outcomes. Delays may result in later entry into the health system with advanced disease, more extensive and expensive care, burdening resources. Late stage breast cancer likely results in poorer health outcomes or early death.Purpose and Aims: The purpose of this research is to describe contextual characteristics at the health delivery level as well as individual characteristics of women with abnormal mammography, and their association with use of follow- up health services. Particularly, this research examines the differences between women who are early and late responders after an abnormal mammogram.Population: The participants were a convenience sample of 380 women who participated in mobile breast cancer screening. A subset of women with inconclusive or abnormal mammogram findings was the focus of analysis.Methods: This research utilized a descriptive design with quantitative data collection through participant survey at mobile mammogram screening events in multiple urban and rural Arizona sites. Participants requiring further health care were followed by chart review. Analysis of correlations with the outcome variable: time to first follow- up appointment for recommended health care in women with abnormal mammograms was conducted.Findings: Data indicated the time to the first follow- up appointment ranged from 1- 110 days with follow- up for 77.4% of participants within 60 days, 6.5% within 60-90 days, and 16.1% without follow- up after 90 days. Significant relationships between contextual and individual characteristics and follow- up were found. Categories included organizational health system characteristics of geographic location, clinical breast exam and shared case management; individual characteristics of beliefs including value of health care; finance including out of pocket costs and perceptions of financial assistance; perceived needs including breast symptoms; and satisfaction with the last health visit and mammograms in general.Implications: Recognition of barriers to follow- up after breast cancer screening is important for development of interventions to improve outcomes and has implications for screening and treatment management programs, community health centers and private practice. Health disparity related to screening without adequate options for access to health care is ethically untenable. Nurses are well positioned to reduce barriers to health care.

The colony formation assay (CFA) is the gold standard for measuring cytotoxic effects on cells. To increase efficiency, the CFA was converted to a 96-well format using an automated colony counting algorithm. The 96-well CFA was validated using ionizing radiation (IR) on the FaDu and A549 cancer cell lines. Its ability to evaluate combination therapies was investigated using cisplatin and IR. The 96-well CFA was transferred to a robotic platform for evaluation as a high-throughput screen (HTS) readout for the discovery of novel anti-cancer compounds, and radiosensitizers. Screening yielded eight putative anti-cancer hits, and five putative radiosensitizing hits. Secondary screening confirmed 6/8 anti-cancer compounds, and 0/5 radiosensitizing compounds. Thus, the 96-well CFA can be adopted as an alternative assay to the 6-well CFA in the evaluation of cytotoxicity in vitro, providing a possible readout to be utilized in HTS for discovering anti-cancer compounds, but with limited applicability in discovering radiosensitizers.

The aim of the study was to investigate how schools identify primary school children with early reading difficulties and what kind of early intervention special education teachers use to support these students. This study also investigates early interventions that special education teachers con­sider to be effective. Ten special education teachers were interviewed regarding early intervention. The result showed that the schools use several screenings from within the age group of 6-9 year olds. The special education teachers can at an early stage identify primary school children with difficulties in their reading and they use a range of different interventions. It is found in this study that an intense period of individual tuition is the intervention that most special education teachers think leads to good results for primary school children with difficulties in their early reading. That finding is also supported in previous research. It is clear that special education teachers and special education teachers need more time for early intervention. The teachers' opportunities for doing this should be reviewed.

Syftet med denna uppsats är att beskriva hur två skolor med likartade förutsättningar från samma skolområde, arbetat med resultaten av den obligatoriska kommunala screeningen i åk 2 vårterminen 2016. Studien avser också att klargöra hur kommunikationen angående screeningen mellan de olika nivåerna fungerar, till exempel mellan kommunens centrala tjänstemän och rektor, samt vidare ut till verksamheten. Avsikten är även att belysa hur den årliga screeningen påverkar specialpedagogers arbete, samt hur de olika skolornas arbete skiljer sig åt. Vi har undersökt detta ur ett sociokulturellt perspektiv och har använt intervjuer och dokumentstudier som datasamlingsmetod. Vi har intervjuat nio personer i olika positioner inom kommunen för att få en så heltäckande bild av processen som möjligt. Även ett stort antal informella samtal med personer som har beröring med ämnet har skett och har fördjupat förståelsen av det som undersökts. De dokument som studerats har varit instruktioner till genomförandet av screeningen (från kommunens intranät) samt statistik från Skolverkets databas SIRIS. Resultatet visade att arbetet med screeningen skiljer sig åt mellan de två skolorna, framför allt i efterarbetet med resultaten och påverkan på specialpedagogens arbete. Den ena skolan genomför ett arbete med träningstillfällen som sedan avslutas med ett nytt testtillfälle. Allt detta sker på uppdrag av rektor. På den andra skolan genomför ansvarig pedagog ett självständigt arbete med fokus på samarbete eftersom hen anser att det är vad gruppen främst behöver. Dessa skillnader i arbetssätt menar vi hör samman med olikheter i kontinuitet i personalsituationen samt ledningens inställning till resultat. Studien har också visat på brister i kommunikationen mellan de olika organisationsnivåerna och en tveksamhet till materialets utformning. Detta har vi tolkat som att screeningen som fenomen är ifrågasatt, utifrån aktuell läroplans synsätt och modern forskning kring bedömning.