Class of 2009 Abstract / OBJECTIVES: The purpose of this study was to determine the perceptions of the general public in Arizona regarding random drug screening and obtaining and maintaining health care benefits. METHODS: Individuals entering and leaving a grocery store in Chandler, Arizona on one day were invited to voluntarily participate in the study by reviewing a subject disclosure form and completing an 11 item questionnaire. The Likert scale varied from 1 = Strongly Disagree to 6 = Strongly Agree. Individuals were eligible to participate if they were adults and living in Arizona. RESULTS: One hundred adults participated in the study. The results of the study showed general agreement for passing a drug test for illegal substances before being hired (5.0 ± 1.5, mean ± SD), and if testing positive for illegal drugs, being allowed to enroll in a drug treatment program to keep their health care benefits (4.8 ±1.6). Respondents agreed that employers should require random drug testing to maintain employment (4.4 ± 1.8). However, respondents neither agreed or disagreed that people should be required to pass a drug test to be eligible to receive health care benefits (3.9 ± 2.0) or that they should be required to pass a periodic drug test to maintain their health care benefits (3.7 ± 2.0). CONSLUSIONS: Respondents agreed with drug testing to obtain and maintain employment; however, respondents neither agreed or disagreed with the concept of obtaining or maintaining health care benefits with passing a drug test for illegal substances.

Biophysical characterisation of protein – ligand interactions can provide vital information to dissect complex biochemical binding mechanisms. C3d has been shown to interact with a number of different protein ligands, namely CD21 SCR1-2, S. aureus Efb-C, S. aureus Ehp, S. aureus Sbi and complement regulatory protein factor H. Although much is known about the relationship of C3d and CD21 in the induction of humoral immunity, the structural aspects of this interaction remained controversial until very recently. The aim of this thesis was to gain a detailed understanding of the C3d/CD21 SCR1-2 interaction using different biophysical methods and to identify potential low molecular weight inhibitors of the interaction. A crystal structure of the C3d/CD21 complex solved by Szakonyi et al. in 2001 indicated the C3d binding site on CD21 was in the SCR2 domain. It did not agree with mutagenesis studies and recent NMR titration experiments show that only residues from the SCR1 domain of CD21 appear to mediate binding under physiologically relevant ionic strength. In the current work, NMR was employed to monitor ligand binding to C3d and to provide residue specific information that reflects a physiologically relevant binding mode to complement the crystallographic model solved by van den Elsen and Isenman in 2011. Microcalorimetric analysis on the site-directed mutagenesis studies also supported a model of hydrophobically- and electrostatically-driven protein-protein interaction for C3d and CD21 SCR1-2. Complement C3d forms a non-specific thioester linkage with antigen, which then binds to CD21 SCR1-2 and coligates with membrane immunoglobulin of the B cell receptor. While the interactions triggers B cell activation and hence the production of antibody under normal circumstances, it has been demonstrated that the interactions also lead to undue B cell activation and auto-antibody production. There is a well established collagen-induced arthritis (CIA) mouse model to support the significance of C3d and CD21 in disease susceptibility. To this end, a high-throughput SPR-based screening platform was set up to screen a fragment library against C3d, so as to identify low molecular weight compounds that could serve as a starting point for drug development programme. Unfortunately, the work did not yield C3d-binding inhibitors and future work could include screening large fragment libraries that are designed to target protein-protein interfaces.

Entamoeba histolytica is an invasive intestinal amoeba morphologically indistinguishable from Entamoeba dispar, a closely related organism that is not able to invade tissues. Differential diagnosis under conventional microscopy is therefore impossible. Reliable tools are needed for clinical diagnosis and for the reevaluation of the prevalence of infection with the invasive species worldwide. Monoclonal Antibody (MAb) 20/7D exhibited promising results when ascites was used to identify cultured isolates of E. histolytica by indirect immunofluorescence assays (IFA), and when used in a Faecal Antigen Capture Enzyme-Linked Immunosorbent Assay (FAC-ELISA) for laboratory diagnosis of amoebic dysentery and colitis. Here, further development of the assay was attempted to increase its sensitivity and use it for detection of asymptomatic carriers of E. histolytica. After purification and subsequent titration in ELISA, MAb 20/7D did not adequately distinguish between crude lysates of cultured E. histolytica and E. dispar trophozoites. MAb 20/7D reacted with a similar soluble antigen of E. histolytica and E. dispar, which confirmed previous observations in western blot analysis under non-reducing conditions. Therefore, the use of the FAC-ELISA for diagnosis in areas where E. dispar is endemic is probably not viable. A nucleic acid detection method was therefore developed. Polymerase Chain Reaction was used to amplify specific tandem sequences in the 24.5 Kb episome of E. histolytica and E. dispar. After PCR, internal sequences of digoxigenin-labelled PCR products were hybridized to specific biotin-labelled probes for E. histolytica or E. dispar and detected in Enzyme- Linked Immunosorbent Assay (ELISA). The Polymerase Chain Reaction Solution- Hybridisation Immunosorbent Assay (PCR-SHELA) was evaluated on samples from travellers returning from the tropics to Barcelona. The sensitivity and specificity were 98% and 100% respectively, when results were compared with microscopy. PCR-SHELA was also useful for differential diagnosis in cases of amoebic abscesses, amoebic dysentery, salmonellosis, ulcerative colitis and in asymptomatic carriage of E. histolytica. The new test gives sensitive and specific differentiation between E. histolytica and E. dispar in clinical specimens and it has proved successful in screening faecal samples in endemic areas for epidemiological purposes.

Magister Artium (Psychology) - MA(Psych) / The diagnosis of adult ADHD is a complex process that requires information from different sources. Instruments are used to screen or diagnose adult ADHD. The aim of the study was to identify instruments measuring adult ADHD from good quality research. This systematic review was executed following the recommended PRISMA steps. A comprehensive search was conducted across identified databases. The SFS scoring system was used to critically appraise for methodological rigour and coherence. Meta-synthesis was used to summarize extracted data from 26 articles included in the final summation. Ethics clearance was issued by the UWC Senate Research Committee. Sixteen instruments measuring adult ADHD were identified. Screening instruments measure core symptoms whereas diagnostic instruments assess all criteria. Fourteen instruments were based on DSM-IV criteria and four were based on DSM-V criteria for adult ADHD including rival explanations for the symptoms. The lack of adoption of DSM-V criteria remains a concern given criticism against DSV-IV criteria for adult ADHD. Overall instruments presented acceptable psychometric properties. However, the performance of the instruments was study dependent. A cautionary note is that these indices must be interpreted carefully. Further research must explore the reasons underlying the lack of adoption of DSM-V criteria in research, and the lack of revision of instruments measuring adult ADHD.

Bekannte Methoden zur Optimierung von FAEP-basierenden Hörscreeningmethoden beruhen darauf, bessere Signal-Rausch-Verhältnisse durch möglichst große Amplituden der evozeirten Potentiale zu schaffen. Große Potentiale können schnell und sicher im Mittelungsverfahren detektiert werden. Sie führen so zu einer Steigerung der Sensitivität und Spezifität und zu einer Verringerung der benötigten Messzeit bei den AEP-basierenden Screeningverfahren. Zu den Ansätzen zur Optimierung der Reizmethodik zählz die hohe Reizratentechnik, die den Nachweis der Antworten über die Bildung von Überlappungspotentialen erlaubt. Die Zeitgang-BERA ermöglicht unter Bildung solcher auditorischer steady-state Potentiale (ASSR) die Überprüfung mehrerer Pegelstufen.Für ein FAEP-basierendes Hörscreening werden Reize mit breitem Frequenzspektrum verwendet, die große Antwortamplituden hervorrufen und große Teile der Kochlea, den Hörnerv und REgionen des Hirnstammes überprüfen. Der WüChirp2005-Reiz ist ein breitbandiger Reiz, bei dem im Gegensatz zum üblich verwendeten Klick die Laufzeitverzögerung der Wanderwellen, die zu einer nicht-synchronen Summenantwort führt, ausgeglichen wurde. Vorliegende Arbeit befasst sich mit dem Einsatz solcher optimierter Reize in Stufenreizen für die Zeitgang-BERA. ZUm direkten Vergleich erfolgen Messdurchgänge mit Klick-Stufenreizen. Untersucht wurden gesunde Neugeborene im Rahmen des universellen Neugeborenen-Hörscreenings der Universitäts-HNO-Klinik Würzburg unter Verwendung des Screeninggerätes MB 11 (MAICO Diagnostic GmbH, Berlin).Einer ersten Untersuchungsgruppe wurden Stufenreize bestehend aus vier aufeinanderfolgenden Reizen ansteigender Pegelwerte (30,40,50,60 dBnHL; Stufenreize I). Die Stufenreize der zweiten Gruppe entsprechen in ihrem Aufbau dem klassischen Zeitgangreiz nach Finkenzeller mit sechs Reizen (10,20,30,40,50,60dBnHL; Stufenreize II).Daraus resultieren unterschiedliche zeitliche Abstände zwischen der Einzelreizen (Stufenreize I: 10,81ms; Stufenreize II: 5ms). Bei den Einzelreizen handelt es sich in einer ersten Messungun normale Klicks (Klick-Stufenreize I bzw.II), in einer Zweiten werden WüChirp2005-Reize (WüChirp2005-Stufenreize I bzw. II) verwendet. Die Laufzeitkorrektur erfolgte nach dem linearen Kochlea-Modell von de Boer und der Frequenz-Transformation nach Greenwood. Der Spektralgehalt beider Einzelreizformen ist identisch (135 Hz bis 8kHz). Bei den Stufenreizen I führte der Einsatz von WüChirp2005-Reizen zu signifikant größeren Wellen V gegenüber Klick-Reizen. Bei der im Neugeborenen-Hörscreening relevanten Pegelstufe 40 dBnHL waren bei 20 der 76 durchgeführten Messungen ausschließlich unter Darbietung der neuen Reize evozierte Potentiale nachzuweisen. Es konnte so u.a. gezeigt werden, dass WÜChirp2005-Reize die Spezifität der Screeningmethode erhöhen kann. Nach der Auswertung und Diskussion der Amplituden der evozierten Potentiale werden die Latenzen der evozierten Wellen V untersucht. / Methods for optimizing auditory brainstem responses (ABR)-based hearing-screeningmethods are based on optimizing the signal-to-noise-ratio by maximize the evoked potential.Large answers can be detected fast and definitely by averaving procedures and lead to increased specifity and sensitivity and also reduce the duration of examination. An appendage to optimize the stimulus methodology is the high-rate-technique. The "Zeitgang"-BERA leads so to auditory steady-state-potentials (ASSP)and makes a chek of several volume levels in only one measurement possible. For a ABR-based hearing-screening are stimuli with large frequency spectrum used wich evoke large amplitudes and chek wide parts of the cochlea, the hearing nerv and also regions of the brainstem. The WüChirp2005-stimulus is a large frequency spectrum signal,in which the delay of the travelling wave in the inner ear is compensated. In contrast to the normal click-stimulus this optimized chirp-signal should lead to larger answer potentials, because they appear much higher synchronisied. This study deals with a combination of WüChirp2005-signals in stimuli for the Zeitgang-BERA. For direct comparison there were accomplished measurements with click-step-stimuli.Measured were healthy newborns which took part in the universal hearing-screening programm in Würzburg. Used was the screening instrument MB11 (MAICO Diagnostics, Berlin). The step-stimuli of a first group are made of four chained stimuli with increasing volume levels (30,40,50,60dBnHL; step-stimuli I). The step-stimuli of a second group accord to the classical Time-step-stimulus (Zeitgang)-signal of Finkenzeller with six signals (10,20,30,40,50,60dBnHL).That results in different time intervals between the several signals (step-stimulus I: 10,81ms; step-stimuli II: 5ms). The step-stimuli are composed in a first measurement of clicks (click-step-stimuli I and II), each newborn was then measured with WüChirp2005-step-stimuli (I and II). The delay of the traveling wave was corrected by cochlea models from de Boer and Greenwood. The frequency spectrum of both click and chirps were identical (135 Hz-8kHz). The results of the step-stimuli of the first group showed significant larger evoked waves V compared to the click-step-stimuli. At 40 dBnHL, which is relevant for hearing screening, answers were in 20 of 76 measurements only after presentation the new, delay-corrected signals were verified, but not after presentation click-step-stimuli. It was able to show that WüChirp2005-signals are able to increase the specitifity of the ABR-screening method. After discussion the amplitudes of the evoked potentials, the study includes also a discussion of the wave V-latencies.

Die vorliegende Arbeit befasst sich mit der Suche nach neuen Leitstrukturen im Bereich der 4-Chinolone mit dem Verfahren der Random Chemistry. Die zugrunde liegende Idee hinter diesem Verfahren besteht darin, neue, potentiell wirksamere Strukturen durch die zufällige Erzeugung einer Substanzbibliothek zu erhalten. Die Substanzbibliothek entsteht dabei durch die Kombination bzw. Reaktion einer mäßig aktiven Ausgangsverbindung mit einem Initiator, der eine Zufallsreaktion in Gang setzt. Vorangegangene Untersuchungen dazu belegten, dass bei der Nutzung von γ-Strahlen als auslösende Spezies vornehmlich durch Radiolyse des Lösungsmittels erzeugte Radikale für die Bildung neuartiger Substanzen verantwortlich waren. Diese als Initiator dienenden Radikale können auch mit herkömmlichen Radikalstartern wie dem Fentons Reagenz erzeugt werden. Fentons Reagenz erweist sich dabei als deutlich kostengünstigere und einfacher zu handhabende Alternative, die ohne jeglichen technischen Aufwand genutzt werden kann. Durch die Reaktion von Wasserstoffperoxid und Eisen(II)-Ionen entstehen Hydroxylradikale, die als hochreaktive Spezies durch die Addition an Doppelbindungen oder durch die Abstraktion von Wasserstoffradikalen und die dadurch ausgelösten Sekundärreaktionen eine Vielzahl neuer Verbindungen erzeugen. Zur Leitstruktursuche wurden als Ausgangssubstanzen zum einen ein Vertreter der 6-Fluor-7-(piperazin-1-yl)-chinolone (1) und zum anderen das 4-Chinolon-Grundgerüst (2) gewählt, um sowohl den Einfluss möglicher Strukturvariationen am Chinolongrundkörper als auch an möglichen Substituenten zu ermitteln. Aus den nach der Umsetzung mit Fentons Reagenz entstandenen Substanzbibliotheken konnten zunächst Fraktionen erhalten werden, die bei der Testung gegen Trypanosoma brucei brucei Aktivitäten mit geringeren IC50-Werten als die Ausgangschinolone zeigten. Die Strukturen der mittels HPLC isolierten Reinsubstanzen wurden aufgeklärt und ihre biologische Wirkung ermittelt. Auf diese Weise konnten ein Chinolonderivat mit N-(2-Aminoethyl)formamid-Rest in Position 7 (Frk1-2c) sowie ein Derivat mit (2-((Methyl-amino)methoxy)ethyl)amino-Rest in Position 7 und einer Hydroxylgruppe in Position 6 (Frk1-1c) identifiziert werden, die eine antitrypanosomale Aktivität mit einem IC50-Wert von 20.69 μM bzw. 17.29 μM aufweisen. Neusynthetisierte Chinolone mit einem amidofunktionalisiertem Piperazinring in Position 7 zeigten eine noch bessere Aktivität gegen Trypanosoma brucei brucei, welche durch Amidierung der Carbonsäure in 3-Stellung noch weiter gesteigert werden konnte. Die Suche nach neuen Leitstrukturen mit Hilfe von Fentons Reagenz offenbarte, dass ein Großteil der in den Substanzbibliotheken gebildeten neuen Strukturen literaturbekannten Metaboliten der Fluorchinolone ähnelt. Da annähernd 50% der möglichen Wirkstoffkandidaten an einer zu geringen Bioverfügbarkeit oder aufgrund ihrer toxischen Metabolite scheitern, ist es sinnvoll bereits in einem möglichst frühen Forschungsstadium auch Parameter wie Resorption, Distribution, Metabolismus, Elimination und Toxikologie (ADMET-Parameter) einer Substanz zu berücksichtigen bzw. zu ermitteln. Deshalb wurde in einem weiteren Teil dieser Arbeit untersucht, ob Fentons Reagenz zur Erzeugung des metabolischen Profils neuer biologisch aktiver Substanzen dienen kann, um damit frühzeitig auch auf mögliche Toxizitäten der Metabolite einer Substanz schließen zu können. Dazu wurden die drei bekannten Antiinfektiva, Cinoxacin, Ciprofloxacin und Linezolid mit Fentons Reagenz umgesetzt und die entstandenen Substanzbibliotheken nach literaturbekannten Metaboliten der jeweiligen Ausgangssubstanz gescreent. Bei einer ausreichenden Löslichkeit der Ausgangssubstanz konnten so vor allem die Metabolite erzeugt werden, die durch Hydroxylierung, Oxidation oder Hydrolyse auch bei der Verstoffwechslung durch Cytochrom-P450 erhalten werden. Da der Nachweis der Bildung literaturbekannter Metabolite durch Fentons Reagenz gelang, ist es nun möglich, einen Teil der potenziellen Metabolite neuer biologisch aktiver Substanzen bereits im Vorfeld, ohne die Nutzung von humanem Lebergewebe zu identifizieren. / The present work is dealing with the search for new lead structures in the field of 4-quinolones via random chemistry. The underlying concept of this approach was to generate compound libraries by combining an active starting compound with an initiator. Earlier experiments using γ-radiation as initializing species revealed that most of the new generated compounds were products of radical chemistry, built by the radiolysis of the solvent. Thus, the idea arose to replace the gainless γ-irradiation by an alternative radical starter such as Fentons reagent which is cheaper and easier-to-handle. Comparing the application of Fentons reagent with the results of the γ-irradiation revealed that similar compound libraries could be achieved. The reaction of hydrogen peroxide with ferrous ions forms highly reactive hydroxyl radicals which are themselves able to generate new compounds by the addition to double bonds or the abstraction of hydrogen radicals. Numerous randomized chain reactions take place and create a compound library of great diversity. In order to find new lead structures two different starting quinolones, 1 and 2, were chosen, in order to examin the influence of structural variation both at the quinolone framework and at the substituents, like the piperazine ring in position 7. A subsequent bioassay-guided-HPLC-fractionation as a deconvolution strategy of the gained product library revealed fractions with higher activity against Trypanosoma brucei brucei and therefore lower IC50 values than the starting compounds. The structures of the purified compounds were elucidated and their biological activity examined. Thus, a new quinolone derivative was identified bearing an N-(2-aminoethyl)formamide residue in position 7 (Frk1-2c) as well as a derivative with a (2-((methylamino)methoxy)ethyl)-amino residue in position 7 beside a hydroxyl group in position 6 (Frk1-1c), possessing an antitrypanosomal activity with an IC50 value of 20.69 μM and 17.29 μM, respectively. Newly synthesized quinolones with an amido-functionalized piperazine ring in position 7 showed an even better activity against Trypanosoma brucei brucei, which could be further enhanced by an amidation of the carbocyclic acid in position 3. Within the scope of searching for new lead structures with Fentons reagent in the field of anti-infectives it could be ascertained that a great part of the produced substances resemble known metabolites of fluoroquinolones. This is due to the fact, that the underlying chemistry of Fenton's one-electron oxidation is comparable to that of cytochrome-P450, which is the main metabolism enzyme. As almost 50% of possible drug candidates fail due to their low bioavailability or their toxic metabolites, it is necessary to take parameters like absorption, distribution, metabolism, elimination and toxicology (ADMET parameter) into consideration as early as possible during the search for new drugs. Contemplating this fact, a further aim of this work was to explore, whether the conversion with Fentons reagent may serve as a screening tool for the metabolic profile of new biologically active compounds. For this purpose, three structurally different antibiotics, ciprofloxacin, linezolid and cinoxacin were chosen for treatment with Fentons reagent. Subsequently the generated compound libraries were screened for the occurrence of known metabolites of the starting substances. If an adequate solubility of the starting compounds was guaranteed, especially metabolites could be generated that evolve from hydroxylation, oxidation or hydrolyses by the cytochrome enzyme complex. As the generation of known metabolites with Fentons reagent could be verified, it is now feasible to create and identify possible metabolites of new bioactive compounds without using human liver tissue.

Im Rahmen dieser Arbeit wurden verschiedene Inhibitoren der Acetylcholinesterase (AChE) untersucht, die als potentielle Substanzen zur Behandlung von Morbus Alzheimer eingesetzt werden können. Die Hemmwirkung der einzelnen Substanzen wurde mittels Ellman-Test überprüft. Gemeinsames Strukturmerkmal der Substanzklasse, von der im ersten Teil der Arbeit ausgegangen wurde, war das Grundgerüst des AChE-Reaktivators TMB4 [1,1´-Trimethylen-bis(4-Formyl-Pyridiniumbromid)-Dioxim]. Anhand der biologischen Daten konnte beobachtet werden, dass die Art der Substitution die inhibitorische Aktivität der Verbindungen wesentlich beeinflusst. Am wirksamsten von allen Bispyridinium-Derivaten zeigte sich das 2,6-chlorierte Derivat DUO3 (IC50 = 0.34 μM), gefolgt von monobenzyl-substituiertem UNO3, bismethylsubstituiertem TBM und unsubstituiertem TMB4. Weiterhin wurde der Bindungsmodus der DUO-Substanzen im aktiven Zentrum der AChE untersucht. Die Docking-Studien an Substanzen der DUO-Klasse zeigten ein einheitliches Bindungsmodel, welches folgende Wechselwirkungen be-inhaltet: π-π-„stacking“ zwischen dem Benzylring einer DUO-Substanz und dem Trp84 am Grunde der Bindetasche des Enzyms, face-to-face Wechselwirkung (π-π und Kation-π) zwischen dem Pyridiniumring und Trp334 oder Phe331 der aromatischen Furche. Bei 60% der gedockten Strukturen wurde eine face-to-face-Wechselwirkung an der anionischen peripheren Seite (PAS) der AChE-Tasche festgestellt. Weiterhin wurden neue optimierte Inhibitoren entwickelt. Die Bispyridinium-Struktur der DUO-Derivate wurde um den aus der Furche herausragenden Benzylring gekürzt. Als Leitstrukturen dienten die AChE-aktivsten Substanzen DUO3 (2,6-Cl-Derivat) und DUO12 (2-Cl-Derivat) sowie ein bisphthalimidomethyl-substituiertes TMB4-Derivat (WDUO). Die aktivste Verbindung der Pyridinium-Klasse war die Phthalimid-Phenyl-substituierte Substanz 3c (IC50 = 0.073 μM). Ihre inhibitorische Aktivität gegenüber AChE befand sich im Bereich der des Tacrins (IC50 = 0.044 μM). Sie zeigte eine sehr gute AChE-Selektivität; die BChE hemmte sie um den Faktor 34 schwächer. Im Rahmen der vorliegenden Arbeit wurde nicht nur das rationale Design angewandt, um zu viel versprechenden Kandidaten bezüglich klinischen Einsatzes zu gelangen. Auch das Verfahren der „Random Chemistry“ kam zum Einsatz, um neue und interessante Strukturen zu erzeugen, die eventuell bessere Eigenschaften als die Ausgangssubstanz besitzen. Der Grundgedanke dieses Verfahrens liegt in der Ausnutzung der durch gamma-Strahlen (60Co-Quelle) induzierten Radiolyse des Lösungsmittels, welches seine primären Produkte zur chemischen Reaktion mit dem in ihm gelösten Stoff zur Verfügung stellt. Aus den entstandenen Produkten wurden durch spezifische biologische Testung (Inhibition der AChE) positiv reagierende Komponenten herausselektiert. Die Proben wurden zuerst im Ganzen auf ihre Fähigkeit, die AChE zu hemmen, geprüft. Nach der bioaktivitätsgeleiteten Fraktionierung und Subfraktionierung mittels HPLC erwies sich die Tacrin/MeOH-Probe als die, mit dem interessantesten Aktivitätsprofil. Die Charakterisierung der entstandenen Verbindungen bezüglich ihrer Vielfältigkeit erfolgte mittels ESI-Massenspektrometrie und UV-Spektroskopie. Die Substanz mit höchster Hemmwirkung gegenüber AChE (Peak E der Tacrin/MeOH-Probe) wurde nach der Isolierung der Reinheitsprüfung und Strukturaufklärung mittels NMR-, FTIR- und (Tandem)-ESI-Massenspektrometrie zugeführt und auf ihre biologische Wirkung hin untersucht. / This study aimed at developing and biological screening of new potential acetylcholin-esterase (AChE) inhibitors. They represent the most widely used class of therapeutics for treatment of cognitive disorders such as Alzheimer´s Disease and neuromuscular diseases. The inhibitory potency toward acetylcholinesterase was evaluated by means of the spectrophotometric Ellman test. At first, the series of AChE inhibitors of bispyridinium type derived from TMB4 [1,1´-Trimethylene-bis-(4-formyl-pyridinium bromide) dioxime] was tested against AChE. In essence, the substitution pattern influenced the inhibitory potency against the enzyme. The most potent inhibitor of the bispyridinium-class was the 2,6-halogenated compound DUO3 (IC50 = 0.34 μM), followed by monobenzyl substituted (UNO3), bismethyl substituted (TBM), and unsubstituted derivatives of TMB4. This experimental finding could be explained by docking studies whose goal was to thoroughly explore possible binding modi of this type compounds. One major observation was that almost all of the compounds docked display a general binding mode; they are a kind of ditopic inhibitors which bind to both the catalytic and the peripheral sites of the enzyme. The interactions found after docking include π-π-stacking with amino acid residues of the anionic substrate binding site (Trp84), cation-π contacts with Phe331 and Tyr334 of aromatic gorge and the peripheral anionic binding site (Trp279). This type of interactions is already known for other potent AChE-inhibitor donepezil. Moreover, all compounds were potentially able to bind inside the active side gorge, although, not the whole molecule was able to interact with amino acid residues of the enzyme. This “size problem” of the DUO-ligands may be one reason for their reduced activity as compared to other potent AChE-inhibitors e.g. donepezil, tacrine. Using the hypothesis of the study on bisbenzylethers of bispyridinium compounds which were too long to tightly fit into gorge of AChE, shorter compounds of pyridinium type were developed. This new series of compounds was systematically shortened from 2,6-dichloro-benzyl, 2-chlorobenzyl and phthalimidomethyl ditopic compounds and substituted with several moieties on contralateral end of molecule. The most potent pyridinium compound was 3c (IC50 = 0.073 μM), the activity of which was in the same range of inhibition concentration as tacrine (IC50 = 0.044 μM). In addition, the affinity of 3c toward BChE was rather low (IC50 = 2.49 μM), indicating a lower degree of side effects. In addition to the rational drug design new potent AChE-inhibitors were generated through serendipitous but reproducible approach – via random chemistry. In order to achieve this objective, compound libraries were generated by using gamma-irradiation as an initiator of random free radical recombinations in aqueous or alcohol solution of starting compounds. The bioassay-guided-HPLC-fractionation as a deconvolution strategy of the gained product library was employed. Where the activity in a single first round fraction was observed further steps of fractionation were warranted till a potent hit was observed. The biological screening of first round fractions revealed the tacrine/methanol mixture to be the one with most active fractions toward AChE. Hence, further research efforts were focused on separation, deconvolution, isolation and characterization of biologically active principles. HPLC/ESI-MS (high performance liquid chromatography-electrospray ionisation mass spectrometry), NMR and UV spectroscopy were used to determine the origin and character of generated compounds, first of all that of active ones.

PhD, Faculty of Health Sciences, University of the Witwatersrand / HIV/AIDS continues to be one of the greatest health challenges which South Africa faces. The epidemic in children is closely linked to that in women, the prevalence of which continues to grow according to antenatal statistics from the South African Department of Health (DOH). HIV is known to invade the central nervous system at the time of infection, and causes widespead damage. In children, this leads to a well-researched condition known as HIV encephalopathy, which affects all areas of neurodevelopment. The effects of timely initiation of antiretroviral therapy on alleviating the impact of encephalopathy have been well described. Neurodevelopmental delay is a stage four disease indicator according to the World Health Organisation (WHO), and therefore is a criterion for initiation of Highly Active Antiretroviral Therapy (HAART). HAART is often only administered according to the virologic and immunologic status of a child, as standardised neurodevelopmental assessment tools are not widely available in South African clinics. When HAART initation is dependent on immunologic status, it is often too late to prevent encephalopahy. To date, the only means of prevention of this condition is early initation of HAART, which has not been widely available in South Africa. Stringent guidelines for the commencement of this therapy according to the WHO, and the South African Department of Health (DOH) have had to be followed, leading to late initiation of HAART, and widespread central nervous system encephalopathy. Studies which have been carried out in South African clinics have demonstrated the high prevalence of this condition. Once there is evidence of encephalopathy, children should be referred for assessments in all facets of development, and where necessary, for rehabilitation. A standardised developmental screening tool which is suitable for use in a developing country is therefore necessary in order to screen for neurodevelopmental delays to allow for further assessment and referral to rehabilitation services, as well as providing an additional assessment criterion for initiation of HAART. Paediatric HIV clinics in developing countries are understaffed, and children may be seen by junior staff or screened by nurses due to the high numbers of clinic attendees. This often results in neurodevelopment being inadequately assessed and children are therefore not referred for intervention services. A standardised screening tool, which The Development of a Screening Tool to Evaluate Infants who are HIV Positive could be administered by clinic staff in order to ensure correct and timely referral of children for further assessment and intervention is therefore necessary. This is of importance both locally and internationally where a screening tool, which has been developed specifically for this purpose, does not exist. The aim of this study was therefore to evaluate the agreement between the Bayley-III Screening Test and the Bayley Scales of Infant Development (3rd version) in a population of HIV positive infants in order to evaluate its appropriateness for use in South Africa. The Bayley Scales of Infant Development have long been considered the ‘gold standard’ in infant developmental assessment, which is why this tool was chosen to evaluate the Bayley-III Screening Test against. The developmental scores in each facet (cognitive, motor or language) were evaluated to determine which should be included in an assessment tool for this population. Further objectives for the study were to adapt the screening tool to the needs of the population, or to develop a new screening tool should the Bayley-III Screening Test not prove suitable for use in this population. In order to meet the aims and objectives, a cross-sectional study was conducted where 112 HIV positive infants between the ages of six and eighteen months were assessed using the Bayley-III Screening Test and the Bayley Scales of Infant Development (3rd version) (BSID III). The infants were stratified into four age groups namely 6-8 months, 9- 12 months, 13-16 months, and 17-18 months. Children were recruited from Harriet Shezi Children’s Clinic at Chris Hani Baragwanath Hospital in Soweto. The agreement between the Bayley-III Screening Test and the Bayley Scales of Infant Development (3rd version) was analysed using Kappa, for the overall group, and for each age group. Overall agreement between the tools was as follows: K=0.58 for the Cognitive facet, K=0.82 for the Expressive Communication facet, K=0.76 for the Receptive Communication facet, K=0.44 for the Fine Motor facet and K=0.57 for the Gross Motor facet. These values indicate that the Bayley-III Screening Test is therefore not acceptable for clinical use, as excellent agreement (k≥0.75) in all facets would be necessary for this purpose. A new screening tool therefore had to be developed. The infant’s developmental scores from the BSID III were analysed to determine which facets of development were most severely affected, and therefore which facets should be included in a new screening tool. Gross motor function was demonstrated to be the area which was most severely affected, followed by cognitive function. A gross motor screening tool would therefore be suitable for use in this population, as no equipment would be necessary. Gross motor development is the most universally similar aspect of development, which is not completely dependent on cultural or socioeconomic factors which often have an influence on language and cognitive development. Item selection from the BSID III was undertaken to determine which items should be included in a brief screening tool. In each of the four age groups, item selection occurred as follows: Two items which discriminated the At-Risk, from Emerging and Competent groups (less than 20% in the At-Risk group, and 100% in the other groups) were selected. Two items, which discriminated between children in the ‘Emerging’ and ‘Competent’ categories on the BSID III were selected (0-5% of children who were At-Risk obtained credit, 30-50% of the Emerging group obtained credit, and 100% of the Competent group obtained credit). Lastly, two items were selected which discriminated the Competent group from the other two groups (100% or as high as possible in the Competent group, and 0% in the other groups). The new gross motor screening tool was assembled using the selected items, scoring was allocated, and it was tested against the scores obtained on the Gross Motor facet of the BSID III for the initial 112 infants. Agreement between the tools was analysed using Kappa, and refinements were made according to the discrepancies. This was done three times, until the Kappa value revealed excellent agreement between the tools (k = 0.87). A panel of experts was then invited to examine the new gross motor screening tool, and to comment on it, and further adjustments were made accordingly. Preliminary concurrent validity testing of the new gross motor screening tool was then carried out against the Gross Motor facet of the BSID III on 60 children, who were recruited from the Harriet Shezi Children’s Clinic at Chris Hani Baragwanath Hospital in Soweto. Statistical analysis revealed that the agreement between the BSID III and the new screening tool was excellent (k=0.85). The diagnostic properties of the new gross motor screening tool were as follows: sensitivity 97.4%, specificity 85.7%, positive predictive value 92.7%, and negative predictive value 94.7%. These values indicate that the statistical properties of the tool are excellent, and the tool will not be predisposed to underreferrals or over-referrals. Preliminary reliability testing was carried out on 15 children for test-retest/intrarater reliability and 15 children for interrater reliability. Interrater, test-retest and intrarater reliability were excellent (r=1, r=0.98, r=0.98 respectively). Further testing of reliablity and validity should be undertaken in order to establish these properties, and standardisation should also be carried out on healthy children. Given the need for an assessment tool of this nature in South Africa and other developing countries, and the statistical properties thus far, the tool may be used clinically for the purposes for which it was developed.

Aim: To explore potential methods of increasing cervical screening coverage. Methods: Cervical screening defaulters in Dumfries and Galloway were identified in 2012, split into a control (N=64) and 7 intervention groups who were offered multiple screening options including self-collecting a vaginal sample at home. Self-samples were tested for high-risk human papillomavirus (HPV). A total of 3323 were invited to request a kit and 492 were sent a kit directly. Women who declined screening were asked to complete a questionnaire. Colposcopy referrals from defaulters were audited to identify changes over time. Defaulters attending the hospital smear clinic were questioned to ascertain barriers to cervical screening. Results: Among seven intervention groups the proportion responding varied between 32% (25%-38%) and 14% (11%-17%) compared to 6% among controls. One hundred and thirty women were HPV positive on self-sample, 8 of whom had CIN2+ diagnosed. A significantly higher number of defaulters were referred to colposcopy in June-December 2012 (n=51) than in the same period in 2011 (n=17; OR=3.8, 2.1-6.9). Defaulting was more commonly attributed to practical (112/155=72%) than attitudinal barriers (23/115=15%) (RR=4.9, 3.3-8.0). Conclusions: Practical barriers are often the cause of women not attending for cervical screening and offering more options, particularly the option of self- sampling at home, increases screening coverage.

Investigating the psychometric properties of a screening instrument for young children is necessary to ascertain its quality and accuracy. In light of the important role culture plays on human beliefs and parenting styles, a newly translated and adapted test needs to be studied. Evaluating outcomes on a translated version of a test may reveal significant information related to cultural specifications as well as the common nature of child development. The current study examined psychometric properties of the 48-month interval of the Ages and Stages Questionnaires: Social-Emotional Second Edition (ASQ:SE-2) and its Traditional Chinese version (ASQ:SE-TC), using item response theory (IRT). Participants in the U.S. included 3,005 young children/parents dyads; 1,455 dyads were collected to represent a Taiwanese sample. A two-dimensional Rasch Partial Credit Model (2D-RPCM), which was determined to present a better fit than a unidimensional Rasch Partial Credit Model, was used to examine the item fit, item difficulty, reliability, and item information curves to evaluate the psychometric properties on the ASQ:SE and ASQ:SE-TC. Further, differential item functioning was conducted to examine whether items were functioning differently in the two population groups. Lastly, the differences between the distributions of children’s latent traits on the continuum of social and emotional competencies for the U.S. and Taiwanese samples were investigated. Based on findings, the adequacy of psychometric properties is discussed, providing insight into the quality of particular items. Identified differences between the two populations are explored by reviewing literature regarding cultural comparisons of childrearing practices, parenting styles, and cultural beliefs. Future directions for research include examining the cultural equivalence between translated and original versions of other ASQ:SE-2 intervals.