Ziel der Untersuchungen war, zur Klärung der Ursachen einer während der letzten Jahrzehnte erhöhten Frequenz sowohl von PCOS als auch von IO beizutragen. Es war zu ermitteln, ob hormonelle Verschiebungen bei den Patienten nachweisbar und diese durch genetische und epigenetische Faktoren erklärbar sind. Ausgehend von dem Postulat, daß verminderte 21-OH- und 3beta-HSD-Aktivitäten als prädisponierende Faktoren von PCOS und IO angesehen werden, waren hormonanalytische Untersuchungen zur Ermittlung partieller 21-OH- bzw. 3beta-HSD-Defizienzen durchgeführt worden. Den eigenen Erfahrungen und Darstellungen der internationalen Literatur entsprechend befaßt sich ein Teil der Methodik mit der Entwicklung einer neuen, der üblichen 17alfa-OHP-Messung überlegenen Methode zur Ermittlung von 21-OH-Defizienzen durch 21-DOF-Bestimmung nach ACTH-Test im Blutplasma. Wir erhielten bei vier von 21 PCOS-Patientinnen und drei von acht Patienten mit IO erhöhte 21-DOF-, 21-DOF/F- bzw. 17alfa-OHP-Werte nach ACTH-Test, die auf partielle 21-OH-Defizienzen hinweisen. Zusätzlich wurden bei 12 PCOS-Patientinnen erhöhte basale DHEAS- oder DHEAS/F-Werte gefunden, die als Hinweise auf partielle 3beta-HSD-Defizienzen oder 17,20-Lyase-Hyperaktivität gedeutet wurden. In der Stichprobe der IO waren DHEAS oder DHEAS/F-Werte bei vier Patienten erhöht. Da bei vier der 12 Patientinnen mit PCOS und zwei von vier Patienten mit IO genetisch und endokrinologisch gleichzeitig eine partielle 21-OH-Defizienz nachgewiesen wurde, kann bei diesen Patienten eine partielle 3beta-HSD-Defizienz weitgehend ausgeschlossen werden. Es wurden molekulargenetische Untersuchungen für die 14 häufigsten Mutationen in CYP21 bei Cohorten mit AGS, PCOS und IO durchgeführt. Die Untersuchung der AGS-Patienten sollte dazu dienen, ein effizientes und schnelles System der Mutationssuche für diagnostische Zwecke zu etablieren. Es wurden die häufigsten, phänotypisch wirksamen Mutationen in CYP21 bei der Mehrzahl dieser Patientengruppe im homozygoten bzw. compound heterozygoten Zustand gefunden und eine deutliche Genotyp-Phänotyp-Korrelation festgestellt. Auch bei Patientinnen mit PCOS sowie bei IO, bei denen partielle 21-OH-Defizienzen nachweisbar waren, wurden Mutationen in CYP21 gefunden. Die hierbei heterozygot vorliegenden Mutationen waren dieselben, die homozygot oder compound heterozygot bei schweren Formen des AGS gefunden wurden. Es ergab sich eine Korrelation molekulargenetischer und hormonanalytischer Befunde bei AGS, PCOS sowie IO. Allerdings konnten bei der Mehrzahl der Fälle mit PCOS und mit IO weder Mutationen noch hormonelle Auffälligkeiten hinsichtlich partieller 21-OH-Defizienzen gefunden werden. Die jedoch bei vielen Patientinnen gefundenen erhöhten DHEAS- und DHEAS/F-Werte stimmen mit Untersuchungen überein, die parallel starke Zunahmen der Häufigkeit der Hemmung des Enzyms 3ß-HSD bzw. der Aktivierung der 17,20-Lyase bei PCOS-Patientinnen und der Prävalenz des PCOS selbst bei nach 1955 geborenen Frauen und von Spermatogenesestörungen bei nach 1960 geborenen Männern fanden. Die Ursache hierfür wird in der Beeinflussung der adrenalen und gonadalen Steroidhormonsynthese vor allem durch das Umweltteratogen DDT und seine Metaboliten gesehen. Weiterhin wurde der Umweltfaktor Streß diskutiert. Für die Ätiopathogenese der untersuchten Fertilitätsstörungen werden materno-fetale Mechanismen postuliert, worauf unsere sowohl molekulargenetischen als auch hormonanalytischen Befunde hinweisen. Insgesamt bestätigen die Ergebnisse unserer Arbeit die These, daß Leben auf der Interaktion von Genen und Umweltfaktoren beruht und daß Hormone dabei als Mediatoren wirken. In gen- oder umweltbedingten unphysiologischen Konzentrationen können sie während kritischer Entwicklungsphasen des neuroendokrinen Systems als Teratogene wirken und zu lebenslangen Reproduktionsstörungen führen. / This paper describes a mutational and hormonal screening in a cohort of 21 patients ultrasonically diagnosed with PCO. Our data show single heterozygous base pair CYP21 mutations in 4 patients. The four women with PCOS and CYP21 mutations also displayed clear signs of partial 21-hydroxylase deficiency through a significant rise in 21DOF or 17alfa-OHP plasma levels after ACTH stimulation. Azziz et al. have reported several heterozygous mutations in hyperandrogenic women with LO-CAH. Other studies report several heterozygous point mutations in hyperandrogenic woman who, however, were not examined for polycystic ovaries.The correlation between the hormone profiles and genetic screening results found with our patients underscores the latter s usefulness with PCOS patients. In contrast to the hormone profile, genetic screening is not influenced by external factors. The frequency of heterozygous CYP21 mutations is higher (19%) than in the normal population (5-8%), suggesting a link with PCOS in some cases. The ratio of LH/FSH was significantly raised in 43% of the cases. Most importantly, basal plasma DHEA-S levels and DHEA-S/F ratios were clearly increased, higher than the means +2SD in controls. This suggests a partial 3beta-hydroxysteroid dehydrogenase deficiency or 17,20 lyase hyperactivity. Other authors, however, were not able to find mutations in the corresponding genes. This could be explained by the fact that the DDT metabolite o,p DDD is a strong inhibitor of 3beta-HSD, and that DDT and its metabolites may be able to activate the 17,20 lyase, a cytochrome P450 enzyme. Furthermore, DDT has some oestrogen activity, and its perinatal administration can produce a PCOS-like syndrome in rats. Very significantly, there has not only been an approximately fourfold increased prevalence of PCO in women borne since 1955 in eastern Germany, following a massive prenatal exposure to DDT, but also a notable shift in the hormone profiles of those affected. A predominance of 3beta-HSD deficiencies and 17,20 lyase hyperactivity (70%) vs. 21-hydroxylase deficiency (23%) has emerged, in contrast with 21-hydroxylase deficiencies in 70% vs. 3beta-HSD deficiencies or 17,20 lyase hyperactivity in 14% for those born earlier than 1955. Similar results were obtained in this study for women with PCOS born since 1955, suggesting that the prenatal exposure of high amounts of DDT and its metabolites indeed appear to be responsible - at least in part - for the major increase in PCO and PCOS.

Enzymes are ubiquitous in biological systems. They catalyze chemical reactions and are involved in many biochemical processes. The enzyme of interest is Pseudomonas aeruginosa D-arginine dehydrogenase (PaDADH). This flavin-dependent enzyme is composed of approximately 375 amino acid residues and has a broad substrate specificity with D-amino acids. A water recognition motif, observed in roughly 1200 non-redundant protein data bank (PDB) structures, was revealed to be embedded near the active site of PaDADH. This motif coincides with the conformational changes of the enzyme’s gated mechanism. Molecular dynamics simulations were carried out to study the gated properties and structural characteristics of PaDADH in solution. Single amino acid mutations were undertaken to further understand the dynamics of the gated mechanism of this enzyme. In addition, pKa,shift analyses were evaluated to probe for the basic catalytic amino acid residue that is suggested to trigger the catalytic mechanism of PaDADH.

Thesis (MScMedSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Background: The conventional sequence analysis is the most common method used for the detection of drug-resistant mutants. Due to its sensitivity limitations, it is unable to detect these mutants when comprising less than 20% (minor populations) of the total virus population in a sample. However, real-time PCR-based assays offer a rapid, sensitive, specific and easy detection and quantification of such mutants. The HIV-1 variants harbouring the K103N mutation are associated with resistance to nevirapine (NVP) and efavirenz (EFV). The persisting drug-resistant mutants decay slowly to low levels, and therefore they are called minor drug-resistant mutants. Consequently, they affect subsequent treatment with the drugs of the relevant class. Objectives: The objective of this study was to design two TaqMan real-time PCR-based assays called selective-polymerase chain reaction (SPCR), namely the total viral copy SPCR assay and the K103N-SPCR assay. The former detects HIV-1 of subtype C reverse transcriptase sequences, whereas the latter detects K103N drug-resistant variants in these sequences. Design and Methods: In developing the SPCR assays, sets of appropriate primers and probes for the HIV-1 subtype C reverse transcriptase (RT) were developed to use in the K103N-specific reaction and the total copy reaction. Twelve DNA plasmid standards with sequence diversity were constructed for the assay from two HIV-1subtype C samples known to harbour the K103N mutation (AAC or AAT) in our Department‟s Resistance Databank. Their RT regions were amplified, cloned and verified with sequencing. Site-directed mutagenesis was used to induce mutations at 103 amino acid position in some of these clones to generate more standards with either one of the three codons (AAA, AAC and AAT). The two assays were optimized and validated, and a standard curve was generated for each assay using 10-fold serial dilution (5x107-5x100 DNA copy/μL) of a K103N-mutant plasmid standard. The optimized and validated SPCR assays were used to screen 40 nested PCR products of previously genotyped patient samples for minor K103N variants. Results: Two sensitive and reproducible selective real-time PCR (SPCR) assays, with cut-offs of 8.23 and 10.33 and a detection limit of 0.01% for the K103N resistance variants, were successfully developed. The assays detected a prevalence of 25.64-46.15% for the K103N resistance mutation in 39 patient samples. The genotyping (population sequencing) missed 40-53.85% of these variants. Conclusion: In conclusion, sensitive and reliable selective real-time PCR assays to detect and quantify minor K103N variants of HIV-1 in nested PCR products were successfully developed. The assay had a lower detection limit of 0.01%. / AFRIKAANSE OPSOMMING: Agtergrond: Konvensionele volgorde bepaling analise is die mees algemeenste metode wat gebruik word vir die opsporing van middel-weerstandige mutasies, maar weens beperkte sensitiwiteit is dit nie moontlik om hierdie mutante op te spoor wanneer dit minder as 20% (minderheids populasie) van die totale viruspopulasie in `n monster uitmaak nie. Nietemin, kwalitatiewe PKR-gebaseerd toetse bied vinnige, sensitiewe, spesifieke en makliker opsporings en kwantifisering van sulke mutante aan. MIV-1 variante wat die K103N mutasie bevat word geassosieer met weerstand teen nevirapine (NVP) and efavirenz (EFV). Volhoudende middel-weerstandige mutasies vergaan stadig na laer vlakke en word daarom na minderheids middel weerstandige mutasies verwys. Gevolglik affekteer dit opvolgende behandeling met die middel van die relevante klas. Doelwitte: Die doel van die studie was om twee TaqMan kwantifiserende PKR gebaseerde selektiewe polymerase ketting reaksies (SPKR), naamlik totale virale kopie SPKR en K103N-SPKR te ontwikkel. Die voormalige toets het die MIV-1 subtipe C omgekeerde transkriptase volgorde bepaal, waar K103N die middel-weerstand variante in hierdie volgorde opspoor. Ontwerp en Metodes: `n Geskikte stel inleiers en peiler was ontwikkel vir die MIV-1 subtipe C omgekeerde transkriptase (OT) vir gebruik in die K103N-spesifieke en die totaal kopie reaksie. Twaalf DNS plasmied standaarde met volgorde diversiteit was saamgestel vir die toets vanaf twee MIV-1 subtipe C monsters wat volgens ons Departement se weerstand databasis geklassifeer is vir die besit van die K103N mutasie (AAC of AAT). Die OT streke was geamplifiseer, gekloneer en geverifieer deur volgorde bepaling. Punt-gerigte mutagenese is gebruik om `n mutasie by die amino suur posisie 103 van sekere klone te induseer om meer standaarde te genereer wat een van die drie kodons (AAA, AAC en AAT) bevat. Die twee toetse is geoptimiseer en gevalideer en `n standard kurwe is genereer vir elk van die toetse deur die gebruik van tienvoud serie verdunnings (107-1 DNS kopie/μL) van `n algemene K103N-mutante plasmied standard. Die geoptimiseerde en gevalideerde SPKR toets was gebruik om vir die minderheids K103N variante in 40 “nested” PKR produkte van voorheen gegenotipeerde pasiënt te soek. Resultate: Twee sensitiewe en herproduseerbare selektiewe kwantitiewe PKR toetse met `n ΔCt afsnypunt van 8.23 en `n deteksie limiet van 0.006% was ontwikkel vir die K103N weerstand variant. Die toets het `n voorkomsyfer van 25.6 % vir die K103N weerstand mutasie in 40 pasiënt monsters bepaal, waar genotipering (populasie volgorde ) 40% van hierdie variante nie opgespoor het nie. Gevolgtrekking: `n Sensitiewe en betroubare selektiewe kwantitatiewe PKR toets vir die opspoor en kwantifisering van die minderheids K103N variante van MIV-1 in PKR produkte was ontwikkel. Hierdie toets het `n laer opsporings limiet van 0.01%. / Poliomyelitis Research Foundation (PRF) / National Research Fund (NRF) / National Health Laboratory Service Research Trust (NHLS RT)

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Rifampicin resistance in clinical isolates of Mycobacterium tuberculosis develops through selection of bacterial variants harbouring mutations in the rpoB gene. These mutations infer a fitness-cost in the absence of antibiotic pressure, however, fitness-levels of rifampicin-resistant strains can be restored by compensatory mutations in rpoA and rpoC. This study was the first to investigate the epidemiological relevance of these compensatory mutations in clinical M. tuberculosis isolates collected in South Africa. Through targeted DNA sequencing, we demonstrated a strong association between rpoC mutations and transmission, and the rpoB S531L mutation. Our study emphasises the epidemiological relevance of compensatory evolution in response to the emergence of rifampicin resistance, and illustrates how compensatory mutations may be selected as a function of epistatic interactions. Recently a hypothesis has been developed which suggests that the activation of efflux systems through exposure to rifampicin may explain the observed spectrum of rifampicin resistance phenotypes. To elucidate whether rifampicin dependent activation of efflux systems also increases energy production, the RNA expression profiles of candidate energy metabolism genes were investigated. This study demonstrated that rifampicin exposure induced an overall increase in the expression of energy metabolism genes. Our findings suggest that the response to rifampicin is not universal and may depend on other genomic mutations. From these results we conclude that the stress response induced by exposure to rifampicin increases the energy production which fuels efflux activity thereby enabling the cell to extrude rifampicin in an energy dependent manner. This also provides a platform to explain the mechanism by which the newly developed drug, TMC207, increases the rate of culture conversion when used in combination with second-line anti-TB drugs. We propose that inhibition of ATP synthesis by TMC207 will deprive the efflux pumps and transporter genes of energy, which will result in the accumulation of second-line anti-TB drugs within the bacilli, leading to more efficient binding of the second-line drugs to their targets and ultimately to cell death. To identify the genetic basis governing the level of rifampicin resistance, we sequenced the genomes of MDR clinical isolates and in vitro generated rifampicin resistant mutants. Only minor genetic changes in addition to the rpoB mutation were identified in the genomes of in vitro rifampicin resistant mutants which displayed varying levels of resistance. This suggests that these mutants may either use alternative regulatory mechanisms or have acquired SNPs outside the genetic regions investigated in this study to modulate rifampicin resistance levels. In contrast, the genomes of clinical MDR isolates from the Low Copy Clade showed considerable variability in genes involved in cell wall, cellular processes and lipid metabolism, while the genomes from the Beijing Clade displayed variability in genes known to confer drug resistance and compensatory mechanisms. These results suggest that the structure and processes of the cell wall, as well as lipid metabolism plays a critical role in determining the intra-cellular concentration of rifampicin. Finally, this study illustrated the complexity in the physiology of M. tuberculosis resistant to rifampicin, whereby multiple mechanisms are employed by the bacteria to modulate its resistance levels. / AFRIKAANSE OPSOMMING: Rifampisien weerstandigheid in kliniese isolate van Mycobacterium tuberculosis ontwikkel deur die seleksie van bakteriële variante wat mutasies in die rpoB geen het. Alhoewel hierdie mutasies lei tot „n afname in fiksheid van die bakterieë in die teenwoordigheid van antibiotika, kan die fiksheids vlakke van rifampisien weerstandige stamme herstel word deur vergoedende mutasies in rpoA en rpoC. Hierdie is die eerste studie wat die epidemiologiese relevansie van hierdie vergoedende mutasies in kliniese M. tuberculosis isolate wat in Suid-Afrika versamel is, ondersoek. Deur middel van doelgerigte DNA volgordebepaling het ons „n sterk assosiasie tussen rpoC mutasies en transmissie, en die rpoB S31L mutasie getoon. Hierdie studie beklemtoon die epidemiologiese relevansie van regstellende evolusie na aanleiding van die ontwikkeling van rifampisien weerstandigheid en illustreer hoe regstellende mutasies geselekteer mag word as „n funksie van epistatiese interaksies. „n Hipotese is onlangs ontwikkel wat voorstel dat blootstelling aan rifampisien uitvloei sisteme in die bakterium aktiveer, wat moontlik die waargenome spektrum van rifampisien weerstandige fenotipes kan verklaar. Ons het die RNA uitdrukkingsprofiele van kandidaat-energiemetabolisme gene ondersoek om te bepaal of rifampisien afhanklike aktivering van uitvloei sisteme ook energieproduksie verhoog. Hierdie studie demonstreer dat rifampisien-blootstelling „n algehele verhoging in die uitdrukking van energiemetabolisme gene induseer. Ons bevindinge stel voor dat die reaksie van die sel op rifampisien blootstelling nie universeel is nie, en moontlik ook afhanklik is van ander genomiese mutasies. Uit hierdie resultate kan ons aflei dat die stres respons wat geïnduseer word deur rifampisien-blootstelling energieproduksie verhoog, wat weer die uitvloei aktiwiteit aanvuur, en gevolglik die sel in staat stel om rifampisien op „n energie-afhanklike wyse uit te dryf. Dit bied ook „n basis om die meganisme te verklaar waardeur die nuwe middel, TMC207, die tempo van kultuuromskakeling verhoog wanneer dit saam met tweede-linie anti-TB middels gebruik word. Ons stel voor dat die inhibisie van ATP sintese deur TMC207 die uitvloeipompe en transporteerder gene van energie ontneem. Gevolglik veroorsaak dit „n ophoping van tweedelinie anti-TB middels binne-in die bakterium, wat geleentheid bied vir meer effektiewe binding tussen die middels en hulle teikens en uiteindelik seldood veroorsaak. Ons het DNA volgordes bepaal van die genome van MDR kliniese isolate en in vitro selekteerde rifampisienweerstandige mutante om sodoende die genetiese grondslag waarop die vlak van rifampisienweerstandigheid beheer word, te identifiseer. Slegs klein verskille, bo en behalwe die rpoB mutasie, is geïdentifiseer in die genome van in vitro rifampisien weerstandige mutante wat verskillende vlakke van weerstandigheid getoon het. Dit dui aan dat hierdie mutante of ander regulatoriese meganismes gebruik, of hulle het enkelnukleotied polimorfismes buite die genetiese area wat in hierdie studie ondersoek is, waarmee rifampisien weerstandigheid gemoduleer word. In teenstelling hiermee het die genome van kliniese MDR isolate van die “Low Copy Clade” aansienlike variasie getoon in gene wat betrokke is by die selwand, sellulêre prosesse en lipiedmetabolisme. Verder het die genome van die Beijing genotipe variasie in gene getoon wat betrokke is by middelweerstandigheid en regstellende meganismes. Hierdie resultate dui aan dat die struktuur en prosesse van die selwand, asook lipiedmetabolisme, „n kritiese rol speel in die bepaling van die intrasellulêre konsentrasie van rifampisien. Opsommend, hierdie studie toon verskeie meganismes aan wat deur die bakterieë gebruik word om weerstandigheidsvlakke te moduleer en die kompleksiteit van die fisiologie van M. tuberculosis wat weerstandig is teen rifampisien. / The National Research Foundation (NRF) / South African Medical Research Council (MRC) / Harry Crossley Foundation

Η πιο απλή μορφή γενετικής διαφοροποίησης στον άνθρωπο είναι οι μονονουκλεοτιδικοί πολυμορφισμοί (Single Nucleotide Polymorphisms - SNPs). Ο αριθμός αυτού του είδους πολυμορφισμών που έχουν βρεθεί στο ανθρώπινο γονιδίωμα και επηρεάζουν την παραγόμενη πρωτεΐνη αυξάνεται συνεχώς, αλλά η αντιστοίχηση τους σε πιθανές ασθένειες με πειραματικές μεθόδους είναι ασύμφορη από θέμα χρόνου και κόστους. Για αυτό τον λόγο έχουν αναπτυχθεί διάφορες υπολογιστικές μέθοδοι με σκοπό να ταξινομήσουν τους μονονουκλεοτιδικούς πολυμορφισμούς σε παθογόνους και μη. Οι περισσότερες από αυτές τις μεθόδους χρησιμοποιούν ταξινομητές, οι οποίοι παίρνοντας σαν είσοδο ένα σύνολο δομικών, λειτουργικών, ακολουθιακών και εξελικτικών χαρακτηριστικών, επιχειρούν να προβλέψουν αν ένας μονονουκλεοτιδικός πολυμορφισμός είναι παθογόνος ή μη. Για την εκπαίδευση αυτών των ταξινομητών, χρησιμοποιούνται δύο σύνολα μονονουκλεοτιδικών πολυμορφισμών. Το πρώτο αποτελείται από μονονουκλεοτιδικούς πολυμορφισμούς που έχει βρεθεί πειραματικά ότι οδηγούν σε παθογένεια και το δεύτερο από μονονουκλεοτιδικούς πολυμορφισμούς που έχει αποδειχθεί πειραματικά ότι είναι αδρανείς. Οι μέθοδοι αυτές διαφέρουν στα χαρακτηριστικά των μεταλλάξεων που λαμβάνουν υπόψη στην πρόβλεψη τους, καθώς επίσης και στην εκπαίδευση και τη φύση των τεχνικών ταξινόμησης, που χρησιμοποιούν για τη λήψη των αποφάσεων. Το βασικότερο προβλήματα τους ωστόσο έγκειται στο γεγονός ότι καθορίζουν τα χαρακτηριστικά, που θα χρησιμοποιήσουν σαν είσοδο στους ταξινομητές τους με τρόπο εμπειρικό και μάλιστα διαφορετικές μέθοδοι προτείνουν και χρησιμοποιούν διαφορετικά χαρακτηριστικά, χωρίς να τεκμηριώνουν επαρκώς τις αιτίες αυτής της διαφοροποίησης. Δύο ακόμα προβλήματα που δεν έχουν καταφέρει να αντιμετωπίσουν οι υπάρχουσες μεθοδολογίες είναι το πρόβλημα της ανισορροπίας των δύο κλάσεων ταξινόμησης και των ελλιπών τιμών σε πολλά από τα χαρακτηριστικά εισόδου των ταξινομητών, ώστε να επιτυγχάνουν πιο ακριβή και αξιόπιστα αποτελέσματα. Από τα παραπάνω είναι ξεκάθαρο πως υπάρχει μεγάλο περιθώριο βελτίωσης των υπάρχουσων μεθοδολογιών για το συγκεκριμένο πρόβλημα ταξινόμησης. Στην παρούσα διπλωματική εργασία προτείνουμε μια νέα υβριδική μεθοδολογία υπολογιστικής νοημοσύνης, που ξεπερνά πολλά από τα προβλήματα των υπάρχοντων μεθοδολογιών και βελτιώνει με τον τρόπο αυτό την απόδοσή τους. Δύο είναι τα βασικά βήματα που ακολουθήσαμε για την επίτευξη του στόχου αυτού. Πρώτον, συγκεντρώσαμε από τις διαθέσιμες δημόσιες βάσεις δεδομένων, τους μονονουκλεοτιδικούς πολυμορφισμούς που χρησιμοποιήθηκαν για την εκπαίδευση και τον έλεγχο των μοντέλων μηχανικής μάθησης. Συγκεκριμένα, συλλέχθησαν και φιλτραρίστηκαν τα θετικά και αρνητικά σύνολα εκπαίδευσης και ελέγχου, που αποτελούνται από μονονουκλεοτιδικούς πολυμορφισμούς που είτε οδηγούν σε παθογένεια, είτε είναι ουδέτεροι. Για κάθε πολυμορφισμό των δύο συνόλων υπολογίσαμε χρησιμοποιώντας υπάρχοντα διαθέσιμα εργαλεία όσο το δυνατό περισσότερα δομικά, λειτουργικά, ακολουθιακά και εξελικτικά χαρακτηριστικά. Για εκείνα τα χαρακτηριστικά, για τα οποία δεν υπήρχε κάποιο διαθέσιμο εργαλείο υπολογισμού τους, υλοποιήσαμε τον κατάλληλο κώδικα για τον υπολογισμό τους. Το δεύτερο βήμα της διπλωματικής αφορούσε το σχεδιασμό και την υλοποίηση της κατάλληλης υβριδικής μεθόδου για την επίλυση του προβλήματος που μελετάμε. Χρησιμοποιήσαμε μια νέα μέθοδο ταξινόμησης την EnsembleGASVR. Πρόκειται για μια ensemble μεθοδολογία, που συνδυάζει σε ένα ενιαίο πλαίσιο ταξινόμησης οκτώ διαφορετικούς ταξινομητές. Κάθε ένας από αυτούς τους ταξινομητές βασίζεται στον υβριδικό συνδυασμό των Γενετικών Αλγορίθμων και των μοντέλων Παλινδρόμησης Διανυσμάτων Υποστήριξης (nu-Support Vector Regression). Συγκεκριμένα ένας Προσαρμοζόμενος Γενετικός Αλγόριθμος χρησιμοποιείται για να καθοριστεί το βέλτιστο υποσύνολο χαρακτηριστικών, καθώς και οι βέλτιστες τιμές των παραμέτρων των ταξινομητών. Σαν μέθοδο ταξινόμησης των μεταλλάξεων σε ουδέτερες και παθογενείς, προτείνουμε τον nu-SVR ταξινομητή, καθώς παρουσιάζει υψηλή απόδοση, καλή γενίκευση, δεν παγιδεύεται σε τοπικά βέλτιστα, ενώ ταυτόχρονα επιτυγχάνει την ισορροπία μεταξύ της ακρίβειας και της πολυπλοκότητας του μοντέλου. Μάλιστα για να ξεπεράσουμε τα πρόβληματα των ελλιπών τιμών και της ανισορροπίας των δύο κλάσεων ταξινόμησης, αλλά και για να βελτιώσουμε τη συνολική απόδοση της μεθοδολογίας μας, επεκτείναμε τον υβριδικό αλγόριθμο, ώστε να λειτουργεί σαν μία ensemble-συλλογική τεχνική, συνδυάζοντας οκτώ επί μέρους μοντέλα ταξινόμησης. Τα πειραματικά αποτελέσματα της προτεινόμενης μεθοδολογίας ήταν εξαιρετικά ελπιδοφόρα, καθώς η EnsembleGASVR μεθοδολογία υπερτερεί σημαντικά έναντι άλλων ευρέως γνωστών μεθόδων ταξινόμησης παθογενών μεταλλάξεων. / Single Nucleotide Polymorphisms (SNPs) are the most common form of genetic variations in humans. The number of SNPs that have been found in human genome and affect protein functionality is constantly increasing. Finding matches between SNPs and diseases using experimental techniques, is excessive disadvantageous in terms of time and cost. For this reason, several computational methods have been developed. These methods classify polymorphisms as pathogenic and non-pathogenic. Most of them use classifiers, which take as input a set of structural, functional, sequential and evolutionary features and predict whether a single nucleotide polymorphism is pathogenic or neutral. For training these classifiers use two sets of SNPs. The first one consists of SNPs that have been experimentally proven as pathogenic, whereas the second set consists of SNPs that have been experimentally characterized as benign. These methods differ in the classification methods they deploy and in the features they use as inputs. However, the main problem is the determination of an empirically verified set of features for training. Specifically, different methods suggest different feature sets, without adequately documenting the causes of this differentiation. In addition, the existing methodologies do not tackle efficiently the class imbalance problem between positive and negative training sets and the problem of missing values in the datasets. In this thesis a new hybrid computational intelligence methodology is proposed, that overcomes many of the problems of existing methodologies. The proposed method achieves high classification performance and systematizes the selection of relevant features. In the first phase of this study the polymorphisms were gathered from the available public databases and they were used for training and testing of the machine learning models. Specifically, the positive and negative training and test sets were collected and filtered. They consist of single nucleotide polymorphisms that lead to either pathogenesis or are neutral. For each polymorphism of the two sets, using existing available tools, a wide range of structural, functional, sequential and evolutionary features were calculated. For those features for which there was no available tool, the suitable program (code) was developed in order to compute them. In the second step a new embedded hybrid classification method called EnsembleGASVR is designed and implemented. The method uses an ensemble methodology, based on hybrid combination of Genetic Algorithms and nu-Support Vector Regression (nu-SVR) models. An Adaptive Genetic Algorithm is used to determine the optimal subset of features and the optimal values of the parameters of classifiers. We propose the nu-SVR classifier, since it exhibits high performance, good generalization ability, it is not trapped in local optima and achieves a balance between accuracy and complexity of the model. In order to overcome the problem of missing values and class imbalance, we extended the above algorithm to function as a collective ensemble-technique, combining eight individual classification models. In overall, the method achieves 87.45% accuracy, 71.78% sensitivity and 93.16% specificity. These priliminary results are very promising and shows that EnsembleGASVR methodology significantly outperforms other well-known classification methods for pathogenic mutations.

Au cours des dernières années, la génétique a subi une progression phénoménale suite au développement de nouvelles technologies de séquençage. En effet, le séquençage de l’exome entier chez des familles a permis l’identification de nouveaux gènes impliqués pour plusieurs maladies. La neurologie a d’ailleurs bénéficié de ces avancées et plusieurs gènes ont été mis en évidence comme causatifs pour différents désordres neurologiques. Dans ce travail il sera question de deux désordres du mouvement pour lequel nous avons utilisés des technologies de séquençage traditionnelles, en l’occurrence le séquençage par Sanger, ainsi que de nouvelles technologies pour le séquençage de l’exome entier afin d’identifier de nouveaux gènes causatifs. Le premier désordre du mouvement qui sera décrit est l’ataxie, où ne seront abordées que les ataxies de cause génétiques, à transmission récessive. Le premier chapitre relatera les nouvelles mutations qui ont été trouvées chez des canadiens-français souffrant de l’ataxie de Beauce. Il sera aussi question de nouvelles mutations retrouvées dans deux autres populations, confirmant l’implication du gène SYNE1 dans les cas d’ataxie cérébelleuse à travers le monde. Le second chapitre fera la démonstration qu’il est souhaitable d’utiliser le séquençage de l’exome entier dans le but de poser un diagnostic clinique. En effet, il a été possible de trouver la cause génétique d’une famille comportant deux membres atteints d’atrophie congénitale du cervelet, où le symptôme prédominant est l’ataxie. Le séquençage de l’exome a permis la mise en évidence de mutations dans le gène PMM2, déjà connues pour cause le syndrome des glycoprotéines déficientes en hydrates de carbone. Dans un second temps, il sera question d’un autre désordre du mouvement la paraplégie spastique familiale (PSF). Le chapitre 3 relatera les mutations trouvées dans le gène CYP7B1 dans notre cohorte de patients PSF. / Over the past years, genetics has undergone a phenomenal growth due to the development of new sequencing technologies. Indeed, whole exome sequencing in families led to the identification of new genes involved in many diseases. Neurosciences were also able to benefit from these discoveries, where several new genes have been identified in several neurological diseases. This thesis will covered two different movement disorders for which we have used traditional sequencing technology, in this case by Sanger sequencing, combined with whole exome sequencing for new gene discovery. The first movement disorder that is described is ataxia, which will be focused on autosomal recessive mode of inheritance. The first chapter will relate the new mutations found in French-Canadian with ataxia of Beauce. We will also discuss new mutations found in two other populations, confirming the involvement of the gene SYNE1 in worldwide cases of cerebellar ataxia. The second chapter will demonstrate that it is desirable to use whole exome sequencing in order to make a clinical diagnosis. Indeed, it has been possible to find the genetic cause for a family with two members with congenital cerebellar atrophy, which the predominant symptom is ataxia. Exome sequencing allowed the identification of mutations in the PMM2 gene, already known to cause a syndrome leading to glycosylation deficit of glycoprotein. For the second part, we will cover another movement disorder call hereditary spastic paraplegia (HSP). Chapter 3 relates the mutations found in the CYP7B1 gene in our cohort of HSP patients.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

In recent years, the genetic landscape of B-cell derived lymphoid malignancies, including chronic lymphocytic leukemia (CLL), has been rapidly unraveled, identifying recurrent genetic mutations with potential clinical impact. Interestingly, ~30% of all CLL patients can be assigned to more homogeneous subsets based on the expression of a similar or “stereotyped” B-cell receptor (BcR). Considering that biased distribution of genetic mutations was recently indicated in specific stereotyped subsets, in paper I, we screened 565 subset cases, preferentially assigned to clinically aggressive subsets, and confirm the SF3B1 mutational bias in subset #2 (45%), but also report on similarly marked enrichment in subset #3 (46%). In contrast, NOTCH1 mutations were predominantly detected in subsets #1, #8, #59 and #99 (22-34%). This data further highlights a subset-biased acquisition of genetic mutations in the pathogenesis of at least certain subsets. Aberrant NF-κB signaling due to a deletion within the NFKBIE gene previously reported in CLL warranted extended investigation in other lymphoid malignancies. Therefore, in paper II, we screened 1460 patients with various lymphoid malignancies for NFKBIE deletions and reported enrichment in classical Hodgkin lymphoma (27%) and primary mediastinal B-cell lymphoma (PMBL) (23%). NFKBIE-deleted PMBL cases had higher rates of chemorefractoriness and inferior overall survival (OS). NFKBIE-deletion status remained an independent prognostic marker in multivariate analysis. EGR2 mutations were recently reported in advanced stage CLL patients; thus, in paper III we screened 2403 CLL patients for mutations in EGR2. An overall mutational frequency of 3.8% was reported and EGR2 mutations were associated with younger age, advanced stage and del(11q). EGR2 mutational status remained an independent marker of poor outcome in multivariate analysis, both in the screening and validation cohorts. Whole-genome sequencing (WGS) of 70 CLL cases, assigned to poor-prognostic subsets #1 and #2 and indolent subset #4, were investigated in Paper IV and revealed a similar skewing of SF3B1 mutations in subset #2 and NOTCH1 mutations in subset #1 to that reported in Paper I. Additionally, an increased frequency of the recently proposed CLL driver gene RPS15 was observed in subset #1. Finally, novel non-coding mutational biases were detected in both subset #1 and #2 that warrant further investigation.

Introdução: Dentre as neuropatias hereditárias, as neuropatias hereditárias motoras e sensitivas (HMSN), também conhecidas como Doença de Charcot-MarieTooth (CMT) são as mais comuns, podendo acometer 1:2500 pessoas. Elas podem ser classificadas com base nas características clínicas, eletrofisiológicas, padrão de herança e mutação em localização gênica/mutação. Atualmente, mais de 80 genes estão relacionados à CMT, dentre eles o GDAP1 que é responsável pelas formas CMT4A, AR-CMT2, CMTRIA e CMT2K. O gene codifica a proteína GDAP1 que é expressa pelos neurônios do sistema nervoso periférico e central e também pelas células de Schwann. Mutações no GDAP1 geralmente estão relacionados com CMT de herança recessiva, mas autossômica dominante também pode ocorrer. Geralmente, as neuropatias recessivas são de diagnóstico molecular mais difíceis, são mais graves e tem rápida progressão. É necessário que haja um maior número de casos de pacientes com CMT envolvendo mutações no GDAP1, juntamente com os dados clínicos, patológicos e de eletrofisiologia detalhados, para estabelecer uma relação de confiança entre o genótipo e o fenótipo das diferentes formas da doença. O objetivo do trabalho foi investigar mutações no gene GDAP1 em uma amostra da população brasileira com quadro clínico de CMT tanto axonal quanto desmielinizantes. Métodos: Screening mutacional do gene GDAP1 por sequenciamento direto em 100 pacientes com diagnóstico clínico sugestivo de CMT4, CMT2, CMTi e CMT esporádico, onde mutações nos genes MPZ, MFN2 e GJB1 foram previamente excluídos. Resultados: Foram encontradas alterações no GDAP1 em 6 pacientes índices não relacionados, sendo que 1 deles foi homozigoto para a alteração Q163*, 1 heterozigoto composto para as alterações N64S e R125*, dois pacientes não relacionados foram heterozigotos compostos para as alterações P119T e Q163*, 1 paciente em heterozigose para a alteração P119T e 1 paciente em heterozigose para alteração K207T. Dentre essas alterações, as variantes N64S, P119T, K207T ainda não foram descritas na literatura. Conclusão: Os resultados obtidos mostraram que mutações no gene GDAP1 estão presentes em pacientes da população brasileira com fenótipo de CMT2, AR-CMT2 e CMT esporádico. A frequência de ocorrência pode ser considerada alta (3,88%). Ademais, foram encontradas na população de estudo, duas mutações conhecidamente patogênicas (Gln163Ter e Arg125Ter) e três novas variantes (Asn64Ser, Pro119Thr, Lys207Thr) que ainda não haviam sido relacionadas ao fenótipo de CMT. / Introduction: Among the inherited neuropathies, the hereditary motor and sensory neuropathies (HMSN), also known as Charcot-Marie-Tooth disease, are the most common ones and may affect 1:2500 people. They can be classified based on their clinical features, electrophysiology, inheritance pattern and mutation on a gene location/mutation. Today, more than 80 genes have been related to the CMT disease and among them the GDAP1 gene, responsible for the CMT4A, AR-CMT2, CMTRIA and CMT2K forms. This gene codes the GDAP1 protein, which is expressed by neurons from the peripheral and central nervous system and also by Schwann cells. GDAP1 mutations are usually related to recessive inheritance, although autosomal dominant cases can also occur. Most of the times, recessive neuropathies tend to have a more difficult molecular diagnosis, besides being more severe and presenting fast progression. A larger number of patients with CMT related to GDAP1 mutations, along with detailed clinical, pathological and electrophysiological data, is necessary in order to establish a trustful relation between genotype and phenotype in the different forms of this disease. The objective of this research was to investigate mutations in the GDAP1 gene in a brazilian sample of patients with clinical picture of both axonal and demyelinating CMT. Methods: Mutational screening of the GDAP1 gene by direct sequencing of 100 patients presenting suggestive clinical diagnosis of CMT4, CMT2, CMTi and sporadic CMT, where mutations in the MPZ, MFN2 and GJB1 genes have been previously excluded. Results: Alterations in the GDAP1 were found in 6 unrelated index patients, including 1 homozygous for the Q163* alteration, 1 compound heterozygous for the N64S and R125* alterations, 2 compound heterozygous for the P119T and Q163* alterations, 1 heterozygous for the P119T alteration alone and 1 heterozygous for the K207T alteration. Among these alterations, the variants N64S, P119T, K207T haven\'t been described in the literature yet. Conclusion: The results obtained showed that mutations in GDAP1 gene are present in patients of brazilian population with phenotype of CMT2, AR-CMT2 and sporadic CMT. The occurrence frequency can be considered high (3,88%). Furthermore, were found in the studied population two mutations known as pathogenic (Gln163Ter and Arg125Ter) and three news variants (Asn64Ser, Pro119Thr, Lys207Thr), that had not been related to the CMT phenotype.

Os microRNAs são pequenos RNAs importantes na modulação da expressão gênica. O carcinoma papilífero de tireoide (CPT) é responsável pela maior parte dos casos de câncer de tireoide. Mutações somáticas ativam as vias de MAPK e PI3K/AKT e exercem papel importante no desenvolvimento do CPT. Acredita-se que miRNAs regulem genes associados a essas vias. A expressão de miRNAs foi avaliada em amostras parafinadas de CPT caracterizadas quanto à presença da mutação BRAFV600E. A expressão de miRNAs variou devido à heterogeneidade dos tecidos emblocados e entre pacientes, dados importantes quando microRNAs são avaliados como biomarcadores. Microarranjos de DNA mostraram diferenças na expressão de miR-16, miR-19a, miR-21, miR146a, miR-146b, miR-221 e miR-222 entre amostras positivas e negativas para BRAFV600E, indicando um possível papel na modulação de vias influenciadas por esta mutação. Quando amostras adicionais foram analisadas, apenas miR-146b apresentou expressão diferencial entre os dois grupos, ressaltando a variabilidade entre pacientes quanto à expressão de miRNAs. / MicroRNAs are small non-coding RNAs that regulate protein-coding genes. Papillary thyroid cancer (PTC) accounts for most of the thyroid cancer cases. Somatic mutations activate MAPK and PI3K/AKT pathways and play a leading role in PTC. MiRNAs may contribute to the regulation of these pathways. We studied the expression of miRNAs in formalin-fixed, paraffin-embedded (FFPE) PTC samples submitted to mutation characterization. Our results show that miRNA expression varies due to embedded sample heterogeneity and that there is great variability in miRNA expression among patients, both important issues when miRNA expression is evaluated as a biomarker. DNA microarray experiments compared BRAFV600E positive and negative samples. MiR-16, miR-19a miR-21, miR146a, MiR-146b, miR-221 and miR-222 were deregulated, indicating a possible implication in BRAF-related pathways. When additional samples were evaluated, only miR-146b presented consistent variation in expression, highlighting variability among patients