Self-assembled monolayers on gold and glass substrates are employed as templates to direct the crystal growth and polymorphism of active pharmaceutical ingredients. Orthogonal approaches are used to control polymorphism either through complementary hydrogen-bonding interactions or through repulsive interactions.

When Darwin introduced natural selection in 1859 as a key mechanism of evolution, little was known about the underlying cause of variation within a species. Today we know that this variation is caused by the acquired genomic differences between individuals. Polymorphism, defined as the existence of multiple alleles or forms at a genomic locus, is the technical term used for such genetic variations. Polymorphism, along with reproduction and inheritance of genetic traits, is a necessary condition for natural selection and is crucial in understanding how species evolve and adapt. Many questions regarding polymorphism, such as why certain species are more polymorphic than others or how different organisms tend to favor some types of polymorphism among others, when solved, have the potential to shed light on important problems in human medicine and disease research. Some of these studies require more diverse species and/or individuals to be sequenced. Of particular interest are species with the highest rates of polymorphisms. For instance, the sequencing of the sea squirt genome lead to exciting studies that would not be possible to conduct on species that possess lower levels of polymorphism. Such studies form the motivation of this thesis. Sequencing of genomes is, nonetheless, subject to its own research. Recent advances in DNA sequencing technology enabled researchers to lead an unprecedented amount of sequencing projects. These improvements in cost and abundance of sequencing revived a greater interest in advancing the algorithms and tools used for genome assembly. A majority of these tools, however, have no or little support for highly polymorphic genomes; which, we believe, require specialized methods. In this thesis, we look at challenges imposed by polymorphism on genome assembly and develop methods for polymorphic genome assembly via an overview of current and past methods. Though we borrow fundamental ideas from the literature, we introduce several novel concepts that can be useful not only for assembly of highly polymorphic genomes but also genome assembly and analysis in general.

Kawasaki disease (KD) is an acute febrile vasculitic syndrome of unknown etiology that preferentially affects the coronary artery. Several lines of evidence point to an important role of genetic variation in KD susceptibility. Acute KD is associated with systemic immune activation, including elevated serum levels of IL-1, IL-4, IL-6, IL-8, IL-10, TNF£\, and decreased serum levels of TGF£], as well as a variety of other cytokines that are believed to play important roles in the onset of KD. This study aims to investigate the association of 14 various polymorphisms of nine cytokine genes (IL-1A, IL-1B, IL-1RN, IL-4, IL-6, IL-8, IL-10, TNFA and TGFB) with the risk of KD. A total of 213 KD children and 226 sex-matched infection disease-free adult controls were recruited in the hospital-based case-control study. The polymorphisms of IL-1 RN intron2 VNTR and IL-4 intron3 VNTR were determined by PCR. The polymorphisms of IL-1B T-31C and IL-1B C-511T were determined by PCR-RFLP. The other single nucleotide polymorphisms (SNPs) of IL-1A C-889T, IL-4 T-590C, IL-6 G-174C, IL-8 T-251A, IL-8 C+781T, IL-10 A-1082G, IL-10 T-819C, IL-10 A-592C, TNFA G-308A and TGFB C-509T were determined by TaqMan real-time PCR method. We found that there were no associations between the 14 polymorphisms and risk of KD, either in the single locus genetypic analysis or in the multiple loci haplotypic analysis. However, in the combined analysis, subjects with four and five ¡§at risk¡¨ genotypes combined from IL-1A, IL-1B, IL-1RN, IL-4, IL-6, IL-8, IL-10, TNFA and TGFB genetic polymorphisms were with 1.85 fold risk of KD as compared to those with less or equl to four ¡§at risk¡¨ genotypes (AOR=1.85; 95% CI, 1.16-2.97; P=0.011). Additionally, subjects with six and seven ¡§at risk¡¨ genetic polymorphisms were with 2.34 fold risk as compared to those with less or equal to four ¡§at risk¡¨ genotypes (AOR=2.34; 95% CI, 1.45-3.76; P=0.001). In conclusion, no association was found between cytokine genetic polymorphisms and KD risk, except in the combined analysis.

The rapid development of high-throughput sequencing technology provides a new chance to extend the scale and resolution of genomic research. How to efficiently and accurately call genetic variants in single base level (germline single nucleotide polymorphisms (SNPs) or somatic single nucleotide variants (SNVs)) is the fundamental challenge in sequencing data analysis, because these variants reported to influence transcriptional regulation, alternative splicing, non-coding RNA regulation and protein coding. Many applications have been developed to tackle this challenge. However, the shallow depth and cellular heterogeneity make those tools cannot attain satisfactory accuracy, and the huge volume of sequencing data itself cause this process inefficient. In this dissertation, firstly the performance of prevalent reads aligners and SNP callers for second-generation sequencing (SGS) is evaluated. And due to the high GC-content, the significantly lower coverage and poorer SNP calling performance in the regulatory regions of human genome by SGS is investigated. To enhance the capability to call SNPs, especially within the lower-depth regions, a fast and accurate SNP detection (FaSD) program that uses a binomial distribution based algorithm and a mutation probability is proposed. Based on the comparison with popular software and benchmarked by SNP arrays and high-depth sequencing data, it is demonstrated that FaSD has the best SNP calling accuracy in the aspects of genotype concordance rate and AUC. Furthermore, FaSD can finish SNP calling within four hours for 10X human genome SGS data on a standard desktop computer. Lastly, combined with the joint genotype likelihoods, an updated version of FaSD is proposed to call the cancerous somatic SNVs between paired tumor and normal samples. With extensive assessments on various types of cancer, it is demonstrated that no matter benchmarked by the known somatic SNVs and germline SNPs from database, or somatic SNVs called from higher-depth data, FaSD-somatic has the best overall performance. Inherited and improved from FaSD, FaSD-somatic is also the fastest somatic SNV caller among current programs, and can finish calling somatic mutations within 14 hours for 50X paired tumor and normal samples on normal server. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Several active pharmaceutical ingredients (API) exhibit polymorphism i.e. the ability of a solid material to exist in more than one form or crystal structure (polymorphs). The polymorphs differ somewhat in physical and, sometimes, chemical properties, although their solutions and vapours are identical. Among the different physical properties that are affected by polymorphism are solubility and dissolution rate which have great impact on the bioavailability of the drug. Frequently an API polymorph transforms to another, more thermodynamically stable, phase. When such transformation takes place then a pharmaceutical industry faces problems related to bioavailability of an API as well as litigation problems due to patent protection of specific polymorphs. The pharmaceutical substance that was studied in this current work was, Aripiprazole, an antipsychotic drug. API crystallizes in a large number of polymorphic and solvatomorphic phases. Μore specific 9 polymorphic and 9 solvatomorphic phases have been positively identified, from which Form III and Form V are currently tested as possible candidates for drug formulations. The objective of the present work was to establish methods capable of identification and quantification of Apiprazole crystal forms and their possible transformation to hydrate phase in Aripiprazole powders as well as in the tested tablets. Stability test were also performed on Aripiprazole crystal forms III and V. The analytical techniques applied were X-ray Powder Diffraction (XRPD), Infrared spectroscopy (IR) and Raman spectroscopy. Calibration lines of mixtures of API Form III and Monohydrate were constructed. Results show that all three techniques were capable of identifying and quantifying the monohydrate phase however XRPD is probably the method of choice due to the lower detection limit (0.34%) of monohydrate in Phase III-monohydrate mixtures. Quantitative analysis of these polymorphs in the presence of excipients (tablets) is difficult due to the rather low API percentage in tablets (8%) and the high detection limits of monohydrate in mixtures of III and monohydrate. An effort to increase the low API content was made by dissolving in water a large percentage of excipients at alkaline pH. Even though the stability tests show that no transformation of pure Phase III occurs under these conditions, in tablets with some presence of monohydrate, rapid transformation of the remaining Phase III was observed during the dissolution process of the excipients. Thus, the only way was to apply directly the analytical techniques that were used for the pure mixtures to tablets. It was found that Raman and IR spectra cannot be used due to heavy overlapping with excipients bands. By increasing the scan time the identification of anhydrate phase and the hydrate was possible by using XRPD. In order to avoid any possible transformation of both Form III and Form V (stability tests show transformation to hydrate phase) it was decided to build the calibration line using only the hydrate phase and the placebo. The constructed calibration line was constructed exhibit low detection limit (0.1156%). Based on this calibration line and the calibration lines that were build using pure Form III and hydrate mixtures a methodology was proposed and the polymorphs were determined in finished formulations. / Αρκετές ενεργές φαρμακευτικές ουσίες εμφανίζουν πολυμορφισμό, δηλαδή την ικανότητα μιας στερεής ένωσης να υπάρχει σε παραπάνω από μια κρυσταλλική φάση (πολύμορφα). Τα πολύμορφα διαφέρουν σε φυσικές, και σε μερικές περιπτώσεις, χημικές ιδιότητες παρόλο που τα διαλύματα και οι ατμοί τους είναι πανομοιότυποι. Μεταξύ των διαφόρων φυσικών ιδιοτήτων που επηρεάζει ο πολυμορφισμός είναι η διαλυτότητα και ο ρυθμός διάλυσης, οι οποίες έχουν μεγάλη επίδραση στην βιοδιαθεσιμότητα του φαρμάκου. Συχνά ένα πολύμορφο μιας φαρμακευτικής ουσίας μετατρέπεται σε ένα άλλο πολύμορφο το οποίο είναι θερμοδυναμικά πιο σταθερό. Όταν ένας τέτοιος μετασχηματισμός λαμβάνει χώρα, μια φαρμακευτική βιομηχανία είναι πιθανόν να αντιμετωπίσει προβλήματα που σχετίζονται με την βιοδιαθεσιμότητα της φαρμακευτικής ουσίας καθώς επίσης και νομικά προβλήματα που αφορούν τα διπλώματα ευρεσιτεχνίας των συγκεκριμένων πολυμόρφων. Η φαρμακευτική ουσία που μελετήθηκε στην συγκεκριμένη εργασία ήταν ένα αντιψυχωσικό φάρμακο, η αριπιπραζόλη. Η αριπιπραζόλη κρυσταλλώνεται σε ένα μεγάλο αριθμό πολυμορφικών και επιδιάλυτων φάσεων (solvatomorphic- ένυδρων φάσεων αλλά και φάσεων με διάφορα άλλα μόρια διαλυτών ενσωματωμένα στη δραστική). Πιο συγκεκριμένα έχουν ταυτοποιηθεί 9 πολυμορφικές και 9 επιδιάλυτες φάσεις, εκ των οποίων η Φάση ΙΙΙ και η Φάση V εξετάζονται επί του παρόντος σαν υποψήφιες για την παρασκευή φαρμακευτικών σκευασμάτων. Σκοπός της συγκεκριμένης ερευνητικής προσπάθειας ήταν η ανάπτυξη μεθοδολογιών για τον ποιοτικό και ποσοτικό προσδιορισμό των διαφόρων φάσεων της αριπιπραζόλης και την πιθανή μετατροπή τους στην ένυδρη φάση σε μίγματα καθαρών δραστικών ουσιών καθώς επίσης και στα εξεταζόμενα δισκία. Επίσης διενεργήθηκε έλεγχος σταθερότητας στις πολυμορφικές φάσεις ΙΙΙ και V. Οι αναλυτικές τεχνικές που χρησιμοποιήθηκαν στην παρούσα εργασία είναι η Περίθλαση Κόνεως Ακτινών Χ (XRPD), η Φασματοσκοπία Υπέρυθρου (IR) και η Φασματοσκοπία Raman. Κατασκευάστηκαν καμπύλες βαθμονόμησης μιγμάτων Φάσης ΙΙΙ και Μονοένυδρης Φάσης αριπιπραζόλης. Τα αποτελέσματα έδειξαν ότι και οι τρεις τεχνικές είναι μπορούν να χρησιμοποιηθούν για τον ποιοτικό και τον ποσοτικό προσδιορισμό της ένυδρης φάσης, η μέθοδος όμως της Περίθλασης Ακτινών Χ είναι πιθανώς καλύτερη εξαιτίας των χαμηλότερων ορίων ανίχνευσης (0,34%) της ένυδρης φάσης σε μίγματα Μονοένυδρης Φάσης-Φάσης ΙΙΙ. Η ποσοτική ανάλυση των πολυμορφικών φάσεων αυτών σε δισκία δηλαδή παρουσία εκδόχων κατέστη πολύ δύσκολη εξαιτίας του σχετικά μικρού ποσοστού δραστικής ουσίας στο δισκίο (8%) και των υψηλών ορίων ανίχνευσης της μονοένυδρης φάσης σε μίγματα αυτής με την φάση ΙΙΙ. Έγινε προσπάθεια να αυξηθεί η ποσότητα της δραστικής ουσίας στο δισκίο διαλύοντας σε νερό ένα μεγάλο ποσοστό των εκδόχων σε αλκαλικό pH. Παρόλο που ο έλεγχος σταθερότητας έδειξε ότι δεν λαμβάνει χώρα μετατροπή της καθαρής Φάσης ΙΙΙ σε μονοένυδρη φάση κάτω από αυτές τις συνθήκες, στα δισκία με κάποια παρουσία μονοένυδρης φάσης λαμβάνει χώρα ταχεία μετατροπή της εναπομείνουσας φάσης ΙΙΙ κατά την διαδικασία διάλυσης των εκδόχων. Κατ' επέκταση ο μόνος τρόπος ήταν η εφαρμογή των αναλυτικών τεχνικών στα δισκία χωρίς περαιτέρω επεξεργασία τους. Βρέθηκε ότι τα φάσματα Raman και IR δεν μπορούν να χρησιμοποιηθούν λόγω των μεγάλων αλληλοεπικαλύψεων των κορυφών τους με τις κορυφές των εκδοχών. Ο προσδιορισμός της ένυδρης φάσης και της άνυδρης φάσης κατέστη δυνατός αυξάνοντας το χρόνο σάρωσης στην Περίθλαση Ακτινών Χ (XRPD). Προκείμενου να αποφευχθεί οποιαδήποτε μετατροπή των δυο Φάσεων ΙΙΙ και V (ο έλεγχος σταθερότητας έδειξε μετατροπή στην ένυδρη φάση), αποφασίστηκε να δημιουργηθεί καμπύλη βαθμονόμησης χρησιμοποιώντας μόνο την ένυδρη φάση και τα μίγμα των εκδόχων (placebo). Η καμπύλη βαθμονόμησης που κατασκευάστηκε παρουσιάζει χαμηλά όρια ανίχνευσης (0,12 %). Με βάση αυτήν την καμπύλη βαθμονόμησης σε συνδυασμό και με τις καμπύλες που δημιουργήθηκαν χρησιμοποιώντας μίγματα της Φάσης ΙΙΙ και της ένυδρης Φάσης, προτάθηκε μια μεθοδολογία με την οποία προσδιορίζονται τα πολύμορφα στα τελικά σκευάσματα.

No description available.

Background: Significant interest in homocysteine exists due to its established role in embryogenesis, cardiovascular disease and neurotoxicity. This research investigates total plasma homocysteine in the context of carcinogenesis among healthy individuals aged 20 to 50 years. It is hypothesized that during this timeframe, elevated total plasma homocysteine (tHcy) concentration implicates a breakdown of the methionine-homocyteine biosynthesis pathway, in which folate deficiency, oxidative stress and altered DNA methylation capacity are potential consequences relevant to cancer etiology. Purpose: The overall purpose of this research is to identify novel genetic predispositions to a biomarker of cancer risk (tHcy). Interactions with dietary and genetic factors that act on this pathway are explored. Methods: The study population consisted of 284 healthy male and female volunteers recruited in Kingston, Ontario and Halifax, Nova Scotia between 2006 and 2008. Specifically, polymorphisms under consideration included: i) the thymidylate synthase enhancer region (TSER) tandem repeat polymorphism and ii) the GC single nucleotide polymorphism (G/C SNP) both found on the 5’untranslated region (UTR) of the TS gene, and iii) the 6 base pair deletion at base pair 1494 (TS1494del6) found on the 3’UTR. TS polymorphisms were categorized based on either 5’ or 3’ location and were dichotomized to either high or low TS expression. Gene-gene interactions between polymorphisms in TS and methylenetetrahydrofolate reductase (MTHFR C677T) on tHcy concentration were also analyzed. In addition, gene-diet interactions between serum folate and vitamin B12 status were examined. Results: Mean tHcy concentration for this study population was 8.65 µmol/L (standard deviation=1.96 µmol/L). After adjustment for confounders, higher mean tHcy levels of 0.48 μmol/L and 0.46 μmol/L were observed for the main effects of 5’polymorphisms (5’High) (p=0.04) and 3’polymorphism (3’High) (p=0.05), respectively. The largest difference in mean tHcy concentration was observed for the joint effects of TS polymorphisms (µ=0.74 μmol/L, p=0.11). Gene-gene interaction was observed between TS and MTHFR polymorphisms on tHcy concentrations (p<0.01). Conclusions: The findings of this research provide evidence of an association between TS polymorphisms and tHcy concentrations. These results suggest that TS polymorphisms, independent of dietary factors, may lead to elevated tHcy levels and potentially contribute to cancer development. / Thesis (Master, Community Health & Epidemiology) -- Queen's University, 2008-08-21 16:05:02.797

No description available.

When Darwin introduced natural selection in 1859 as a key mechanism of evolution, little was known about the underlying cause of variation within a species. Today we know that this variation is caused by the acquired genomic differences between individuals. Polymorphism, defined as the existence of multiple alleles or forms at a genomic locus, is the technical term used for such genetic variations. Polymorphism, along with reproduction and inheritance of genetic traits, is a necessary condition for natural selection and is crucial in understanding how species evolve and adapt. Many questions regarding polymorphism, such as why certain species are more polymorphic than others or how different organisms tend to favor some types of polymorphism among others, when solved, have the potential to shed light on important problems in human medicine and disease research. Some of these studies require more diverse species and/or individuals to be sequenced. Of particular interest are species with the highest rates of polymorphisms. For instance, the sequencing of the sea squirt genome lead to exciting studies that would not be possible to conduct on species that possess lower levels of polymorphism. Such studies form the motivation of this thesis. Sequencing of genomes is, nonetheless, subject to its own research. Recent advances in DNA sequencing technology enabled researchers to lead an unprecedented amount of sequencing projects. These improvements in cost and abundance of sequencing revived a greater interest in advancing the algorithms and tools used for genome assembly. A majority of these tools, however, have no or little support for highly polymorphic genomes; which, we believe, require specialized methods. In this thesis, we look at challenges imposed by polymorphism on genome assembly and develop methods for polymorphic genome assembly via an overview of current and past methods. Though we borrow fundamental ideas from the literature, we introduce several novel concepts that can be useful not only for assembly of highly polymorphic genomes but also genome assembly and analysis in general.

Thesis ( Ph.D.) -- McMaster University, 1997. / Computer disk in pocket. Includes bibliographical references. Also available via World Wide Web.