Thèse (Ph. D.) -- Université Laval, 1998. / Bibliogr.: f. 195-232. Publié aussi en version électronique.

Thèse (M.Sc.) -- Universoté Laval, 1997. / Bibliogr.: f. 79-85. Publié aussi en version électronique.

Η αποαδενυλίωση είναι συνήθως στάδιο που καθορίζει το ρυθμό της αποικοδόμησης και καταστολής της μετάφρασης του mRNA. Το γεγονός αυτό καθιστά την αποαδενυλίωση ως το κυριότερο σημείο ελέγχου για τις δύο αυτές διαδικασίες. Οι αποαδενυλάσες είναι εξωριβονουκλεάσες εξαρτώμενες από Mg2+ που υδρολύουν το mRNA με κατεύθυνση 3’-5’, παράγοντας 5’-AMP. Ο αριθμός των γνωστών αποαδενυλασών έχει επεκταθεί πρόσφατα, κυρίως μέσω βιοχημικών και γενετικών μελετών. Όλες οι γνωστές αποαδενυλάσες ανήκουν σε μία από της δύο ομάδες, την DEDD ή την exonuclease– endonuclease–phosphatase (EEP) υπέρ-οικογένεια. ∆εν είναι ακόμη πλήρες κατανοητό το πλεονέκτημα της ύπαρξης τόσων πολλών αποαδενυλασών. Υποψήφιες αποαδενυλάσες έχουν ταυτοποιηθεί με χρήση προγραμμάτων βιοπληροφορικής, ωστόσο η δράση τους δεν έχει αποδειχθεί, όπως η PNLDC1. Αρχική βιοπληροφορική ανάλυση του γονιδίου pnldc1 έδειξε ότι κωδικοποιεί μια πρωτεΐνη η οποία παρουσιάζει ομολογία με την PARN με πετιδικό σήμα που σχετίζεται με μια διαμεμβρανική περιοχή της πρωτεΐνης. Επιπρόσθετα φαίνεται ότι ο υποκινητής του γονιδίου περιέχει νησίδες CpG. Με μοντελοποίηση με βάση την ομολογία με την ανθρώπινη PARN της περιοχής με δράση νουκλεάσης προβλέφθηκε μία δομή που περιέχει ενεργό κέντρο με τα χαρακτηριστικά κατάλοιπα DEDD να κατευθύνονται σωστά για να αλληλεπιδρούν με τα ιόντα Mg2+ και να υποστηρίζουν ενζυμική δράση. Το γονίδιο pnldc1 κλωνοποίηθηκε και η ανασυνδυασμένη πρωτεΐνη καθαρίσθηκε με χρωματογραφία συγγένειας. Πειράματα αποαδενυλίωσης έδειξαν ότι η PNLDC1_1 είναι μία αποαδενυλάση με υψηλή συγγένεια γα το υπόστρωμα poly(A) αλλά χαμηλή Vmax σε σχέση με την PARN. Η έκφραση του pnldc1 μπορεί να ρυθμιστεί μέσω μεθυλίωσης του υποκινητή. Ύστερα από χορήγηση του απομεθυλιωτικού 5-Aza- Deoxycytidine, η έκφραση του pnldc1 αυξήθηκε 4 φορές στις κυτταρικές σειρές HaCaT και HEK 293 T ενώ τα επίπεδα των PARN και CNOT7 mRNA παρέμειναν αμετάβλητα, υποδεικνύοντας πιθανόν έναν μοναδικό ρόλο της νέας αποαδενυλάσης στην γονιδιακή έκφραση κατά την διάρκεια της εμβρυογένεσης και διαφοροποίησης. Η αποαδενυλάση PAN2 είναι μέλος της οικογένειας των DEDD νουκλεασών και ξεκινάει την βράχυνση του poly(A) απομακρύνοντας σχεδόν την μισή ουρά μέχρι άλλες αποαδενυλάσες να αποικοδομούν την υπόλοιπη. Παρόλο που ο ρόλος της PAN2 στην αποικοδόμηση του mRNA είναι διευκρινισμένος ο βιολογικός ρόλος του ενζύμου δεν έχει αποσαφηνισθεί. Μετά από αποσιώπηση της PΑΝ2 παρατηρήθηκε σημαντική μείωση στα επίπεδα έκφρασης των mRNA PARN, Papola, CBP20 και MYC ενώ δεν παρατηρήθηκε σημαντική αλλαγή στα επίπεδα EIF4E και CNOT7. Τα αποτελέσματα μας υποδεικνύουν ότι η PAN2 εμπλέκεται στην γονιδιακή έκφραση πιθανόν με το να διεγείρει την αποικοδόμηση των ήδη υπαρχόντων μορίων mRNA επιτρέποντας την παραγωγή νέων. / Deadenylation is often a rate-limiting step for mRNA decay and translational silencing, making it an ideal control point for both processes. Deadenylases are Mg2+ -dependent exoribonucleases that hydrolyse RNA in the 3′→5′ direction, which results in release of 5′- AMP. The number of known deadenylases has recently expanded, mainly through biochemical and genetics studies. All known deadenylases belong to one of two groups, the DEDD or the exonuclease–endonuclease–phosphatase (EEP) superfamilies. The advantage of having such a range of deadenylases is not yet completely understood. Putative deadenylases have been identified through bioinformatics, but their functions has not yet been established such as the mammalian PNLDC1 which is a Poly(A)-specific Ribonuclease homolog (termed PARN-like). Preliminary bioinformatic analysis of the pnldc1 gene revealed that it encodes a PARN-similar protein with a signal peptide which corresponds to a trans-membrane region. In addition several CpG islands within the gene’s promoter were identified. Homology modeling of the nuclease domain with its human PARN homolog predicted a structure that forms an active site which contains the characteristic DEDD residues orientated correctly to interact with Mg2+ ions to support enzymatic activity. Pnldc1 was cloned and the recombinant protein was purified via affinity chromatography. Deadenylation assays showed that PNLDC1_1 is a deadenylase with high affinity for poly(A) substrates but relatively low Vmax compared to PARN. Interestingly, pnldc1 expression can be regulated through promoter methylation. Following treatment with the demethylating agent, 5-Aza-Deoxycytidine, pnldc1 expression was increased 4-fold in both HaCaT and HEK 293 T cell lines, whereas PARN and CNOT7 mRNA expression was unchanged, thus implying a possible unique role of this new deadenylase in gene expression during embryogenesis and differentiation. PAN2 deadenylase, a member of the DEDD nucleases initiates poly(A) degradation removing almost half of the tail when other nucleases degrade the rest of the tail. Although the role of PAN2 in mRNA degradation has been revealed, its biologic function has not been studied. On knockdown of PAN2 a significant reduction was observed in the levels of PARN, Papola, CBP20 and MYC, but no significant change changes were observed in the levels of EIF4E and CNOT7 mRNA expression. Our results indicate that PAN2 is involved in gene expression, this probably by triggering the degradation of the current messengers allowing production of new messengers.

Despite the considerable expansion of knowledge in the development of pancreatic cancer, there has been little progress made in facilitating an early diagnosis of this disease and predicting an accurate response to treatment. We aim to translate this knowledge to clinical practice by using a prospective database of precursor cystic lesions in pancreas cancer, assessing the use of over-expressed genes in pancreatic juice as a surrogate marker of these pancreas cancer and finally, downstream of these changes at the genetic level, use metabolomic techniques to look for biomarkers in pancreas cancer in serum. In the first study, we investigate the natural history of pancreatic cystic neoplasms, specifically IPMNs, using a prospectively collected database to examine the profiles and outcomes of main duct IPMN, branch duct IPMN and cystic lesions measuring less than 3 cm in size. A total of 99 patients with suspected pancreatic cystic tumours were enrolled over 3 years. Median follow-up was 24 months (range 0 – 124). Cystic tumours comprised of 13 MD-IPMN, 40 BD-IPMN, 11 MCN and 8 adenocarcinomas among others. The complete cohort showed an overall risk of adenocarcinoma of 8%. Main duct IPMN showed a cumulative risk of 46% with evidence of progression of disease in a further 23%. The associated mortality in MD-IPMN was related to the underlying adenocarcinoma and was 38% in our group. The incidence of adenocarcinoma in branch duct IPMN was 11% with disease progression seen 13.8%. Evidence of extra-pancreatic malignancies was seen in 37.7% of patients with IPMN. In the second study, we explore the feasibility of gene expression profiling from RNA isolated from matched pancreatic juice and tumour tissue in patients with pancreatic cancer and pancreatic cystic tumours. RNA was isolated and Poly(A) PCR was used to globally amplify the RNA. RT-PCR was used to measure expression levels of 18 genes common to both pancreas cancer and pancreatic cystic tumours. Spearman’s rank correlation test was used to examine the relationship of gene expression between pancreatic juice and tissue. One gene out of eighteen, MSLN (p<0.008), showed significant correlation in the expression levels between paired pancreatic juice and tissue samples in pancreas cancer. In the cystic tumour group, only one gene MMP-7 (p<0.01), showed a significant correlation between paired juice and tissue samples. When the whole cohort was analysed for the false discovery rate, these genes did not exhibit statistically significant correlation between the samples. RNA analysis of pancreatic juice is feasible using the Poly(A) cDNA technique and correlation of gene expression is shown to exist, albeit with low sensitivity, indicating its potential use in clinical practice with small tissue and juice samples. In the final study, we performed a literature review on the use of metabolomics in pancreas cancer. We performed metabolic profiling of serum samples from selected cancer patients and noncancerous controls using UPHLC-MS to generate and compare the metabolic profiles in serum samples from a cohort of patients with pancreas cancer, ampullary cancer and endocrine cancer. Thirty nine serum samples (including 19 pancreatic cancers, 9 ampullary cancers and 5 endocrine cancers) and 21 matched HUSERMET controls were analysed using Ultra high performance liquid chromatography mass spectrometry (UHPLC-MS) in both positive and negative ESI modes. The output was generated as a data matrix of mass spectral features with related accurate m/z and retention time pairs. The data was then signal corrected and individual peaks were normalised and the resultant spectra were compared against a metabolite reference library and analysed using univariate and multivariate statistical tests. We found a disparity in the metabolite peaks between the cases and controls on PCA that did not permit the accurate interpretation of the data in the case study set compared to the control set. No obvious reason other than metabolite degradation during storage could account for this difference. PC-DFA analysis of metabolite peaks between pancreas cancer, ampullary cancer and endocrine cancer showed significant difference between endocrine cancers and the other two groups. Significant ESI positive metabolites included those involved in lipid pathways and metabolites involved in glucose metabolism. There is encouraging scope for studies using prospective controls to identify and develop metabolic biomarkers in pancreas cancer.

Táto práca sa zaoberá zvlákňovaním biopolymérov z taveniny. V teoretickej časti sú zhrnuté doterajšie poznatky o zvlákňovaní poly(3-hydroxy butyrátu) (PHB) a poly(mliečnej kyseliny) (PLA). Následne boli zvláknené polymérne zmesi na báze PHB s rôznym zložením, čo umožnilo štúdium vplyvu PLA, zmäkčovadiel a ich množstva na vlastnosti pripravených vlákien. Boli použité tri komerčné zmäkčovadlá (ATBC, PEG, A6) a dva experimentálne syntetizované. Zvláknenie bolo prevedené na troch rôznych zvlákňovacích linkách. Konvenčné zvlákňovacie linky s odťahovými rýchlosťami nad 150 m/min sa preukázali ako nevhodné pre spracovanie našich zmesí. Kvôli nedostatočnej pevnosti taveniny sú potrebné nízke odťahové rýchlosti. Boli pripravené vlákna s dĺžiacim pomerom 6,4; ktoré boli následne analyzované pomocou GPC, MDSC a ťahovej skúšky. Na základe nameraných teplôt skelného prechodu zmäkčovadlá ATBC a PEG vykázali lepšiu schopnosť zmäkčiť skúmané PLA/PHB zmesi. Vlákna obsahujúce A6 vykazovali najvyššiu pevnosť v ťahu (250 MPa) a modul pružnosti (2,7 GPa). Nakoniec bol skúmaný vplyv starnutia, tepla a vriacej vody na mechanické vlastnosti týchto vlákien.

Since the discovery of organic semiconductors (OSCs) over four decades ago, the field of organic electronics has broken our misconceptions regarding the possibilities of modern electronics. The synthetic toolkit of organic chemistry enables the creation of a limitless number of unique OSCs that can be specifically tailored and engineered with the specific and desired properties for unique applications. The rapid adoption of modern information systems, “Internet of Things,” in which smart devices and sensors ubiquitously collect and exchange data has resulted in a need for low-cost sensors to be deployed everywhere from the monitoring of food supply chains, environmental conditions, to human health. Organic thin-film transistors (OTFTs) are a necessary component to support these technologies. However, their mass adoption will require reduction in cost and improved compatibility with low voltage generating printed batteries or flexible and ultrathin photovoltaics. This thesis is focused on the development of high performing solid state polymer electrolytes to be employed as the gating medium in OTFTs. The choice of conventional gating materials often leads to a tradeoff between high capacitance, operating speed and material softness. For example liquid electrolyte gating materials have high capacitance but low operating speed and are liquid at room temperature which is unacceptable for many electronics application. Polymer gating materials often have lower capacitance but fast operating conditions and solid at room temperature. In this thesis we establish structure property relationships which aid in the design of novel block copolymer-based gating materials which simultaneously enable the increase in capacitance and switching speed while remaining solid at room temperature. In the first study I established a styrene-based ionic liquid monomer can be using as a controlling monomer in the nitroxide mediated copolymerization of methacrylates. The second study then focuses on the integration of these materials into OTFT devices; the morphology (block vs random copolymers) effect on device performance is assessed. The last study builds on the findings of the previous study and further explores the structural elements of block copolymers on device performance. The work presented here outlines the development of advanced poly(ionic liquid) based solid electrolyte materials that enables both reduced operating voltages and fast switching. Finally, we establish structure-property relationships that relate the molecular architecture to OTFT device performance paving the way for the adoption of a new generation of high performing, printable and flexible electronics.

Teoretická část této diplomové práce popisuje vlastnosti a možnosti modifikace poly(3-hydroxybutyrátu) (PHB) a amorfní poly(mléčné kyseliny) (PLA) a jejich směsí. V experimentální části je studována reaktivita Joncrylu, Raschigu a fosfitových činidel trifenylfosfitu, tris(nonylfenyl) fosfitu a difenylisodecylfosfitu s čistým PLA a PHB. Raschig, oligomerní aditivum na bázi polykarbodiimidu, prokázal v množství 2hm. % zvýšení viskozity taveniny obou polymerů, a proto byl použit k přípravě směsí o pěti hmotnostních poměrech PHB/(PHB+PLA). Vzorky s Raschigem a odpovídajícími nereaktivními vzorky byly studovány pomocí reologie, gelové permeační chromatografie a modulované diferenční kompenzační kalorimetrie. Výsledky naznačily reakce Raschigu v PHB/PLA směsích vedoucí k rozvětveným strukturám. Rychlost reakce však není nedostatečná ke kompenzaci poklesu viskozity v důsledku degradace při zpracování. Následně zůstává nezreagované množství Raschigu v matrici. Na základě těchto zjištění se dospělo k závěru, že Raschig se chová spíše jako relativně účinný stabilizátor reologických vlastností, než jako činidlo pro záměrnou modifikaci struktury směsí PHB/PLA. Za účelem studia mechanických vlastností těchto směsí byly ve dvoušnekovém extrudéru připraveny vzorky plastifikovány acetyltributylcitrátem.

Thesis advisor: Jeffery A. Byers / This dissertation discusses the development of a family of iron complexes and their role in the synthesis of degradable polymers. Chapter one will introduce the different areas of redox-switchable polymerization. In chapter two the synthesis of block copolymers containing a polyester and polyether block is presented. The application redox-switchable polymerization to form a copolymer with two fundamentally distinct backbone functionalities and their characterization is discussed. In chapter three the synthesis of a degradable cross-linked polymer through a novel redox-triggered cross linking event is summarized. In chapter four, a detailed mechanistic study of iron-complex catalyzed epoxide polymerization is examined and a unique mechanistic scheme is proposed. Lastly, in chapter five the synthesis and characterization of a formally iron(I) complex is presented. This complex shows remarkable catalytic activity towards ring-opening polymerization. / Thesis (PhD) — Boston College, 2018. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Changes in the synthesis and activity of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are associated with myocardial remodeling. Here we measured the expression and activity of MMPs and TIMPs, and tested the hypothesis that increased MMP activity plays a proapoptotic role in β-adrenergic receptor (β-AR)-stimulated apoptosis of adult rat ventricular myocytes (ARVMs). β-AR stimulation (isoproterenol, 24 h) increased mRNA levels of MMP-2 and TIMP-1 while it decreased TIMP-2 mRNA levels as analyzed by real-time PCR. Western blot analysis, immunocytochemical analysis, in-gel zymography, and MMP-2 activity assay confirmed β-AR-stimulated increases in MMP-2 protein-levels and activity. Inhibition of MMPs using GM-6001 (a broad-spectrum inhibitor of MMPs), SB3CT (inhibitor of MMP-2), and purified TIMP-2 inhibited β-AR-stimulated apoptosis as determined by TdT-mediated dUTP nick end labeling staining. Treatment with active MMP-2 alone increased the number of apoptotic cells. This increase in MMP-2-mediated apoptosis was inhibited by GM-6001 and SB3CT pretreatment. Coimmunoprecipitation studies indicated increased physical association of MMP-2 with β1-integrins after β-AR stimulation. Inhibition of MMP-2 using SB3CT or stimulation of β1-integrin signaling using laminin inhibited the increased association of MMP-2 with β1- integrins. β-AR stimulation increased poly-ADP-ribose-polymerase cleavage, which was inhibited by inhibition of MMP-2. These data suggest the following: 1) β-AR stimulation increases MMP-2 expression and activity and inhibits TIMP-2 expression; 2) inhibition of MMPs, most likely MMP-2, inhibits β-AR-stimulated apoptosis; and 3) the apoptotic effects of MMP-2 may be mediated, at least in part, via its interaction with β1 integrins and poly-ADP-ribose-polymerase cleavage.

There are numerous RNAs transcribed in the cell that are not directly involved in protein translation. Maintaining proper levels of RNA is crucial for cell viability, making RNA surveillance an essential process (equivalent to regulating protein levels). Mtr4 is an essential RNA helicase that activates exosome-mediated 3'-5' turnover in RNA processing mechanisms. Mtr4 has several binding partners, with the most prominent one being the complex Trf4/5-Air1/2-Mtr4 polyadenylating (TRAMP) complex. The polyadenylation and unwinding activity of TRAMP is modulated by a sensing mechanism in Mtr4 that detects both length and identity of 3'-end poly(A) tails. While it has been known that Mtr4 has an unwinding preference for substrates with a 3' poly(A) tail and a length of approximately 5 nucleotides, the mechanistic detail is unclear. It is also unclear what structural features of Mtr4 contribute to this sensing function. By using x-ray crystal structures of Mtr4, a ratchet helix was identified to interact with RNA substrates. Significant conservation of this ratchet helix along the RNA binding path was observed, similar to conservation patterns throughout Ski2-like and DEAH/RHA-box helicases. Structural characterization revealed a novel arch domain shown to bind structured RNAs, which may aid in cooperative RNA recognition in conjunction with the ratchet helix. In this thesis we demonstrate that the conserved residues at the third (R1030) and fourth (E1033) turns of the Mtr4 ratchet helix uniquely influence RNA unwinding rates. Furthermore, when mutated, ratchet helix positions confer slow growth phenotypes to Saccharomyces cerevisiae and are synthetically lethal in an Mtr4-archless background. The unwinding activity of these mutants when in the TRAMP complex alters the unwinding rates of Mtr4, and in some instances recovers substrate specificity. Our findings demonstrate the importance of R1030 and E1033 for helicase activity, and additionally link the arch domain of Mtr4 in essential unwinding events.