Η πρωτεΐνη Tip60 (Tat-interactive protein, 60 kDa) είναι μέλος της οικογένειας πρωτεϊνών MYST και αρχικά προσδιορίστηκε ως αλληλεπιδρώσα πρωτεΐνη με την HIV-1 ΤΑΤ πρωτεΐνη. Πολλά από τα μέλη της οικογένειας MYST μεταξύ αυτών και η Tip60, δρουν ως ακετυλοτρανσφεράσες ιστονών, γεγονός που υποδηλώνει πιθανούς ρόλους στην αναδιαμόρφωση της χρωματίνης και στην γονιδιακή ρύθμιση. Στη παρούσα διπλωματική, στόχος ήταν η μελέτη του προφίλ έκφρασης της ακετυλοτρανσφεράσης Τip60 σε πληθυσμούς Τ βοηθητικών παρθενικών (CD3+CD4+CD45RA+) και Τ βοηθητικών μνημονικών (CD3+CD4+CD45RO+) λεμφοκυττάρων καθώς και η δράση της στην μεταγραφή του HIV-1 και της IL-2. Οι λόγοι που μας οδήγησαν στην μελέτη της Τip60 προκύπτουν από πρόσφατα αποτελέσματα του εργαστηρίου μας, που δείχνουν ότι υπάρχει μια πιθανή αλληλεπίδραση της Tip60 με τον παράγοντα Εts-2, ο οποίος συμμετέχει στη ρύθμιση της μεταγραφής τόσο του γονιδίου της IL-2 όσο και του HIV-1. Επιπλέον η IL-2 και ο HIV-1 παρουσιάζουν κοινή μεταγραφική ρύθμιση που ελέγχεται από την πρόσδεση μεταγραφικών παραγόντων σε κοινά cis στοιχεία. Επίσης, η Tip60 είναι μια Τat-αλληλεπιδρώσα πρωτεΐνη που κωδικοποιείται από το γονίδιο Tat του HIV-1. Τέλος το γεγονός ότι ο HIV-1 παραμένει σε λανθάνουσα κατάσταση στα εν ηρεμία Τ βοηθητικά λεμφοκύτταρα καθώς και ότι ο παράγοντας Εts-2, που πιθανόν αλληλεπιδρά με την Tip60, συμμετέχει στη καταστολή της έκφρασης του ιού, υποδηλώνει μια πιθανή συμμετοχή της Tip60 σε αυτή τη διαδικασία. Αρχικά ελέγξαμε την έκφραση της Tip60 σε υποπληθυσμούς μονοπύρηνων κυττάρων περιφερικού αίματος (PBMCs). Η υψηλότερη έκφραση παρατηρήθηκε στα Τ βοηθητικά λεμφοκύτταρα (CD4+). Για να εξακριβώσουμε το αν και πώς εκφράζεται στους υποπληθυσμούς των Τ λεμφοκυττάρων, απομονώσαμε Τ βοηθητικά μνημονικά λεμφοκύτταρα (CD3+CD4+CD45RO+) από ολικό αίμα ενηλίκων και Τ βοηθητικά παρθενικά λεμφοκύτταρα (CD3+CD4+CD45RA+) από αίμα νεογνών (ομφαλίου λώρου). Τα αποτελέσματά μας έδειξαν ότι σε συνθήκες μη ενεργοποίησης η Τip60 εκφράζεται περισσότερο στα μνημονικά Τh λεμφοκύτταρα σε σχέση με τα παρθενικά Τh λεμφοκύτταρα (στα οποία η έκφραση του παράγοντα παραμένει ουσιαστικά αμετάβλητη). Μετά από την ενεργοποίηση των κυττάρων με PMA/IONO (P/I) η έκφραση της Tip60 αυξάνεται σημαντικά στα μνημονικά Th λεμφοκύτταρα ενώ στα παρθενικά Th παραμένει ουσιαστικά αμετάβλητη. Για να εντοπίσουμε μια λευχαιμική κυτταρική σειρά κατάλληλη για να χρησιμοποιηθεί για πειράματα διαμολύνσεων και ανοσοφθορισμού, ελέγξαμε την έκφραση της Tip60 σε εννέα λευχαιμικές κυτταρικές σειρές και είδαμε ότι η Τ λεμφοκυτταρική λευχαιμική σειρά Jurkat παρουσίαζε την υψηλότερη έκφραση της Τip60. Η κυτταρική σειρά Jurkat παρουσιάζει επίσης παρόμοιο μοτίβο έκφρασης σε επίπεδο mRNA με τα Τ βοηθητικά παρθενικά λεμφοκύτταρα. Την έκφραση της Tip60 την ελέγξαμε και σε πρωτεϊνικό επίπεδο με τη μέθοδο Western blot σε ολικά πρωτεϊνικά εκχυλίσματα που απομονώσαμε από κύτταρα Jurkat. Για να ελέγξουμε τον εντοπισμό της Τip60 στα Jurkat, προχωρήσαμε σε πειράματα ανοσοφθορισμού. Τα αποτελέσματα έδειξαν, ότι σε συνθήκες μη ενεργοποίησης (CM) και ύστερα από ενεργοποίηση των κυττάρων (P/I) η Τip60 εντοπίζεται στον πυρήνα των κυττάρων Jurkat. Επίσης η ποσότητα της πρωτεΐνης Τip60 δεν αλλάζει ύστερα από την ενεργοποίηση των κυττάρων με μιτογόνα, αποτέλεσμα που συμφωνεί με τα πειράματα Western blot. Προκειμένου να ελέγξουμε τη δράση της Τip60 στη μεταγραφική δραστηριότητα του HIV-1 και της IL-2 προχωρήσαμε σε πειράματα διαμόλυνσης Jurkat κυττάρων. Αρχικά διαμολύναμε τα κύτταρα με αυξανόμενες ποσότητες πλασμιδίου υπερέκφρασης της Τip60 (pCMX-tip60) και ελέγξαμε την έκφραση της IL-2 σε μεταγραφικό επίπεδο. Παρατηρήθηκε ότι αυξανομένης της ποσότητας του pCMX-tip60, αυξανόταν και η έκφραση του IL-2 mRNA. Μέσω πειραμάτων CAT-assays σε κύτταρα Jurkat, τα οποία συνδιαμολύναμε με αυξανόμενες ποσότητες πλασμιδίου pCMX-tip60 και πλασμιδίου αναφοράς CAT, που βρίσκεται κάτω από τον έλεγχο του HIV-1-LTR, διαπιστώσαμε ότι η υπερέκφραση της Τip60 αυξάνει και τη μεταγραφική ενεργότητα του υποκινητή του HIV-1. Προκειμένου να διαπιστώσουμε εάν η Tip60 δρα απευθείας στον υποκινητή του HIV-1 και της IL-2 μέσω της πρόσδεσής της σε αυτόν είτε άμεσα, είτε έμμεσα μέσω συμπλόκου με άλλες πρωτεΐνες, προχωρήσαμε σε πειράματα ανοσοκρατακρήμνισης χρωματίνης (ChIP assays) του υποκινητή της IL-2. Παράλληλα επειδή δεν υπάρχει γνωστή θέση πρόσδεσης της Tip60 απευθείας στην LTR αλληλουχία του HIV-1 και στον υποκινητή της IL-2 αναζητήσαμε την πρωτεΐνη με την οποία θα μπορούσε να αλληλεπιδρά προκειμένου να προσδεθεί στα παραπάνω. Με πειράματα συνανοσοκατακρήμνισης διαπιστώσαμε ότι υπάρχει αλληλεπίδραση ανάμεσα στην Tip60 και στον παράγοντα Ets-2, ο οποίος προσδένεται άμεσα στην LTR αλληλουχία του HIV-1 και στον υποκινητή της IL-2 και έτσι επαληθεύσαμε τις αρχικές υποθέσεις για την αλληλεπίδραση τους. Τα πειράματα που πραγματοποιήσαμε μας οδήγησαν στα παρακάτω συμπεράσματα: Υπάρχει διαφορική έκφραση της Tip60 ανάμεσα στα Τ βοηθητικά παρθενικά και μνημονικά λεμφοκύτταρα, αφού σε συνθήκες μη ενεργοποίησης (CM) η έκφραση της Tip60 είναι μεγαλύτερη στα μνημονικά σε σχέση με τα παρθενικά Τh λεμφοκύτταρα. Η ενεργοποίηση των κυττάρων με μιτογόνα προκαλεί επαγωγή της έκφρασης της Tip60 στα μνημονικά Τh λεμφοκύτταρα, ενώ στα παρθενικά δεν προκαλεί καμία ουσιαστική αλλαγή. Η πρωτεΐνη Tip60 δρα σαν συνενεργοποιητής της μεταγραφής του ιού HIV-1 και της IL-2 μιας και προκαλεί επαγωγή στην μεταγραφική τους δραστηριότητα. Η πρωτεΐνη Tip60 αλληλεπιδρά in vivo με τον παράγοντα Εts-2 και παρουσιάζει το ίδιο πρότυπο πρόσδεσης με αυτόν, στον υποκινητή της IL-2 στην κυτταρική σειρά Jurkat. / Tip60 (Tat-interactive protein, 60 kDa) is a member of the MYST protein family and was initially identified as an interacting protein with the HIV-1 TAT protein. Several members of MYST family, Tip60 among them, act as histone acetyltransferases, suggesting their possible roles in chromatin remodeling and gene regulation. We chose to study Tip60 because it is a Tat-interactive protein and Tat is encoded by the Tat gene of HIV-1. Recent results from our laboratory suggest that there is a possible interaction between Tip60 and Εts-2 proteins, which is involved in the regulation of the transcription of both IL-2 and HIV-1. In addition, the transcription of IL-2 and HIV-1 is regulated by common transcription factors that act through binding to common cis-trans elements. We also know that the HIV-1 virus is transcriptionally active in activated CD4 T cells, and inactive in naïve CD4 T cells and that HIV-1 expression is blocked in naive Th cells by the transcriptional factor Ets-2. So the possible interaction between Tip60 and Εts-2 protein led us to study the Tip60 protein. The main purpose of this study was to determine the expression profile of Tip60 acetyltransferase in naive T helper (CD3+CD4+CD45RA+) and memory T helper (CD3+CD4+CD45RO+) lymphocytes and its effect on the transcription of HIV-1 and IL-2 (due to their common transcriptional regulation in T helper cells). The expression of Tip60 mRNA was measured in subpopulations of peripheral blood mononuclear cells (PBMCs) and the highest levels were observed in Th lymphocytes. We therefore isolated from human peripheral blood and cord blood, CD3+CD4+CD45RO+ and CD3+CD4+CD45RA+ Th lymphocytes and we examined the expression of Tip60 at the transcriptional level by RT-PCR. The results showed that Tip60 mRNA expression levels were higher in CD3+CD4+CD45RO+ compared to CD3+CD4+CD45RA+ Th cells. Following cell activation with PMA/IONO (P/I), the transcriptional expression of Tip60 was increased in memory Th cells, whereas it remained unchanged in naive Th cells. To identify a suitable cell line for transfection and immunofluorescence experiments, we measured Tip60 expression in nine cell lines and we observed that the T lymphocytic leukemia Jurkat presented the highest expression of Tip60 mRNA. Jurkat cells also presented a similar pattern of Tip60 mRNA expression levels with naïve Th lymphocytes. In addition to determine the localization of Tip60 into the cells, we proceeded in immunofluorescence experiments. As a result, we observed that when cells were cultured in CM media +/- P/I, Tip60 was identified in the nucleus of Jurkat cells. The amount of Tip60 protein remained unchanged after cell activation (which was also determined by Western blot experiments). To study the effect of Tip60 in transcription of HIV-1 and IL-2 we transfected Jurkat cells with increasing amounts of a Tip60 overexpressing vector (pCMX-tip60) and measured the expression of IL-2 and HIV-1 at the transcriptional level. Overexpression of Tip60, induced the transcription of IL-2 and also increased the transcriptional activity of HIV-1. Co-transfection experiments in Jurkat cells with increasing amounts of PCMX-tip60, led to a gradual increase of HIV1-LTR-CAT Finally, we studied if Tip60 acts directly on the promoter of HIV-1 and IL-2 exercising its effect directly or through complexing with other proteins. According to the literature there is no Tip60 binding site in the promoter of HIV-1 and IL-2, so we searched for a protein that could interact with Tip60. We performed co-immunoprecipitation experiments that showed that there was an interaction between Tip60 and Ets-2, a factor that binds directly both on the LTR of HIV-1 and the IL-2 promoter. In this work we suggest that Tip60 is expressed differentially in naïve and memory helper T cells and participates in the transcriptional activation of both HIV-1 LTR and IL-2. Also Tip60 protein interacts in vivo with Ets-2 protein and Tip60 binding activity is similar with Ets-2 binding activity to the ARRE-2 element of the IL-2 promoter in Jurkat cell line.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1972. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

An introduction to nucleoside analogues containing heterocyclic sugar mimics and their synthesis is presented. This includes various ring sizes and different heteroatom combinations. concentrated on work between 1995-2000. The synthesis of novel nucleoside analogues from nitroimidazole precursors has been investigated. The regioisorners I-vinyl-4-nitroimidazole and I-vinyl-S-nitroimidazole have been synthesised from the readily available 4/S-nitroimidazole. Also synthesised from 4/S-nitroimidazoIe IS 3-vinyl-imidazo[ 4' ,S' :S,6]pyrido[2,3- d]pyrimidin-8-one, the pyridine stretched analogue of 3-vinylinosine. 1,3-Dipolar cycloaddition reactions of these three molecules are studied, with stabilised and unstabilised 1,3-dipolar compounds. to produce heterocyclic nucleoside analogues. Structures of these cycloadducts are investigated using nrnr studies to determine the regiochemistry of the reactions. This nrnr evidence is supported my MO calculations. Further studies have established synthetic routes to the pyridine stretched analogues of 2'-deoxyadenosine and 2'-deoxyinosine from deoxyribose and 4/5- nitroimidazole, involving directing the coupling of a chlorosugar to the sodium salt of 4-nitroimidazoIe to yield a maximum of the S-nitroimidazole isomer product

The objective of this project is synthesizing bipyridine-derived ligands in order to study DNA conformational bending. The synthesis of bipyridine derivatives has been investigated. 6,6’-Dibromo-2,2’- bipyridine and small scale of 6,6’-diformyl-2,2’-bipyridine have been successfully synthesized in the laboratory. The synthesis of large amount of a direct precursor to 6,6’-diformyl-2,2’- bipyridine in an multiple step way has been achieved. The synthesis of mono functionalized pyrene derivatives and of 1,6-dissymmetrically functionalized pyrene derivatives has been heavily studied. Successfully methods have been reported in this thesis. The complete assembly of bipyridine and pyrene units into the final ligands and their model has also been studied. Palladium borylation and Suzuki-Miyaura cross-coupling have been used to successfully connect the bipyridine with pyrene units. In addition to Suzuki-Miyaura methodology, the direct coupling of N,N’-dioxide-2,2’- bipyridine with aromatic bromides under palladium catalysis has been investigated. This method could be an alternative way to access to mono-substituted 6-bipyridines, symmetrically or even asymmetrically 6,6’-disubstituted-2,2’-bipyridine derivatives. / Thesis (Master, Chemistry) -- Queen's University, 2009-09-06 01:06:41.646

Made available in DSpace on 2014-06-12T23:04:27Z (GMT). No. of bitstreams: 2 arquivo8877_1.pdf: 1460374 bytes, checksum: c08f2c8b72d50467c3068b2ce9762c97 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2007 / A proteína cerbB-2 tem sido bastante estudada não só por sua importância como fator prognóstico dos carcinomas da mama, mas sobretudo por ser um indicador para terapias com esquemas quimioterápicos. Para eliminar a subjetividade da interpretação do método imunohistoquímico convencional, este estudo tem como objetivo quantificar, morfometricamente, a imunomarcação da proteína cerbB-2 expressa em tumores de mama. Fragmentos de tecido mamário normal (n=10) e com doença tumoral (carcinoma ductal invasivo, CDI, n=51; fibroadenoma, n=11) foram fixados em formalina, submetidos à rotina histológica para inclusão em parafina. Cortes histológicos (4mm), corados em hematoxilina e eosina foram examinados para confirmar o diagnóstico. Os cortes foram incubados com solução de anticorpos por uma por uma hora em temperatura ambiente. A marcação foi visualizada após incubação com diaminobenzidina (DAB) e peróxido de hidrogênio. A análise morfométrica foi realizada utilizando uma estação de análise digital de imagens através do software de análise OPTIMAS®. A partir dos resultados obtidos pode-se concluir que a superexpressão do cerbB-2 em casos de CDI é um fenômeno condizente com o estágio de proliferação das células neoplásicas e quando analisados os casos de fibroadenoma, este marcador não exibiu qualquer correlação ou padrão específico, ao contrário apresentaram resultados semelhantes ao tecido mamário normal. Não houve diferenças significativas entre os diferentes scores qualitativos e a análise morfométrica digital, o que no mínimo demonstra a necessidade de estudos mais acurados a fim de resolver esta dificuldade de interpretação

The oncogenic protein Bcl-2 is overexpressed in many cancers and prevents cells from undergoing apoptosis in response to traditional chemotherapeutic agents. Recent research has focussed on the development of novel agents that can disrupt the function of Bcl-2 and trigger apoptosis in cancer cells. The isothiocyanates are a class of naturally-occurring phytochemical with potential for development as both chemopreventive and chemotherapeutic agents. This thesis investigated the ability of the isothiocyanates to induce apoptosis in cells that overexpressed Bcl-2. Initially, phenethyl isothiocyanate was shown to be cytotoxic to the Jurkat Tlymphoma cell line with an LD50 of 7.4 µM. Bcl-2 expression had little protective effect, and even greater than 50-fold overexpression only increased the LD50 to 15.1 µM. Morphological and biochemical assays indicated that death still occurred by apoptosis despite overexpression of Bcl-2. A variety of other isothiocyanates were also screened for cytotoxic activity. While the isothiocyanate moiety was crucial for induction of apoptosis, the chemistry of the side chain attached to the isothiocyanate moiety also profoundly influenced the ability of an isothiocyanate to kill Bcl-2 overexpressing cells. The aromatic isothiocyanates were generally far more cytotoxic than aliphatic isothiocyanates. However, within the aromatic isothiocyanates tested in this study the length of the carbon linker group, between the phenyl ring and the isothiocyanate moiety, also influenced cytotoxic activity. Phenethyl isothiocyanate was identified as the most promising compound when targeting cells that overexpressed Bcl-2. Given that minor structural alterations significantly altered cytotoxic activity it is hypothesised that specific interactions with cellular targets may mediate induction of apoptosis by the isothiocyanates. Finally, using a sensitive proteomic technique to label oxidised thiol proteins a preliminary investigation of the targets of the isothiocyanates was performed. A number of thiol proteins were selectively modified following exposure to phenethyl isothiocyanate. One thiol protein that consistently changed was identified as mitochondrial peroxiredoxin-3. Changes to the oxidation state of peroxiredoxin-3 occurred well before activation of apoptosis and may play a role in mediating induction of apoptosis in cells that overexpress Bcl-2. The results of this thesis have provided a platform to permit further investigation of the chemotherapeutic potential of the isothiocyanates and investigation of the mechanisms that allow the isothiocyanates to induce apoptosis in cells that overexpress the oncogene Bcl-2. In the future, the identification of primary targets of the isothiocyanates may aid the design and testing of novel anticancer drugs, and it will also provide novel insight into the regulation of apoptosis.

No description available.

No description available.

GLP-2 is a potent intestinotrophic peptide that can increase mucosal growth, intestinal blood flow, and nutrient absorption when administered exogenously. We aimed to delineate the effects of endogenous GLP-2R signalling in conditions of nutrient deprivation and excess. Using a mouse with a targeted genetic deletion of the Glp2r gene (Glp2r-/-), we addressed the hypothesis that the known GLP-2R is required for intestinal adaptation to nutrient deprivation and excess. In Chapter 2, we demonstrate that Glp2r−/− mice fasted for 24 hours and re-fed for 24 hours failed to increase intestinal growth and jejunal crypt cell proliferation compared to littermate Glp2r+/+ mice. Administration of EGF to Glp2r−/− during the re-feeding period rescued this re-feeding defect. Wildtype mice re-fed for 30, 90, and 180 minutes following a 24 hour fast displayed increased jejunal mRNA levels of the ErbB ligands amphiregulin, epiregulin and HB-EGF. Treatment with the pan ErbB inhibitor CI-1033 inhibited induction of these ErbB ligands in jejunum of mice in association with prevention of crypt cell proliferation. Re-feeding also caused an increase in jejunal p-Akt levels and treatment with CI-1033 prevented increased p-Akt levels. Moreover, re-fed Glp2r−/− mice failed to increase ErbB ligands or p-Akt levels 90 minutes following re-feeding when compared to Glp2r+/+ littermates. Therefore, the GLP-2R is essential for re-feeding induced intestinal adaptation by activating the ErbB network and p-Akt to increase crypt cell proliferation. In Chapter 3, we show that the known GLP-2R is not required for intestinal adaptation to a perceived nutrient deprivation challenge (STZ-induced diabetes) or chronic nutrient excess (high-fat diet induced glucose intolerance). Although exogenous GLP-2 administration has been previously shown to stimulate glucagon secretion, glucose homeostasis was normal in STZ-diabetic and high fat fed Glp2r−/− mice. We also developed a third model of diabetes and glucose intolerance: ob/ob: Glp2r−/−. In the absence of GLP-2R signalling, ob/ob mice display improved oral but impaired intraperitoneal glucose tolerance, elevated fed and fasted glucose levels, increased circulating glucagon, decreased beta cell and increased alpha cell mass. Taken together, these results suggest that endogenous GLP-2R signalling is essential for intestinal and islet adaptation to conditions of nutrient deprivation and excess.

The herpes simplex virus type 2 (HSV-2) UL21 protein is conserved between all members of the Alphaherpesvirinae subfamily. Although UL21 is essential for virus propagation in HSV-2, its function in viral replication is poorly understood. Cells infected with HSV-2 strains lacking UL21 exhibit an approximate two-hour delay in viral gene expression that cannot be explained by a defect in virus entry or capsid engagement with, or movement along, microtubules. However, we noted a defect in the ability of UL21 knockout (KO21) capsids to associate with the nucleus after infection. We found that the delay in viral gene expression was not directly due to the absence of UL21 insofar as cells stably expressing UL21 could not complement the delay in gene expression. We suggest that the KO21 delay in gene expression is due to alterations in virion composition and that in the absence of UL21, a key virion component required for the timely delivery of capsids to the nucleus fails to be packaged into virions. Interestingly, at late times after infection, levels of viral proteins in KO21 infected cells reach wild-type levels, indicating that a secondary function is responsible for the essential nature of UL21. We found that at late times post-infection KO21 infected cells accumulated capsids in the nucleus but these fail to reach the cytoplasm and mature into infectious virions. Thus, we hypothesize that the essential function of UL21 is to facilitate capsid trafficking from the nucleus to the cytoplasm. Moreover, the viral glycoproteins gD and gC were retained in the endoplasmic reticulum, and were under-glycosylated in KO21 infected cells. It is therefore possible that the absence of UL21 prevents the targeting of glycoproteins to the inner nuclear membrane, preventing the formation or function of the nuclear egress complex. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2013-09-10 16:20:32.162