Thesis (M.Sc. (Biochemistry)) --University of Limpopo, 2004 / Refer to the document / National Research Foundation (NRF) and the UNlN Research Commitee

Zur Identifizierung geeigneter Routinemarker für die Prognose von Ependymompatienten führten wir immunhistochemische Untersuchungen und statistische Auswertungen an Ependymomen und Daten von 32 Erwachsenen und 23 pädiatrischen Patienten durch. Davon wurden bei drei Tumoren auch Rezidive untersucht, so dass insgesamt 59 Ependymome in die Untersuchung eingeschlossen wurden. Im Einzelnen handelte es sich um 11 myxopapilläre Ependymome, 6 Subependymome, 19 Ependymome und 23 anaplastische Ependymome. Die größten Fallgruppen bildeten pädiatrische Patienten unter drei Jahren und Erwachsene zwischen 50 und 70 Jahren. Bei Kindern war mit 45,8% die infratentorielle, bei Erwachsenen mit 65% die spinale Tumorlokalisation am häufigsten. Die untersuchten spinalen Ependymome entsprachen zu gleichen Teilen myxopapillären Ependymomen WHO Grad I und Ependymomen WHO Grad II. In supratentorieller Lage fanden sich mit 67% überwiegend anaplastische Ependymome WHO Grad III. Auch bei den infratentoriell gelegenen Ependymomen waren mit 63% die Mehrzahl anaplastische Ependymome, daneben fanden sich 29,6% Ependymome WHO Grad II. Beim Vergleich des von uns definierten und bestimmten Ki67-Scores als Zeichen für die Ependymomproliferation und der immunhistochemischen Positivität für HCK fiel nach Anwendung des Chi-Quadrat-Tests mit p=0,067 ein deutlicher Trend zu schwächerer punktförmiger Positivität bei höherem Ki67-Score auf. Dieser Trend setzte sich in der Erwachsenengruppe separat fort, während er in der Kindergruppe allein nicht nachweisbar war. In der Erwachsenengruppe war mit 28% ein deutlicher Anteil myxopapillärer Ependymome vorhanden, welche bei den Kindern nur 8% ausmachten.Möglicherweise spielt die veränderte HCK-Expression in der Subgruppe der myxopapillären Ependymome eine Rolle. Unsere Untersuchungen zeigten außerdem mit p=0,057 einen deutlichen Trend zu längerem Überleben bei immunohistochemischer DBC1-Negativität. Die Multivarianzanalyse mittels Cox-Regression wies eine Positivität für DBC1 als unabhängigen Risikofaktor für eine kürzere Überlebenszeit nach. Des Weiteren konnte eine mit p=0,013 signifikante Korrelation zwischen immunhistochemischer Positivität für DBC1 und höherem Ki67-Score gezeigt werden. Auch mit höherem WHO-Grad korrelierte die DBC1-Positivität mit p=0,009. Besonders infratentoriell gelegene Ependymome zeigten DBC1-Reaktivität. Hier treten bekannterweise häufiger anaplastische Ependymome mit höherem Proliferationsindex auf. Unsere Ergebnisse legen somit die Eignung des Markers DBC1 als immunhistochemische Routineuntersuchung für die Beurteilung der vom Resektionsstatus unabhängigen Prognose und Überlebenszeit von Ependymompatienten nahe. / Immunohistochemical markers for prognosis and proliferation in ependymomas of children and adults

A dieta pode regular a proliferação e diferenciação no epitélio gástrico de ratos durante o período pós-natal. Avaliamos o efeito tardio do desmame precoce na massa corpórea, proliferação (imunomarcação para Ki-67) e diferenciação celular (immunoblot de Mist 1 e pepsinogênio C) aos 15, 18, 30 e 60 dias. Ratos Wistar foram divididos nos grupos: desmame precoce (DP) aos 15 dias e desmamados aos 21 dias (A). Os resultados mostraram: massa corpórea aos 18 (p<0,01) e 21 (p<0,05) dias no grupo DP menor do que no A. Aos 60 dias, os valores se equiparam, porém há diferença entre machos e fêmeas. O índice de proliferação, a espessura da mucosa e profundidade da glândula foram superiores aos 18 dias no grupo DP (p<0,05), havendo resultado oposto aos 30 dias e nenhuma resposta aos 60 dias; houve variação no conteúdo de Mist 1 (p<0,05) e pepsinogênio C (p<0,01) nos animais DP aos 18 dias. Assim, O DP alterou o ganho de peso corporal e crescimento do estômago logo após o início da ingesta de ração, e tal reposta foi compensada na idade adulta. / Diet can regulate proliferation and differentiation in gastric epithelium of rats during the postnatal period. We evaluated the late effects of early weaning on body mass, cell proliferation (Ki-67 immunolabeling) and differentiation (Mist 1 and pepsinogen C by immunoblot) at 15, 18, 30 and 60 days. Wistar rats were divided into groups: early-weaned (EW) at 15 days, and suckling and weaned at 21 days (S). The results showed that: at 18 (p<0,01) and 21 (p<0,05) days, body mass was lower in EW than in S. At 60 days, values were similar between S and EW, however we registered a difference between males and females. The proliferation index, mucosal thickness and gland depth were higher in EW (vs. S) at 18 days (p<0,05), being reversed at 30 days, whereas no change detected at 60 days. We found a variation in Mist 1 (p<0,05) and pepsinogen C (p<0,01) at 18 days. Thus, EW altered body weight gain and stomach growth soon after its onset, and part of responses were compensated in adulthood.

Doctor of Philosophy (PhD) / It is well recognised that both genetic and environmental factors determine an individual’s predisposition to asthma. In recent years, the airway smooth muscle (ASM) cell has come to the attention of researchers to, not merely be a contractile cell of the airway, but one that orchestrates events affecting airway remodelling and proliferation. Experiments described in this thesis have, for the first time, examined genes that are associated with various aspects of the pathogenesis of asthma by using the candidate gene approach and a genome wide search. Genes have not only been identified to be differentially expressed in ASM cells derived from asthmatic and non-asthmatic participants, but have also been linked with a functional consequence of asthma. The three genes found to be differentially regulated between ASM cells derived from asthmatic and non-asthmatic participants were Peroxisome Proliferator-Activated Receptor- gamma (PPARγ), mimecan and fibulin-1. Expression of the anti-proliferative transcriptional factor PPARγ, found by the candidate gene approach, was elevated in ASM cells derived from asthmatic participants. Whilst elevated, the anti-proliferative effect of PPARγ was absent in ASM cells derived from asthmatic participants. By microarray analysis, mimecan, an anti-proliferative agent was identified. Mimecan levels, although not different basally in ASM cells, were upregulated by transforming growth factor β (TGFβ) only in asthmatic derived ASM cells. Silencing mimecan, by the use of specific oligonucleotides, increased proliferation of ASM cells. This suggested that by increasing mimecan expression, the proliferation of ASM cells may be halted. Fibulin-1, also found by microarray analysis and the final gene examined in this thesis, was found in elevated levels in BAL fluid, serum and ASM cells obtained from asthmatic participants. In addition, ASM cells derived from asthmatic participants, for the first time were shown to have faster wound healing rates compared with nonasthmatics. The elevated fibulin-1 levels in ASM cells derived from asthmatic participants, in the presence of TGFβ, were demonstrated to contribute to this increased wound healing. Specifically, fibulin-1 was found to affect wound healing by increasing proliferation rather than migration. The current available treatments for asthma, target the contractility and inflammatory conditions in the airway. Through this thesis, novel genes discovered to be associated with proliferation may be potential therapeutic targets to treat asthma. In particular, the fibulin-1 gene is outstandingly promising, as it was shown that silencing fibulin-1 resulted in slower wound healing rates through decreased cell proliferation, to possibly inhibit the airway remodelling observed in asthma, and furthermore, corticosteroid therapy was demonstrated not to affect to this gene.

Thesis (M. Phil.)--University of Hong Kong, 2007. / Also available online.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Thesis (M. Phil.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

E6-associated protein (E6-AP), which was originally identified as an ubiquitin-protein ligase, also functions as a co-activator that enhances the hormone-dependent transactivation of estrogen (ER) and progesterone (PR) receptors. To investigate the in vivo role of E6-AP in mammary gland development, we generated transgenic mouse lines that specifically overexpress either wild-type human E6-AP (E6-APWT) or the ubiquitin-protein ligase defective mutant E6-AP (E6-APC833S) in the mammary gland. Here we show that overexpression of E6-APWT results in impaired mammary gland development. In contrast, overexpression of E6-APC833S or loss of E6-AP (E6-APKO) increases lateral branching and alveolus-like protuberances in the mammary gland. We also show that the mammary phenotypes observed in the E6-AP transgenic and knockout mice are in large part due to the alteration of PR-B protein levels. RNAi-mediated knockdown of E6-AP in T47D breast cancer cells increased PR-B protein levels and stability. In vitro ubiquitination assay using purified E6-AP and PR-B reinforce these conclusions and demonstrate that E6-AP promotes PR-B turnover in an ubiquitin-dependent manner. Furthermore, we also show that E6-AP regulates progesterone-induced Wnt-4 expression by modulating the steady state level of PR-B in both mice and in human breast cancer cells. This novel mechanism appears to regulate normal physiology of the mammary gland and its dysregulation may prove to contribute importantly to mammary cancer development and progression. To test this hypothesis, we examined the E6-AP transgenic mice for tumor formation over a period of 6, 9, 12, 18 and 24 months. Our data shows that, unlike the E6-APWT mice that show normal phenotype, the E6-APC833S mice develop mammary hyperplasia at high penetrance (80%); with a median latency of 18 months. Our findings indicate that the inactivation of the E3-ligase function of E6-AP is sufficient to initiate the process of mammary tumor development. These findings strongly suggest that E6-AP may act as a tumor suppressor by down regulating the ER-alpha, PR-B and thereby their signaling pathways.

La crête neurale (CN) est une structure transitoire apparaissant en bordure de la plaque neurale chez les embryons de vertébrés. Au cours du développement embryonnaire, les cellules de la CN prolifèrent, subissent une transition épithélio-mésenchymateuse, migrent et se différencient en de nombreux types cellulaires tels que des neurones et cellules gliales du système nerveux périphérique, des mélanocytes, des cellules musculaires lisses ou des élements du squelette cranio-facial. Afin de mieux comprendre les mécanismes moléculaires contrôlant la prolifération et la spécification des cellules de la CN, nous avons étudié le rôle de deux facteurs de transcription, Hairy2 et Stat3, via des expériences de perte et gain de fonction chez l’embryon de xénope. Le gène Hairy2 code pour un facteur de transcription bHLH-O répresseur. Il est exprimé précocement au niveau de la bordure de la plaque neurale incluant la CN présomptive. Nous avons montré que Hairy2 est requis pour la prolifération des cellules de la CN en aval de signaux FGFs et qu’il maintient les cellules dans un état indifférencié en réprimant l’expression précoce des gènes spécifiques de la CN. Hairy2 réprime aussi la transcription du gène Id3 codant pour un facteur HLH essentiel à la prolifération des cellules de la CN. Id3 affecte également Hairy2. Nous avons observé que la protéine Id3 interagit physiquement avec Hairy2 et bloque son activité, démontrant que les interactions entre Hairy2 et Id3 jouent un rôle important dans la prolifération et la spécification des cellules de la CN. Afin de comprendre le mode d’action de Hairy2 dans la CN, nous avons comparé les propriétés de la protéine Hairy2 sauvage à celle d’une version mutée de la protéine incapable de lier l’ADN. Nos résultats ont montré que Hairy2 fonctionne selon deux mécanismes distincts. La capacité de Hairy2 à promouvoir la survie et la maintenance des cellules progénitrices de la CN dans un état non spécifié et indifférencié est dépendante de sa liaison à l’ADN. A l’inverse, sa capacité à stimuler la prolifération cellulaire et l’expression des gènes spécifiques de la CN est indépendante de sa liaison à l’ADN mais nécessite l’activation du ligand du récepteur Notch, Delta1. De plus, nous avons également montré que la capacité de Hairy2 d’induire Delta1 dans la CN requiert Stat3. Le gène Stat3 code pour un facteur de transcription latent dans le cytoplasme pouvant être activé par de nombreux signaux extracellulaires. Nos résultats ont montré que Stat3 joue un rôle crucial dans la prolifération cellulaire et dans l’expression des gènes de la bordure de la plaque neurale et de la CN. Stat3 est phosphorylé directement par la voie de signalisation FGF via FGFR4 et est requis in vivo en aval de FGFR4. Nous avons aussi montré que Hairy2 et Id3 sont des régulateurs positifs et négatifs de l’activité de Stat3 qui facilite et inhibe la formation du complexe Stat3-FGFR4, respectivement. De plus, Stat3 contrôle la transcription des gènes Hairy2 et Id3 de manière dose dépendante. Nous avons observé que Hairy2 est activé à faible dose et Id3 à forte dose de Stat3, suggérant que Stat3 s’auto-régule de manière indirecte via l’activation d’une boucle de rétro-contrôle positive (Hairy2) et une négative (Id3). Stat3 régule également de manière dose dépendante la prolifération et la différenciation des cellules de la CN. Une faible activité de Stat3 stimule la prolifération cellulaire et l’expression des gènes spécifiques de la CN tandis qu’une forte activité de Stat3 ralentit le cycle cellulaire, inhibe l’expression des gènes de la CN et maintient les cellules de l’ectoderme dans un état non spécifié et indifférencié. En conclusion, nous montrons pour la première fois que Stat3, en aval des FGFs et sous le rétro-contrôle de Hairy2 et Id3, joue un rôle essentiel dans la coordination de la progression du cycle cellulaire et de la spécification de la CN au cours du développement embryonnaire du xénope.

The pancreas is an endodermally derived organ consisting of three major cell lineages. The endocrine cells, organised into the Islets of Langerhans, regulate blood glucose homeostasis by producing and secreting hormones such as glucagon and insulin into the bloodstream. The major part of the pancreas consists however of acinar cells that produce digestive enzymes that are transported via a highly branched ductal system to the duodenum where they function in breakdown of food. Early in pancreas development a dorsal and ventral evagination of the foregut epithelium appear, resulting in the formation of the dorsal and ventral pancreatic bud. These pancreatic buds subsequently grow, branch and differentiate to form the mature pancreas via a process controlled by intrinsic factors, such as transcription factors, and extracellular signals. Insulin promoter factor 1 (Ipf1), also known as Pdx1 (for Pancreatic duodenal homeobox gene 1), is required for pancreas development. Although the evagination of pancreatic buds still occurs in Ipf1/Pdx1 mutant mice, the subsequent proliferation, branching and differentiation is impaired, resulting in complete pancreatic agenesis. Gene array profiling identified several candidate Ipf1/Pdx1 target genes, including FgfR2IIIb, ErbB3, Ptf1a/p48, Pax6 and Nkx6.1, in pancreatic progenitor cells. Together these genes provide a mechanistic explanation for the pancreatic growth arrest observed in Ipf1/Pdx1 deficient mice. In addition, Spondin1, which has not previously been described in the pancreas, was identified to be regulated by Ipf1/Pdx1. The spatial and temporal expression pattern of Spondin1 defines Spondin1 as a marker for early pancreatic progenitor cells. The Notch signalling pathway controls cell type specification and differentiation during pancreas development. The Fringe family of proteins have previously been shown to regulate Notch signalling by altering the interaction between Notch receptors and their ligands, hence affecting the cellular response. Manic Fringe (MFng) is transiently expressed in pancreatic pro-endocrine cells between E9.5 and E14.5. The expression of MFng is regulated by Ngn3, which may suggest a role for MFng in pro-endocrine cell maturation. The lack of a pancreatic phenotype in transgenic mice overexpressing MFng in the pancreatic epithelium and in MFng null mutant mice, however, provide evidence that MFng is dispensable for the specification, differentiation and function of the adult pancreas. Inhibitors of DNA binding (Id) proteins are generally known as inhibitors of differentiation, a feature they mainly perform by forming non-functional heterodimers with bHLH proteins, thereby inhibiting downstream targets of the bHLH proteins. Id proteins also promote cell proliferation by interacting with the cell cycle machinery. In the developing pancreas Id2 and Id3 are co-expressed in an overlapping manner during the period of massive proliferation and expansion of the pancreatic epithelium, suggestive of a role for the Id proteins during these processes. In addition, Id4 expression is also detected in the embryonic pancreas, albeit at lower levels. Gain- and loss- of- function analyses suggest however that specification, differentiation and function of the adult pancreas are largely independent of Id function.