Dissertationen zum Thema „Teeth Growth Molecular aspects“

The purpose of this study was to compare the growth response elicited by an acute bout of resistance exercise (RE) conducted on a traditional weight stack device (WS) and a flywheel device (FW). Eight recreationally trained males (25 ± 9 y, 77 ± 27 kg) performed 4 sets of 7 repetitions of bilateral knee extension on each exercise device separated by 7 days. Muscle biopsies were obtained from the vastus lateralis at rest and 4 hrs post-exercise to examine the expression of selected myogenic and proteolytic genes. RE increased (P < 0.05) mRNA expression of Myogenin (3.6 vs. 3.6 fold), and MyoD (2.2 vs. 2.0 fold) and decreased (P < 0.05) expression of Myostatin (1.4 vs. 1.5 fold) to a similar degree on both exercise devices. There was no change in the expression of Atrogin-1, MuRF-1 or MRF4 following RE on either device. The only device mediated difference in the expression of the selected genes was observed in Atrogin-1 which was lower following RE on the FW versus the WS device. The current data shows that in the initial hrs following RE, use of the FW is as effective as the traditional resistance training devices (WS) in promoting the induction of genes involved with muscle remodeling and growth.<br>School of Physical Education, Sport, and Exercise Science

The human pancreatic cancer cell line T3M4, is known to produce transforming growth factor-alpha (TGF-alpha); as well as overexpress the receptor for this ligand, epidermal growth factor (EGF) receptor. TGF-alpha messenger RNA (mRNA) levels were assayed using northern blot, after addition of epidermal growth factor or TGF-alpha. The level of TGF-alpha mRNA was found to increase 2-fold at 2 hours and then return to near basal levels at 10 hours, after treatment with either ligand. Both ligands were also equipotent in a 2 hour dose response assay, with half maximal stimulation seen at 1 nM and maximal stimulation reached at 4 nM. Furthermore, there appeared to be a 2-fold increase in TGF-alpha transcription as determined by nuclear runoff experiments. Induction of TGF-alpha mRNA coupled with the overexpression of the EGF receptor, may result in a potent autocrine cycle; which may be found in other cancers.

Addenda inserted in back. Includes bibliographical references (leaves 139-160) Assesses the importance of amino acids 221 to 236 of bIGFBP-2 for IGF binding activity, by creating amino acid substitutions.

Thesis (MSc)--University of Stellenbosch, 2003.<br>ENGLISH ABSTRACT: Yeasts, like most organisms, have to survive in highly variable and hostile environments. Survival therefore requires adaptation to the changing external conditions. On the molecular level, specific adaptation to specific environmental conditions requires the yeast to be able: (i) to sense all relevant environmental parameters; (ii) to relay the perceived signals to the interior of the cell via signal transduction networks; and (iii) to implement a specific molecular response by modifying enzyme activities and by regulating transcription of the appropriate genes. The availability of nutrients is one of the major trophic factors for all unicellular organisms, including yeast. Saccharomyces cerevisiae senses the nutritional composition of the media and implements a specific developmental choice in response to the level of essential nutrients. In conditions in which ample nutrients are available, S. cerevisiae will divide mitotically and populate the growth environment. If the nutrients are exhausted, diploid S. cerevisiae cells can undergo meiosis, which produces four ascospores encased in an ascus. These ascospores are robust and provide the yeast with a means to survive adverse environmental conditions. The ascospores can lie dormant for extended periods of time until the onset of favourable growth conditions, upon which the spores will germinate, mate and give rise to a new yeast population. However, S. cerevisiae has a third developmental option, referred to as pseudohyphal and invasive growth. In growth conditions in which nutrients are limited, but not exhausted, the yeast can undergo a morphological switch, altering its budding pattern and forming chains of elongated cells that can penetrate the growth substrate to forage for nutrients. The focus of this study was on elements of the signal transduction networks regulating invasive growth in S. cerevisiae. Some components of the signal transduction pathways are well characterised, while several transcription factors that are regulated via these pathways remain poorly studied. In this study, the RMEt gene was identified for its ability to enhance starch degradation and invasive growth when present on a multiple copy plasmid. Rme1 p had previously been identified as a repressor of meiosis and, for this reason, the literature review focuses on the regulation of the meiotic process. In particular, the review focuses on the factors governing entry into meiosis in response to nutrient starvation and ploidy. Also, the transcriptional regulation of the master initiator of meiosis, IMEt, and the action of Ime1 p are included in the review. The experimental part of the study entailed a genetic analysis of the role of Rme1 p in invasive growth and starch metabolism. Epistasis analysis was conducted of Rme1 p and elements of the MAP Kinase module, as well as of the transcription factors, Mss11p, Msn1p/Mss10p, Tec1p, Phd1p and F108p. Rme1p is known to bind to the promoter of CLN2, a G1-cyclin, and enhances its expression. Therefore, the cell cyclins CLN1 and CLN2 were included in the study. The study revealed that Rme1 p functions independently or downstream of the MAP Kinase cascade and does not require Cln1 p or Cln2p to induce invasive growth. FL011/MUC1 encodes a cell wall protein that is required for invasive growth. Like the above-mentioned factors, Rme1 p requires FL011 to induce invasive growth. We identified an Rme1 p binding site in the promoter of FL011. Overexpression of Rme1p was able to induce FL01t expression, despite deletions of mss11, msn1, ttos, tee1 and phd1. In the inverse experiment, these factors were able to induce FL011 expression in an rme1 deleted strain. This would indicate that Rme1 p does not function in a hierarchical signalling system with these factors, but could function in a more general role to modify transcription.<br>AFRIKAANSE OPSOMMING: Die natuur is hoogs veranderlik en alle organismes, insluitende gis, moet by die omgewing kan aanpas om te kan oorleef. Baie eksterne faktore beïnvloed die ontwikkeling van die gissel. Vir die gis om by spesifieke omgewingstoestande aan te pas, moet die gis op 'n molekulêre vlak: (i) al die omgewingsparameters waarneem; (ii) die waargenome omgewingsparameters as seine na die selkern deur middel van seintransduksieweë gelei; en (iii) transkripsie van gene aktiveer of onderdruk en ensiemaktiwiteit reguleer om sodoende die gepaste molekulêre respons te implementeer. Die beskikbaarheid van voedingstowwe in die omgewing is een van die belangrikste omgewingseine wat eensellige organismes moet kan waarneem. Saccharomyces cerevisiae kan spesifieke ontwikkelingsopsies, na gelang van die voedingstowwe wat beskikbaar is, uitoefen. In groeiomstandighede waar daar 'n oorvloed van voedingstowwe is, verdeel S. cerevisiae d.m.v. mitose en vesprei dit deur die omgewing. Sodra die voedingstowwe uitgeput is, word mitose onderdruk. Diploïede S. cerevisiae inisieer meiose, wat aanleiding tot die vorming van vier spore gee. Hierdie spore bevat slegs die helfte van die ouer se chromosome en kan gevolglik met 'n ander spoor paar om weer 'n diploïede gissel te vorm. Die spore is bestand teen strawwe omgewingstoestande en kan vir lang tye oorleef. Wanneer die spoor aan gunstige groeitoestande blootgestel word, ontkiem dit om aan 'n nuwe giskolonie oorsprong te gee. S. cerevisiae het egter 'n derde ontwikkelingsopsie, naamlik pseudohife-differensiëring. Wanneer die beskikbaarheid van voedingstowwe in die omgewing afneem, maar nog nie uitgeput is nie, ondergaan die gis 'n morfologiese verandering. Hierdie verandering word gekenmerk deur selverlenging, nl. botselle wat slegs aan die een punt van die gissel vorm en dogterselle wat aan die moerderselle geheg bly. Dit lei tot die vorming van kettings van selle wat van die giskolonie af weggroei. Voorts kan die selkettings ook die groeisubstraat binnedring. Dit staan as penetrasie-groei bekend en laat die gis toe om na nuwe voedingsbronne te soek. Hierdie studie het op die elemente van seintransduksieweë, wat by penetrasiegroei betrokke is, gefokus. Sekere komponente van die seintransduksieweë is reeds goed gekarakteriseer, terwyl ander komponente nog grootliks onbekend is. In hierdie studie, word 'n rol vir RME1 in die verbetering van styselafbraak en penetrasiegroei geïdentifiseer. Aangesien Rme1 p voorheen as 'n onderdrukker van meiose geïdentifiseer is, is 'n litetaruurstudie oor die inisiasie van meiose saamgestel. Die faktore wat meiose induseer, naamlik 'n gebrek aan voedingstowwe en die sel se ploïedie, word bespreek. Die regulering van die meester inisieerder van meiosie, IME1, asook die proteïene waarmee Ime1p reageer, is ook in die studie ingesluit. Die eksperimentele deel van die studie behels die genetiese analise van Rme1 p tydens penetrasiegroei en styselhidroliese. 'n Epistase-analise tussen Rme1 p en elemente van die MAP-Kinasemodule, asook van die transkripsie faktore Mss11 p, Msn1p/Mss10p, Tec1p, Phd1p en F108p, is onderneem. Rme1p is bekend om aan die promotor van CLN2 te bind en transkripsie te induseer. Daarom is die selsikliene CLN1 en CLN2 in die studie ingesluit. Die studie dui daarop dat Rme1 ponafhanklik van die MAP-Kinasemodule funksioneer en nie Cln1 p en Cln2p benodig om penetrasiegroei te induseer nie. FL011/MUC1 kodeer vir 'n selwandproteïen wat noodsaaklik vir pentrasiegroei is. Soos in die geval van die bogenoemde faktore, benodig Rme1 p FL011 om penetrasiegroei te kan induseer. Ten spyte van mss11-, msn1-, ttos-, tec1- en phd1- delesies, kan ooruitdrukking van Rme1p die transkripsie van FL011 induseer. In die omgekeerde eksperiment kon die bogenoemde faktore FL011-transkripsie ten spyte van 'n rme1 delesie induseer. Die resultate dui daarop dat Rme1 p nie in 'n hiërargiese pad funksioneer nie, maar dat dit waarskynlik 'n meer algemene rol deur transkripsiemodifisering vervul.

[Truncated abstract] For this thesis, the molecular biology of cancer was approached from two directions. Firstly, an investigation was conducted on the role of growth factor receptor-bound protein 7 (Grb7) in breast cancer. Grb7 is an adapter molecule that binds to a variety of proteins, including the growth factor receptor and proto-oncogene, ErbB2, and mediates signalling to downstream pathways. It has been linked to cell migration and an invasive phenotype, and is of interest as a therapeutic target. To investigate the role of Grb7 in breast cancer, preliminary experiments were performed that, firstly, determined the expression of wild-type Grb7 and a splice variant, Grb7V, in a range of cell lines, and secondly, aided the development of a protocol for treating cells with short interfering RNA (siRNA) against Grb7 and the ErbB ligand, heregulin (HRG), in a cell system appropriate for measuring the functional outcomes. Using this protocol in conjunction with CellTitre (CT) proliferation assays, it was demonstrated that Grb7 does not play a role in the proliferation of either unstimulated or HRG-stimulated SK-BR-3 breast cancer cells. Furthermore, using the protocol in conjunction with Boyden chamber migration assays, it was shown that inhibition of Grb7 expression has a slight stimulatory effect on HRG-stimulated SK-BR-3 cell migration. Thus, Grb7 was found to play only a minor role in the migration of SK-BR-3 cells, suggesting that it is not an ideal anti-cancer target for breast cancers modelled by this cell system. Concurrently, a second investigation was conducted, which similarly sought insight into the molecular biology of cancer, but adopted a more strategic approach. ... These results provide evidence for a biologically significant role for the miR-7-mediated regulation of EGFR expression. A microarray experiment was also performed to identify genes that were down-regulated following treatment with miR-7 compared to NS precursor. Of 248 down-regulated genes, including EGFR, 37 promising new miR-7 target candidates were identified. Functional clustering of down-regulated genes and promising target candidates suggested that miR-7 may have functionally-related targets involved in processes including cell motility and brain-associated functions. This investigation thus yielded a program capable of accurately predicting a miRNA target not predicted by any other target prediction program, verified a previously unknown miRNA:target interaction with functional consequences in cancer cells and provided the first steps towards investigating miR-7-mediated regulation in greater depth. Furthermore, EGFR was, to our knowledge, the first example of a verified miRNA target with target sites that are not conserved across mammals, an observation with important implications for computational target prediction and the evolution of miRNA regulatory systems. In addition, the demonstrated growth inhibitory and cytotoxic effects of miR-7 on lung cancer cells raise the possibility of a miR-7-based therapeutic for the treatment of EGFR-overexpressing tumours.

La malignité des astrocytomes est établie sur base de critères morphologiques définis au sein de la classification de l’Organisation Mondiale de la Santé (OMS). Ce système de gradation, qui s’échelonne de I à IV, constitue actuellement l’outil pronostique le plus fiable. Par facilité, les cliniciens regroupent les astrocytomes de grade I (astrocytomes pilocytiques) et les astrocytomes diffus de grade II sous le terme d’« Astrocytomes de bas-grade » par opposition aux astrocytomes de haut-grade, constitués des astrocytomes anaplasiques (grade III) et des glioblastomes (GBM ;grade IV). Cette terminologie conduit à des prises en charge cliniques inadéquates car elle englobe des tumeurs très différentes en terme d’agressivité :les astrocytomes de grade I, majoritairement non infiltrants, non évolutifs et indolents et les astrocytomes diffus de grade II, toujours infiltrants et évolutifs, progressant systématiquement en astrocytomes de haut-grade et entraînant le plus souvent le décès prématuré du patient. Bien que ces tumeurs soient définies par la classification de l’OMS comme des entités clinicopathologiques distinctes, peu de données sont disponibles dans la littérature pour expliquer leurs particularités biologiques et la pratique quotidienne montre que les différencier peut être difficile. <p><p>Le but des études entreprises au cours de ce travail de thèse est d’apporter une contribution à la compréhension des mécanismes de tumorigenèse qui différencient l’astrocytome de grade I des astrocytomes diffus (grade II-IV), de manière à identifier des voies biologiques qui permettraient, au moins en partie, d’expliquer ces différences de comportement. <p><p>Au cours de la première partie de ce travail, nous avons caractérisé les profils d’expression génomique des astrocytomes de grade I et de grade II, en comparant les données d’expression de gènes (évaluées par des technologies de micropuces d’ADN) de travaux publiés entre 2000 et 2005. L’expression des gènes identifiés a été validée par des analyses de RT-PCR quantitative sur une série indépendante d’astrocytomes de grade I, II et IV. Les fonctions biologiques des protéines codées par chacun de ces gènes ont fait l’objet de recherches bibliographiques détaillées afin de proposer un modèle permettant d’approcher les différences de comportement de ces tumeurs. Cette analyse nous a permis d’identifier TIMP4 (tissue inhibitor of metalloproteinases 4) et IGFBP2 (insulin-like growth factor binding protein 2) comme gènes candidats pour améliorer la caractérisation biologique et clinique des astrocytomes de grade I par rapport aux astrocytomes diffus. TIMP4 et IGFBP2 codent respectivement pour un inhibiteur endogène des métalloprotéinases matricielles (MMPs) et une protéine de liaison capable d’inhiber l’action des « insulin-like growth factors » (IGFs, dont IGFI et IGFII), des facteurs impliqués dans la croissance et la migration des astrocytes normaux et tumoraux. <p><p>Sur base de la surexpression de TIMP4 et d’IGFBP2 dans les astrocytomes de grade I, en comparaison aux astrocytomes diffus de grade II, nous avons posé l’hypothèse suivante :« L’absence d’agressivité des astrocytomes de grade I, en comparaison aux astrocytomes diffus (grade II-IV) pourrait en partie être liée à l’inhibition par TIMP-4 de la protéolyse des complexes IGFBP2-IGFII au sein de ces tumeurs ». Cette protéolyse, qui diminue l’affinité d’IGFBP2 pour IGFII, pourrait contribuer à libérer IGFII dans la matrice extracellulaire (MEC), favoriser la liaison d’IGFII à son récepteur IGF-IR et stimuler la croissance et la migration des cellules astrocytaires tumorales. Pour tester cette hypothèse, nous avons réalisé différentes analyses biochimiques afin i) de caractériser les actions protéolytiques de MMP-2, MMP-9 et MT1-MMP sur le complexe IGFBP2-IGFII, ii) d’identifier la libération d’IGFII lors du clivage de ce complexe, et iii) d’étudier l’action inhibitrice de TIMP-4. A l’aide d’un modèle cellulaire in vitro (lignée astrocytaire tumorale LN229), nous avons ensuite observé l’influence de la protéolyse du complexe IGFBP2-IGFII sur la croissance et la motilité cellulaire. Cette étude a montré :(1) la protéolyse du complexe IGFBP2-IGFII par MMP-9, (2) l’inhibition partielle de cette protéolyse par TIMP-4, (3) la libération d’IGFII résultant de cette protéolyse et (4) les effets stimulants de la libération d’IGFII sur la croissance et la motilité des cellules LN229. Cette étude souligne le rôle important de la protéolyse des complexes IGFBP2-IGFII dans l’agressivité des astrocytomes diffus. Elle confirme les effets stimulants propres d’IGFII, d’IGFBP2 et de MMP-9 sur la motilité et/ou la croissance des cellules astrocytaires tumorales. Enfin, elle identifie un rôle inhibiteur potentiel de TIMP-4 sur la protéolyse du complexe IGFBP2-IGFII, qui pourrait contribuer à expliquer le caractère plus indolent des astrocytomes de grade I en comparaison aux astrocytomes diffus.<p><p>\<br>Doctorat en Sciences médicales<br>info:eu-repo/semantics/nonPublished

Liver progenitor cells (LPCs) play a major role in the regeneration process following chronic liver damage. LPCs can differentiate into hepatocytes and cholangiocytes and thus are capable of replenishing the damaged liver. Due to their plasticity and robust nature in culture systems, they are promising candidates for use in cell therapy. However, to be able to use LPCs as tissue regenerating stem cell-like cells in the clinic, we need to fully understand how they are controlled. Although a strong association between LPCs and inflammation has been shown in many chronic liver diseases, the role of the immune system in LPC-mediated hepatic regeneration is poorly understood. We hypothesise that specific immune cells and mediators are needed to induce the LPC compartment, and that these are common to the LPC response in different injury settings. Therefore, the present study focused on the characterisation of the inflammatory environment in the LPC response, which generates this niche. The aims of this study were (i) to identify the immune cells that are important for the LPC response, (ii) to define the cytokine profile and (iii) to determine the role of the cytokine producing cells during liver regeneration. To study hepatic inflammation following liver injury, a diet-induced model of liver injury (choline-deficient, ethionine-supplemented diet, CDE diet) was compared to two transgenic mouse models of immune-mediated hepatitis (Met-Kb, 178.3). Although all three models are characterised by hepatitis, histological analysis revealed that LPCs were only detectable in the CDE and Met-Kb livers. In the 178.3 model, livers regenerated from proliferating hepatocytes. An LPC response could not be induced in these mice even when liver damage was made more severe. In the other two models, LPC numbers increased over time showing the highest numbers one week after the peak of liver injury. LPCs were often found in close proximity to inflammatory cells, in particular macrophages.

Un des aspects les plus marquants de l'évolution des grands singes est l'augmentation relative du volume du cerveau, et en particulier du néocortex, qui culmine chez Homo sapiens. La microcéphalie primaire est une anomalie congénitale du développement cérébral humain caractérisée par un cerveau normalement formé mais de petit volume. Il en existe une forme isolée, non syndromique, dont la majorité des cas sont d'origine génétique et transmis sur le mode autosomique récessif (MCPH), qui constituent donc un modèle génétiquement simple qui résulte de l'altération d'un seul gène, essentiel dans le développement volumique du cerveau. Une consanguinité parentale est présente dans la majorité des cas, ce qui permet une approche puissante de localisation génomique de la mutation responsable, nommée cartographie d'homozygotie. A ce jour, huit gènes causant cette anomalie ont déjà été identifiés :BRIT1 (MCPH1), ASPM (MCPH5), CDK5RAP2 (MCPH3) et CENPJ (MCPH6), et plus récemment, STIL (MCPH7), CEP152 (MCPH9), WDR62 (MCPH2) et CEP135 (MCPH8). Tous ces gènes jouent un rôle au niveau du cycle cellulaire. Nous avons tenté, au cours de ce doctorat, d’identifier et de caractériser un nouveau gène du locus MCPH4 cartographié au laboratoire et situé sur le bras long du chromosome 15. <p>Dans trois familles MCPH4 originaires de villages voisins du Maroc rural, nous avons affiné la zone de liaison à un segment de 3,7cM, contenant un haplotype commun sur une longueur de 2,7cM suggérant un déséquilibre de liaison autour d’une mutation ancestrale. Le LOD score combiné dans les trois familles était supérieur à 6. Parmi les gènes contenus dans cette région, nous avons sélectionné des candidats que nous avons ensuite analysés par séquençage direct de l’ADN de nos patients. Parmi ces gènes, CASC5 présentait un variant, homozygote chez nos patients, hétérozygote chez leurs parents sains et absent chez 150 contrôles non apparentés. Nous avons utilisé la technologie 454 de séquençage à haut débit de Roche pour séquencer les gènes de l’intervalle de 2.7Mb en une fois. Parmi les mutations identifiées, nous n’avons trouvé qu’une seule variation exonique inconnue qui correspondait à la variation faux-sens déjà identifiée dans le gène CASC5. CASC5 est une protéine centromérique requise pour l’alignement des chromosomes à la métaphase et pour le point de contrôle métaphasique de la progression mitotique. Il était donc potentiellement un très bon candidat causal de la microcéphalie primaire. CASC5 lie directement MIS12, BUB1, BUBR1 et Zwint-1, et fait partie du réseau KMN du kinétochore. Il est nécessaire à l’ancrage des centromères chromosomiques au fuseau mitotique, et est requis pour le contrôle du cycle cellulaire au niveau du Spindle-Assembly Checkpoint.<p>Nous avons ensuite confirmé que la mutation génère un défaut d’épissage chez nos patients consistant en la perte partielle de l’exon 18 dans l’ARNm. La perte de cet exon conduit à un déphasage du cadre de lecture provoquant l’apparition d’un codon STOP prématuré dans l’exon 19. Ceci prédit donc la formation d’une protéine tronquée, ou absente après dégradation par le mécanisme cellulaire de dégradation des ARNm non-sens. Par Western-Blotting nous avons pu révéler, en lignée lymphoblastoïdes, la protéine CASC5 endogène chez tous nos patients, y compris, à notre surprise, chez les sujets atteints. <p>Il est décrit dans la littérature qu’un knockdown de CASC5 provoque un mauvais alignement des chromosomes, une entrée prématurée en mitose et la formation de micronoyaux, conséquence d’un mauvais alignement des chromosomes pendant la métaphase. Les différentes études menées sur le phénotype cellulaire de nos patients en lignées lymphoblastoïdes n’ont pu révéler ces défauts. Notre hypothèse est que l’allèle muté est hypomorphe et que le phénotype cellulaire décrit en boites de culture ne s’observerait in vivo que dans certaines cellules du cerveau en cours de développement.<p>En parallèle de ces travaux, nous avons également contribué à l’identification de la cause d’une microcéphalie primaire syndromique, associée à un diabète insulino-requérant précoce, tansmis sur le mode récessif autosomique et identifié dans une famille d’origine marocaine. Notre laboratoire avait localisé la mutation dans une région de 3 cM du chromosome 4. Parmi les 39 gènes compris dans cette région, nous en avons sélectionné et séquencé plusieurs. Aucun n’a cependant montré de mutation. Un séquençage de l'exome complet de l’un de nos patients, a permis de mettre en évidence une mutation non-sens homozygote dans un gène de l’intervalle critique de liaison. La mutation ségrège avec le phénotype autosomique récessif chez les malades, leurs parents et leurs germains asymptomatiques. L’abondance du transcrit de ce gène a été mesurée en lignées lymphoblastoïdes de patients :il est présent en quantité similaire chez les patients et chez un contrôle non apparenté. <p>En conclusion, notre travail a permis l’identification d’un nouveau gène muté chez des patients atteints de microcéphalie primaire, CASC5, avec un haut degré de preuve de causalité de cette mutation, impliquant ainsi une protéine du réseau KMN du kinétochore dans le développement volumique du cerveau humain. Nous avons par ailleurs contribué à l’identification d’un nouveau gène causant microcéphalie primaire et diabète juvénile, dont le mécanisme biologique est en cours d’investigation.<p><br>Doctorat en Sciences biomédicales et pharmaceutiques<br>info:eu-repo/semantics/nonPublished

[Truncated abstract] The pro-inflammatory cytokine tumour necrosis factor (TNF) has a critical role in skeletal muscle atrophy. The catabolic effect of TNF is partially due to abrogation of the anabolic insulin-like growth factor 1 (IGF-1) signalling pathway. However, the precise signalling events that lead to the loss of myofibrillar protein following activation of TNF receptor are unknown. The over arching aim of the study is to determine the mechanisms of by which TNF induces atrophy in differentiated muscles cells. To achieve this aim a series of experiments were performed to: 1) investigate the molecular events that lead to TNF mediated myofibre atrophy, 2) determine to what extent c-Jun N-terminal Kinase (JNK) signalling plays a part in TNF induced myotube atrophy, and in TNF-mediated inhibition of IGF-1 induced hypertrophy, and 3) use inhibitors of JNK to block the catabolic effects of TNF. 1) To investigate the molecular events that lead to TNF mediated myofibre atrophy, the experiments were conducted using C2C12 mouse myotube cultures and primary myotube cultures derived from FVB mice, and transgenic mice which over-express Class 2 IGF-1 Ea in skeletal muscles (IGF:C2). The treatment of mature C2C12 and FVB primary myotubes (respectively at 7 and 4 days after fusion medium) with 10 ng/mL of TNF for 3 days resulted in statistically significant myotube atrophy (decreased mean width). The observed TNF-mediated atrophy has not previously been demonstrated in tissue cultured myotubes. In contrast, addition of IGF-1 (20 ng/ml) to 7 day C2C12 myotubes for 3 days resulted in significant hypertrophy. ... The most suitable inhibitor was TAT-TIJIP and was thus used in subsequent studies. Inhibition of JNK activity by TAT-TIJIP was confirmed indirectly by detecting nuclear translocation of c- Jun, which is a downstream target of phosphorylated JNK. Immunohistochemical analyses showed nuclear localisation and phosphorylation of c-Jun in TNF treated myotubes. Nuclear localisation and phosphorylation of c-Jun was not observed in cultures pre-treated with TAT-TIJIP before TNF treatment, nor in the untreated control myotubes. 3) The use of JNK inhibitors to block the catabolic effects of TNF was tested using C2C12 and primary myotube cultures. Pre-treatment of C2C12 and primary FVB myotubes with the JNK inhibitor TAT-TIJIP, 30 min before TNF administration (for 3 days) prevented myotube atrophy. The mean width of myotubes pre-treated with TATTIJIP prior to TNF treatment closely resembled that of the control myotubes. Administration of TNF in combination with TAT-TIJIP for 3 days to C2C12 myotubes prevented myotube atrophy and unexpectedly resulted in hypertrophy when compared to the mean widths of untreated and TAT-TIJIP treated myotubes. This trend was also demonstrated in the FVB primary cultures. These combined results strongly support the role of JNK in TNF-mediated atrophy. Preliminary studies were carried out in vivo using the mdx mouse model of muscular dystrophy, TAT-TIJIP was administered via intraperitoneal injection to the mice for 3 days at a dose of 10 mg/ml, however the results form this study are inconclusive. These novel observations are of considerable interest to the field of muscle wasting because they demonstrate for the first time TNF-mediated myotube atrophy, the role of JNK in situations of TNF induced muscle atrophy, and explore the use of JNK inhibitors to prevent muscle atrophy.

Aims to observe changes in the expression of syndecan-1 in both the developing epithelium of the rat oral mucosa, and in epithelial cell rests of Malassez in the developing periodontium of normal rat molars, from late crown development through to early eruption.<br>Thesis (M.D.S.) -- University of Adelaide, School of Dentistry, 2001

by Simon Chan Siu Hoi.<br>Spine title varies.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 1997.<br>Includes bibliographical references (leaves 130-148).<br>Acknowledgments --- p.i<br>Table of Contents --- p.ii<br>List of Abbreviations --- p.ix<br>List of Figures --- p.xiii<br>List of Tables --- p.xvi<br>Page<br>Chapter Chapter 1 --- Introduction --- p.1<br>Chapter 1.1 --- Growth Hormone --- p.1<br>Chapter 1.2 --- Growth Hormone Receptor --- p.3<br>Chapter 1.2.1 --- Cytokine/Hematopoietin Receptor Superfamily --- p.3<br>Chapter 1.2.2 --- Tissue Distribution of GHR --- p.6<br>Chapter 1.2.3 --- Biosynthesis and Degradation of GHR --- p.7<br>Chapter 1.2.4 --- Regulation of GHR Level --- p.8<br>Chapter 1.2.5 --- The GHR Protein --- p.10<br>Chapter 1.2.6 --- The GHR Gene --- p.15<br>Chapter 1.2.7 --- GHR Dimerization --- p.16<br>Chapter 1.2.8 --- Mechanism of Signaling by GHR --- p.19<br>Chapter 1.2.9 --- GH Binding Protein --- p.21<br>Chapter 1.2.10 --- GHR Related Dwarfism --- p.23<br>Chapter 1.3 --- Objectives of the Present Investigation --- p.25<br>Chapter Chapter 2 --- Materials and Methods --- p.27<br>Chapter 2.1 --- Fish Growth Hormone Radioactive Labeling --- p.27<br>Chapter 2.1.1 --- Preparation of Iodogen-Coated Tubes --- p.27<br>Chapter 2.1.2 --- Packing of the Sephadex G-75 Column --- p.28<br>Chapter 2.1.3 --- Iodination of brGH and Purification of the Iodinated brGH --- p.28<br>Chapter 2.1.4 --- Determination of the Specific Radioactivity and Percentage of 125I Incorporation --- p.29<br>Chapter 2.1.5 --- Reagents and Buffers Used --- p.30<br>Chapter 2.2 --- Integrity of 125I-brGH --- p.30<br>Chapter 2.2.1 --- HPLC of brGH --- p.31<br>Chapter 2.2.2 --- HPLC of 125I-brGH after Iodination --- p.31<br>Chapter 2.2.3 --- HPLC of 125I-brGH after Receptor Binding --- p.31<br>Chapter 2.3 --- Preparation of Membranes from Fish Tissues --- p.32<br>Chapter 2.3.1 --- Preparation of Snakehead Fish Liver Membranes --- p.32<br>Chapter 2.3.2 --- Reagents and Buffers Used --- p.33<br>Chapter 2.4 --- Protein Determination of Membrane Preparations --- p.34<br>Chapter 2.4.1 --- The BCA Protein Reaction Scheme --- p.34<br>Chapter 2.4.2 --- BCA Protein Determination Protocol --- p.34<br>Chapter 2.5 --- Receptor Binding Studies --- p.35<br>Chapter 2.5.1 --- Association and Dissociation Studies --- p.36<br>Chapter 2.5.2 --- pH Dependence Study --- p.36<br>Chapter 2.5.3 --- Membrane Protein Dependence Study --- p.37<br>Chapter 2.5.4 --- Ca2+ Dependence Study --- p.37<br>Chapter 2.5.5 --- Tissue Distribution Study --- p.37<br>Chapter 2.5.6 --- Displacement and Specificity Studies --- p.38<br>Chapter 2.5.7 --- Dithiothreitol (DTT) Dependence Study --- p.39<br>Chapter 2.5.8 --- p-Chloromercuribenzene Sulfonate (PCMBS) Pretreatment: Dose Dependence Study --- p.39<br>Chapter 2.5.9 --- Scatchard Analysis of the PCMBS Pretreated and Control Snakehead Fish Liver Membranes --- p.40<br>Chapter 2.5.10 --- Reversibility of the PCMBS Effect --- p.40<br>Chapter 2.5.11 --- Reagents and Buffers Used --- p.41<br>Chapter 2.6 --- Crosslinking Studies --- p.41<br>Chapter 2.6.1 --- Crosslinking Performed on Snakehead Fish Liver Membranes --- p.41<br>Chapter 2.6.2 --- Crosslinking Performed on Solubilized Snakehead Fish Liver Membranes --- p.42<br>Chapter 2.6.3 --- Gel Filtration Chromatography of the Crosslinked Comp)lexes --- p.43<br>Chapter 2.6.4 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) of the Crosslinked Complexes --- p.43<br>Chapter 2.6.5 --- Reagents and Buffers Used --- p.45<br>Chapter 2.7 --- Western Blot Analysis of Snakehead Fish Liver GHR --- p.46<br>Chapter 2.7.1 --- SDS-PAGE of Snakehead Fish Liver Membranes --- p.46<br>Chapter 2.7.2 --- Transfer of Proteins onto Polyvinylidene Fluoride (PVDF) Membrane --- p.46<br>Chapter 2.7.3 --- Antibody Development of PVDF Membrane --- p.47<br>Chapter 2.7.4 --- Reagents and Buffers Used --- p.48<br>Chapter 2.8 --- Solubilization of Snakehead Fish Liver Membranes and Solubilized Receptor Binding Studies --- p.48<br>Chapter 2.8.1 --- Solubilization of Snakehead Fish Liver Membranes --- p.49<br>Chapter 2.8.2 --- Solubilized Receptor Binding Assay --- p.49<br>Chapter 2.8.3 --- "Solubilization of Snakehead Fish Liver Membranes: Detergent Concentration, pH, Temperature and Time Dependence" --- p.50<br>Chapter 2.8.4 --- Solubilized Receptor Binding Study: Interference of Detergent --- p.50<br>Chapter 2.8.5 --- Reagents and Buffers Used --- p.51<br>Chapter 2.9 --- Purification of Snakehead Fish Liver GHR by Affinity Chromatography --- p.51<br>Chapter 2.9.1 --- Affinity Column Preparation --- p.52<br>Chapter 2.9.2 --- Snakehead Fish Liver GHR Purification --- p.52<br>Chapter 2.9.3 --- Reagents and Buffers Used --- p.53<br>Chapter Chapter 3 --- Results: fGH Labeling and Integrity Determination --- p.54<br>Chapter 3.1 --- Introduction --- p.54<br>Chapter 3.2 --- Experimental Results --- p.55<br>Chapter 3.2.1 --- Iodination of fGH --- p.55<br>Chapter 3.2.2 --- Integrity of 125I-fGH --- p.55<br>Chapter 3.3 --- Discussion --- p.61<br>Chapter Chapter 4 --- Results: Membrane Receptor Binding Studies --- p.62<br>Chapter 4.1 --- Introduction --- p.62<br>Chapter 4.2 --- Experimental Results --- p.63<br>Chapter 4.2.1 --- Optimal Conditions for Snakehead Fish Liver Membrane GHR Binding --- p.64<br>Chapter 4.2.1.1 --- Association and Dissociation Studies --- p.64<br>Chapter 4.2.1.2 --- pH Dependence Study --- p.67<br>Chapter 4.2.1.3 --- Membrane Protein Dependence Study --- p.70<br>Chapter 4.2.1.4 --- Ca2+ Dependence Study --- p.73<br>Chapter 4.2.2 --- Localization and Specificity of Snakehead Fish GHR --- p.76<br>Chapter 4.2.2.1 --- Tissue Distribution Study --- p.76<br>Chapter 4.2.2.2 --- Displacement and Specificity Studies --- p.78<br>Chapter 4.2.3 --- Effects of Sulfhydryl Group Reducing and Oxidizing Agents on GHR Binding --- p.81<br>Chapter 4.2.3.1 --- Effect of DTT: Concentration Dependence Study --- p.81<br>Chapter 4.2.3.2 --- Effect of PCMBS: Concentration Dependence Study --- p.84<br>Chapter 4.2.3.3 --- Scatchard Analysis of Control and PCMBS- pretreated Membranes --- p.86<br>Chapter 4.2.3.4 --- Reversibility of the PCMBS Effect --- p.88<br>Chapter 4.3 --- Discussion --- p.90<br>Chapter 4.3.1 --- Optimal Conditions for Snakehead Fish Liver Membrane GHR Binding --- p.90<br>Chapter 4.3.2 --- Localization and Specificity of Snakehead Fish GHR --- p.93<br>Chapter 4.3.3 --- Effects of Sulfhydryl Group Reducing and Oxidizing Agents on GHR Binding --- p.96<br>Chapter Chapter 5 --- Results: Crosslinking and Western Blot Analysis --- p.101<br>Chapter 5.1 --- Introduction --- p.101<br>Chapter 5.1.1 --- Crosslinking Studies --- p.101<br>Chapter 5.1.2 --- Western Blot Analysis --- p.103<br>Chapter 5.2 --- Experimental Results --- p.104<br>Chapter 5.2.1 --- Crosslinking Studies --- p.104<br>Chapter 5.2.2 --- Western Blot Analysis --- p.105<br>Chapter 5.3 --- Discussion --- p.112<br>Chapter Chapter 6 --- Results: Affinity Purification of Snakehead Fish Liver GHR --- p.115<br>Chapter 6.1 --- Introduction --- p.115<br>Chapter 6.1.1 --- Membrane Solubilization and Solubilized GHR Binding Studies --- p.115<br>Chapter 6.1.2 --- Affinity Purification of Solubilized Snakehead Fish Liver GHR --- p.116<br>Chapter 6.2 --- Exp erimental Results --- p.117<br>Chapter 6.2.1 --- Solubilization of Snakehead Fish Liver Membranes --- p.117<br>Chapter 6.2.2 --- Interference of Detergents in the Solubilized Receptor Binding Assay --- p.118<br>Chapter 6.2.3 --- Affinity Purification of Solubilized Snakehead Fish Liver GHR --- p.120<br>Chapter 6.3 --- Discussion --- p.122<br>Chapter Chapter 7 --- General Discussion --- p.125<br>References --- p.130

Another important gene, heat shock factor (MeHSF) was also cloned using homology based PCR because it was suggested to participate in the transcriptional regulation of many essential components of ovarian maturation including vitellogenin gene and several proteins for hormones metabolism. The complete cDNA sequence of MeHSF was 2211 bp in length, which encoded a 622 amino acid protein. The translated MeHSF protein shared high similarity with those of other species, especially in the N terminal region. RT-PCR showed that MeHSF was universally expressed in most of the female tissues investigated including ovary, central nervous system, heart, gill, gut and muscle. However, its expression was not detectable in eyestalk and hepatopancreas. MeHSF was highly expressed in immature ovaries, and decreased dramatically with the progress of ovarian maturation. Since the synthesis of vitellogenin in ovary showed an opposite trend, the result suggested that MeHSF probably functioned as a transcriptional repressor to vitellogenin. Four HSFs isoforms generated from alternative splicing were obtained in immature ovaries, suggesting a possible universal role of HSF in coordinating transcription of different target genes during shrimp ovarian maturation.<br>As an important component of enzymatic scavenger systems, glutathione peroxidases (GPx) play important roles in maintaining the balance between reactive oxygen species (ROS) production and cellular scavenging ability. In this research, a full length GPx gene (MeGPx) which had been identified using RAP-PCR previously was cloned and characterized. MeGPx might play a pivotal role in preventing oocytes from oxidative damage and balancing ROS production. The present data on shrimp GPx provides insights on the regulation of ROS in the ovarian maturation process.<br>Four candidate genes possibly participating in the regulation of ovarian maturation were obtained by random sequencing the libraries, including metallothionein, two zinc finger proteins and member 4 of wingless-type MMTV integration site family (WNT4). The zinc finger protein containing a plant homeodomain, was only expressed in the eyestalk of female with immature ovaries, but not that of female with early mature and mature ovaries. The full length cDNA sequence of shrimp WNT4 gene (MeWNT4) was obtained using RACE technique. RT-PCR showed that the expression of MeWNT4 in eyestalk decreased with the maturation of shrimp ovaries. Interestingly, MeWNT4 was strongly expressed in the central nervous system and gut of both female and male shrimp. It was suggested that WNT4 could antagonize the testis determining factor (SRY), and play an essential role in suppressing the formation of testis, and at the same time, controlling of female development. Thus, the identification WNT4 from crustacean would contribute to our understanding on the sex determination mechanism.<br>In this thesis, two genes, heat shock protein 90 and heat shock factor, possibly playing important roles in shrimp ovarian maturation were identified and characterized.<br>Shrimp farming plays an important economic role in Southeast Asian countries. Yet further development of this industry is seriously restricted by the environmental deterioration and the prevalence of fetal diseases. Moreover, the failure of sexual maturation of cultured female shrimp forms a bottleneck to the further development of shrimp aquaculture. With the aim to produce shrimp without totally depending on the wild stocks, many studies have been focused on the endocrine regulation of shrimp ovarian maturation. In order to enhance our understanding on the molecular events occurred during ovarian maturation, in this research, several candidate genes are identified, and their potential roles in ovarian maturation are studied in the shrimp Metapenaeus ensis.<br>Since heat shock protein 90 gene is one of the essential components of steroid hormone signal cascades in vertebrates, it was cloned and isolated by homology cloning strategy. The complete cDNA sequence of shrimp Hsp90 ( MeHsp90) was 2524 by in length, which encoded a 720 amino acid polypeptide. The MeHsp90 coding region was interrupted by four introns. MeHsp90 was differentially expressed in eyestalk, ovary and hepatopancreas at different ovarian maturation stages, and consistently expressed in other tissues including heart, gill, gut, muscle and central nervous system. In vitro ovary explant assay revealed that MeHsp90 expression in immature ovary could be induced by the addition of exogenous estradio1-17beta. MeHsp90 was highly expressed in pre-vitellogenic oocytes, and its expression decreased with the progress of maturation, and finally stopped in late-vitellogenic oocytes. The co-regulation of MeHsp90 and vitellogenin by estrogen hormone suggested a possible regulatory role of Hsp90 in vitellogenin synthesis of the shrimp.<br>Wu, Long Tao.<br>"May 2008."<br>Adviser: Chu Ka Hou.<br>Source: Dissertation Abstracts International, Volume: 70-03, Section: B, page: 1409.<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 2008.<br>Includes bibliographical references (p. 92-113).<br>Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Abstracts in English and Chinese.<br>School code: 1307.

博士<br>國立中興大學<br>土壤環境科學系所<br>100<br>In the present study, the systematic classification and molecular detection of hydrocarbon degrading and plant growth promoting rhizobacteria were established. The hydrocarbons degrading bacteria were isolated from oil contaminated samples and the degrading capability was analyzed. Dehydrogenase and lipase were used to estimate the microbial activity within oil contaminated soil. The strategy of bioaugmentation was demonstrated that isolates Azospirillum picis CC-TAR-3T, Azospirillum rugosum CC-AFH-6T, Azospirillum formosense CC-Nfb-7T, Novosphingobium olei CC-TPE-1T, and Sphingomonas formonsensis CC-Nfb-2T could increase the enzymatic activity and maintain the stable communities in biotreatmental processes. Besides, the biochemical characteristics and plant promoting ability of PGPR with oil degrading ability were studied. In addition to the abilities to degrade hydrocarbon pollutant of different strains, others have nitrogen fixating, tricalcium phosphate solubilizing, protein decompositing, cellulose decompositing and siderophore producing capabilities. These bacteria were able to be applied to oil-contaminated soil, and the phytoremediation strategy was potential to be combined to integrate the bioremediation processes. The molecular detection technique for the genus Azospirillum was established, the novel designed genus-specific primer pair (Azo494-F/Azo756-R) was successfully used to distinguish the closest related genus Rhodocista spp. and Skermanella spp. With PCR-DGGE, FISH, and real-time PCR technologies, the genus-specific primer was demonstrated with 2.7 pg μL-1 detection limits, containing 6.6 × 102 CFU per gram soil. The detection technique could be used to rapid determinate, identify and develop the novel nature bioresource in the environmental samples.

Copies of author's previously published articles inserted.<br>Bibliography: leaves 175-194.<br>xiii, 195 leaves : ill. ; 30 cm.<br>Eikenella corrodens, a Gram-negative rod, is a normal inhabitant of the human oral cavity. It is one of the most commonly detected cultivable bacteria from sub- and supra-gingival plaque and is often isolated in elevated proportions from sites exhibiting destructive periodontal disease compared with healthy sites. The primary aim of this study was to investigate the metabolism of E. corrodons, with particular reference to energy generation, and to determine the effect of physical environmental parameters on its growth.<br>Thesis (Ph.D.)--University of Adelaide, Dept. of Dentistry, 2000

Indiana University-Purdue University Indianapolis (IUPUI)<br>Mammary ductal epithelial cell growth is controlled by microenvironmental signals in serum under both normal physiological settings and during breast cancer progression. Importantly, the effects of several of these microenvironmental signals are mediated by the activities of the tumor suppressor protein kinases of the Hippo pathway. Canonically, Hippo protein kinases inhibit cellular growth through the phosphorylation and inactivation of the oncogenic transcriptional co-activator Yes-Associated Protein (YAP). This study defines an alternative mechanism whereby Hippo protein kinases induce growth arrest via the phosphorylation of the long isoform of Angiomotin (Amot130). Specifically, serum starvation is found to activate the Hippo protein kinase, Large Tumor Suppressor (LATS), which phosphorylates the adapter protein Amot130 at serine-175. Importantly, wild-type Amot130 potently inhibits mammary epithelial cell growth, unlike the Amot130 serine-175 to alanine mutant, which cannot be phosphorylated at this residue. The growth-arrested phenotype of Amot130 is likely a result of its mechanistic response to LATS signaling. Specifically, LATS activity promotes the association of Amot130 with the ubiquitin ligase Atrophin-1 Interacting Protein 4 (AIP4). As a consequence, the Amot130-AIP4 complex amplifies LATS tumor suppressive signaling by stabilizing LATS protein steady state levels via preventing AIP4-targeted degradation of LATS. Additionally, AIP4 binding to Amot130 leads to the ubiquitination and stabilization of Amot130. In turn, the Amot130-AIP4 complex signals the ubiquitination and degradation of YAP. This inhibition of YAP activity by Amot130 requires both AIP4 and the ability of Amot130 to be phosphorylated by LATS. Together, these findings significantly modify the current view that the phosphorylation of YAP by Hippo protein kinases is sufficient for YAP inhibition and cellular growth arrest. Based upon these results, the inhibition of cellular growth in the absence of serum more accurately involves the stabilization of Amot130 and LATS, which together inhibit YAP activity and mammary epithelial cell growth.

The work presented in this dissertation examines possible modes of action for growth inhibition by anthropogenic endocrine disrupting chemicals (EDCs) as well as endogenous hormones associated with growth in fish. Using the sheepshead minnow (SHM) (Cyprinodon variegatus) as a model, I developed methods to examine perturbations in the endocrine axis controlling fish growth, and also examined effects of EDCs on the whole fish. I used two relatively new techniques to study the endocrine growth axis, quantitative real-time PCR (TaqMan) and differential display analysis. TaqMan analysis is a highly sensitive method to measure specific sequences from a small amount of total RNA using a fluorescent probe and specific primer pairs. I optimized a TaqMan assay for SHM IGF-I to measure hepatic IGF-I mRNA concentrations. in fish injected with hormones known to influence fish growth (GH, T���, E���, insulin, or a carrier control). IGF-I mRNA levels increased in fish injected with GH, T��� and insulin, peaking at 12 h post-injection. IGF-I mRNA levels decreased significantly at 8 h and 12 h post-injection in fish injected with E���, suggesting that pharmacological levels of E��� may affect the GH/IGF-I axis and could have consequences for fish living in waters polluted by EDCs. Differences in growth were observed in fish exposed for 18 weeks to E��� or chlorpyrifos (an organophsophate). Fish exposed to the highest dose of E��� grew larger than controls only during the last week of the experiment. Fish exposed to the lower dose of E��� were not significantly different from controls. The fish exposed to all doses of chloryprifos grew significantly less than controls in a dose-dependent manner. No significant differences were found in hepatic IGF-I mRNA levels in any treatments. To establish patterns of gene up- or down-regulation, I performed differential display analysis on livers of several fish from the previous two experiments. Several genes were identified as being similar to fish including a microsatellite sequence, a choriogenin (vitelline envelope) protein mRNA sequence, a transferrin mRNA sequence and several ribosomal RNA sequences. This technique to evaluate gene expression will become more useful when more fish genes are added to the data bases.<br>Graduation date: 2003

Wong, Wan Man.<br>Thesis submitted in: December 2006.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2007.<br>Includes bibliographical references (leaves 85-98).<br>Abstracts in English and Chinese.<br>Abstract --- p.i<br>Abstract in Chinese --- p.iii<br>Acknowledgements --- p.v<br>Lists of Figures and Tables --- p.vi<br>Table of Abbreviations --- p.xi<br>Table of Contents --- p.xv<br>Chapter Chapter I --- Introduction<br>Chapter 1.1 --- Brain and Reproductive Organ Expressed Gene --- p.1<br>Chapter 1.2 --- Programmed cell death --- p.4<br>Chapter 1.3 --- Limb development in mouse --- p.8<br>Chapter 1.4 --- Role of BRE in apoptosis --- p.12<br>Chapter 1.5 --- Role of programmed cell death in interdigital tissue regression --- p.14<br>Chapter 1.6 --- Aim of study --- p.17<br>Chapter Chpater II --- Materials and methods<br>Chapter 2.1 --- Mice --- p.18<br>Chapter 2.2 --- In-situ hybridization<br>Chapter 2.2.1 --- Histology --- p.18<br>Chapter 2.2.2 --- Preparation of riboprobe for in-situ hybridization --- p.19<br>Chapter 2.2.3 --- In-situ hybridization --- p.20<br>Chapter 2.3 --- Interdigital tissue culture --- p.21<br>Chapter 2.4 --- Gene interference<br>Chapter 2.4.1 --- Construction of Bre-siRNA --- p.22<br>Chapter 2.4.2 --- siRNA transfection of cultured interdigital cells --- p.23<br>Chapter 2.5 --- Semi-quantitative RT-PCR<br>Chapter 2.5.1 --- Sample collection of interdigital cells and explants --- p.23<br>Chapter 2.5.2 --- RNA isolation and extraction --- p.24<br>Chapter 2.5.3 --- Reverse-transcription and cDNA synthesis --- p.25<br>Chapter 2.5.4 --- Polymerase chain reaction --- p.26<br>Chapter 2.6 --- Assay of cell viability by MTT --- p.28<br>Chapter 2.7 --- Comparative proteomics --- p.30<br>Chapter 2.7.1 --- Collection of interdigital cells --- p.30<br>Chapter 2.7.2 --- Preparation of cell lysate --- p.31<br>Chapter 2.7.3 --- Assay of protein concentration in cell lysate --- p.31<br>Chapter 2.7.4 --- Two-dimensional gel electrophoresis --- p.33<br>Chapter 2.7.5 --- Protein identification by mass fingerprinting --- p.36<br>Chapter 2.8 --- Statistical Method --- p.38<br>Chapter Chapter III --- Results<br>Chapter 3.1 --- Spatial and temporal expression of Bre in murine embryonic hindlimbs --- p.39<br>Chapter 3.2 --- Expression of Bre isoforms in interdigital tissues --- p.45<br>Chapter 3.3 --- Silencing of Bre expression by siRNA in interdigital cells --- p.49<br>Chapter 3.4 --- Effect on viability of Bre-silenced interdigital cells by siRNA --- p.51<br>Chapter 3.5 --- Comparative proteomic profile of Bre-silenced interdigital cultured cells --- p.53<br>Chapter 3.6 --- Identification of proteins that were differentially expressed by MALDI- TOF --- p.71<br>Chapter 3.7 --- The mRNA levels of proteins identified that were differentially expressed --- p.74<br>Chapter Chapter IV --- Discussion --- p.77<br>References --- p.85<br>Appendices --- p.99<br>Publication --- p.108

Introduction and aims. South Africa is the epicenter of the HIV/AIDS pandemic. Hypertensive disorders of pregnancy (15.7%) are the second cause of maternal deaths of which pre-eclampsia represents 83%. Normal pregnancy requires a balance between pro- and anti-angiogenic factors to necessitate effective vasculogenesis, angiogenesis and placental development, however, pre-eclampsia is characterised by an excess anti-angiogenic state. The hypoxic placenta releases excess anti-angiogenic factors into the maternal circulation causing endothelial dysfunction. However, there is no data to verify if HIV infection affects pre-eclampsia, or if the angiogenic imbalance is affected. Contradictory data exists on the association between HIV infection and pre-eclampsia. In an attempt to assess the role of HIV infection in pre-eclampsia, this study examined the immunolocalisation of sFlt-1, sEng, PlGF and VEGF in placentae of HIV negative and positive normotensive and pre-eclamptic pregnancies at term using immunohistochemistry (IHC) and immunoelectron microscopy (IEM). Additionally, we estimated the placental expression of sFlt-1, sEng, PlGF and VEGF to verify if the HIV negative differed from the HIV positive cohorts. We further evaluated the maternal serum to determine if variations existed in the circulating levels of these factors in HIV negative and positive normotensive and pre-eclamptic pregnancies. Methods. Following institutional ethical approval and informed consent, placental biopsies and maternal serum were collected post-delivery. For IHC and IEM, 130 and 25 placentae were evaluated, respectively. Following conventional immunohistochemical processing, 5μm sections were immunostained & immunoexpression of the various antibodies were evaluated with the Zeiss Axioscope A1 interfaced with an AxioVision Image analysis software package (version 4.8.3) in combination with the auto-measurement module (Carl Zeiss, Germany). Post-conventional immunoelectron processing, ultra-thin sections were immunolabelled. Sections were post-fixed, contrast enhanced with uranyl acetate and Reynolds lead citrate and viewed on a Jeol 1011 Transmission Electron Microscope. Additionally, the placental expressions of these factors were assessed using RT-PCR. In an attempt to confirm if maternal circulating levels of these factors differed, we quantitatively evaluated these factors in serum from HIV negative normotensives, HIV negative pre-eclamptics, HIV positive normotensives, and HIV positive pre-eclamptics using ELISA techniques. Results and Discussion. The expression of sFlt-1, sEng, PlGF and VEGF was confirmed using immunohistochemistry, RT-PCR and ELISAs. Irrespective of the HIV status, sFlt-1 and sEng was elevated with the concomitant reduction in PlGF in pre-eclamptic compared to normotensive pregnancies. The levels of VEGF were however undetectable across all study groups. It is plausible that this lack of effect of HIV status on the factors under study may be attributed to the treatment regimen as HAART is known to restore the immune response of HIV positive preeclamptic women. However, a concise anti-retroviral treatment history in our study was unavailable. Additionally, this study is novel in that it ultrastructurally immunolocalises sFlt-1, sEng, PlGF and VEGF within the placenta. This immunoelectron localisation data corresponds to our immunohistochemical data. Our study further demonstrates strong immunoreactivity of both placental sFlt-1 and sEng in pre-eclampsia with concurrent elevations in the maternal circulation. A qualitative increase in the occurrence of syncytial knots in the pre-eclamptics compared to the normotensive pregnancies was noted. These observations support the detachment of antixxx angiogenic rich microparticles from syncytial knots in the pre-eclamptics compared to the normotensive pregnancies was noted. These observations support the detachment of antiangiogenic rich microparticles from syncytial knots and their subsequent deportation and elevation in the maternal circulation. Moreover, their consequent antagonistic effects on VEGF, PlGF and TGF-β, disrupts the vascular endothelial maintenance. The strong immunoreactivity of sFlt-1, sEng, PlGF and VEGF was observed in villous endothelial cells. Moreover, a strong sFlt-1 and sEng but a weak PlGF and VEGF immunoreactivity was noted in syncytio- and cytotrophoblasts. This immunoexpression within trophoblasts is suggestive of their autocrine mode of action on normal trophoblast functions including invasion, differentiation and production. It is plausible that the angiogenic imbalance observed in our study, will impact on placental function, by modifying trophoblast activity thereby contributing to abnormal placentation. Conclusion. Our study supports the hypothesis that pre-eclampsia is characterized by an imbalance between pro- and anti-angiogenic factors. Whether the pregnancy is complicated by immune insufficiencies or not, does not affect the role of the anti-angiogenic factors in pre-eclampsia development. Nevertheless, the neutralising effect of HIV infection on the immune system may be insufficient in the development of pre-eclampsia. To our knowledge, the quantification of serum pro-/anti-angiogenic factors in HIV-associated pre-eclampsia is novel. In conclusion, our data reinforces the hypothesis that increased concentrations of sFlt-1 and sEng are involved in the pathogenesis of pre-eclampsia and indicates their possible use as discriminatory factors between diseases.<br>Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2013.

前言：間充質幹細胞容易擴增並且能分化為成骨細胞、軟骨細胞和脂肪細胞，並且能對炎症、感染和損傷做出反應，並且遷移到相應的組織部位。這些特性使間充質幹細胞成為骨骼組織工程學中非常重要的細胞來源。外周血間充質幹細胞是一種存在於血液中的間充質幹細胞，而主要的間充質幹細胞存在與骨髓中，被稱之為骨髓間充質幹細胞。在我們實驗室之前的研究中通過DNA微陣列發現外周血間充質幹細胞中很多基因的表達與骨髓間充質幹細胞有很大區別。這其中的一些基因可能參與調控間充質幹細胞的分化和歸巢，我們從中挑選了三個變化比較明顯的基因--CRBP1, N-cadherin和 SOX11做進一步研究。本研究的目的在於研究CRBP1, N-cadherin和 SOX11在骨髓間充質幹細胞分化和遷移中的作用及相關機理。<br>方法：培養的骨髓間充質幹細胞來源於6-8周大小的SD大鼠。細胞的表型經過多分化潛能測試（成骨分化，成脂分化和成軟骨分化）和流式細胞儀檢驗。克隆大鼠的CRBP1, N-cadherin和SOX11基因到慢病毒載體。而且還設計了針對CRBP1和 N-cadherin的shRNA及非特異性對照shRNA。慢病毒由暫態轉染293FT細胞產生。細胞遷移實驗採用了BD Falcon的細胞遷移系統（cell culture insert）。實驗採用了定量PCR、免疫共沉澱、western雜交和雙螢光報告檢驗。對於體內實驗，細胞經感染帶有不同基因的病毒後，種植到Si-TCP材料並移植到裸鼠皮下。8周後，收集樣品進行組織學和免疫組織學分析。最後，我們建立了大鼠的股骨開放式骨折模型，並在4天后將SOX11基因修飾的間充質幹細胞通過心臟注射打到大鼠體內。4周後，收集股骨骨折樣品並進行microCT、力學測試和組織學分析。<br>結果：CRBP1過表達能夠促進骨髓間充質幹細胞的成骨分化潛能，並能抑制其成脂分化。進一步的機理研究表明CRBP1可以通過與RXRα的蛋白相互作用抑制RXRα誘導的β-catenin降解，從而維持β-catenin和磷酸化-ERK1/2在較高的水準，導致間充質幹細胞成骨能力增強；N-cadherin過表達可以促進間充質幹細胞的遷移，但是卻通過下調β-catenin和磷酸化ERK1/2抑制其成骨分化。過表達SOX11可以通過增強BMP信號通路促進三系分化。SOX11還可以通過啟動CXCR4的表達來促進細胞遷移。最後，在大鼠的股骨開放骨折模型上通過系統注射，我們證明穩定過表達SOX11的間充質幹細胞遷移到骨折部位的數量明顯增加。這些細胞到達骨折部位以後可以起始骨痂的鈣化，促進骨折的修復。<br>結論：本研究證明CRBP1, N-cadherin 和SOX11具有調節骨髓間充質幹細胞遷移和/或分化的功能。這些基因也許會成為幹細胞治療的新靶點。系統注射SOX11基因修飾的骨髓間充質幹細胞對於骨折修復可能具有較好的療效。本研究初步研究了CRBP1, N-cadherin 和SOX11在間充質幹細胞中的作用，為探討以間充質幹細胞為基礎的組織工程的某些新臨床應用提供了一些線索。<br>Introduction: Mesenchymal stem cells (MSCs) can be easily harvested, expanded, and have the capability of differentiating into osteoblasts, chondrocytes and adipocytes, and they can home to various tissues in response to stimuli such as inflammation, infection and injuries. MSCs are therefore valuable cell source for musculoskeletal tissue engineering. Peripheral blood-derived MSCs (PB-MSCs) are one kind of MSCs that reside in peripheral blood, whereas the main source of MSCs is bone marrow-derived MSCs (BM-MSCs). In our previous study, we found many genes were differentially expressed in the PB-MSCs compared to their counterpart BM-MSCs demonstrated by microarray analysis, among which the effects of CRBP1, SOX11 and N-cadherin on MSCs in terms of migration and differentiation are studied.<br>Methods: BM-MSCs and PB-MSCs were cultured from 6-8 weeks SD rats. The phenotypes of MSCs were characterized by tri-lineage (adipo-, osteo- and chondrogenic) differentiation and flow cytometry analysis. The genes encoding rat CRBP1, SOX11 and N-cadherin were cloned into lentiviral vectors respectively. shRNAs targeting CRBP1, N-cadherin, and one nonspecific shRNA were designed. Pseudo-lentivirus was produced by transient transfection of 293FT cells. Cell migration was examined using transwell insert culture system. Quantitative RT-PCR, CO-IP, western blot and dual-luciferase assay were employed in the studies. For in vivo study, MSCs transduced with different genes were seeded on Si-TCP scaffolds and implanted subcutaneously in nude mice. 8 weeks later, the samples were collected for histological and immunohistological analysis. Finally, an open femoral fracture model was established in 8-week old SD rats, SOX11-modified MSCs were injected at four days after fracture. At 4-week after MSCs injection, the femurs were collected for microCT, mechanical test and histological analysis.<br>Results: For CRBP1gene, our results showed that CRBP1 overexpression promoted osteogenic differentiation of BM-MSCs, while inhibited their adipogenic differentiation. We demonstrated that CRBP1 promoted osteogenic differentiation by inhibiting RXRα-induced β-catenin degradation through physical interactions, and maintaining β-catenin and pERK1/2 at higher levels. For N-cadherin gene, we found that N-cadherin overexpression promoted MSCs migration, and suppressed osteogenic potential of MSCs through inhibiting ERK and β-catenin signaling pathways. For SOX11 gene, we demonstrated that SOX11 overexpression enhanced the adipo-, osteo- and chondrogenic differentiation of BM-MSCs, through enhancing BMP signaling pathways. The migration capacity of BM-MSCs was also enhanced when Sox-11 was overexpressed, through activating CXCR4 expression. Finally, in the open femur fracture model we demonstrated that a larger number of SOX11-overexpressing BM-MSCs migrated to the fracture site, initiated earlier callus ossification and improved bone fracture healing quality.<br>Conclusions: This study demonstrated that CRBP1, N-cadherin and SOX11 gene can regulate the migration and/or differentiation potentials of BM-MSCs. These genes may become new therapeutic targets in stem cell therapy applications. Systemic administration of genetically modified SOX11-overexpressing BM-MSCs may be useful in promoting fracture healing. Overall, this study defined some unknown functions of CRBP1, N-cadherin and SOX11 in MSCs and shed the lights on some novel therapeutic implications for MSCs-based tissue engineering.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Xu, Liangliang.<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.<br>Includes bibliographical references (leaves 128-144).<br>Abstract also in Chinese.<br>Declaration --- p.i<br>Abstract --- p.ii<br>摘要 --- p.v<br>Acknowledgements --- p.vii<br>Chapter 1 --- p.1<br>Introduction --- p.1<br>Chapter 1.1 --- Mesenchymal stem cells --- p.2<br>Chapter 1.1.1 --- Characteristics of mesenchymal stem cells --- p.2<br>Chapter 1.1.2 --- Bone marrow- and peripheral blood-derived MSCs --- p.4<br>Chapter 1.1.3 --- Other tissue-derived MSCs --- p.5<br>Chapter 1.2 --- Adipogenesis of MSCs --- p.6<br>Chapter 1.3 --- Chondrogenesis of MSCs --- p.7<br>Chapter 1.4 --- Osteogenesis of MSCs --- p.8<br>Chapter 1.4.1 --- Regulators of osteogenesis --- p.9<br>Chapter 1.4.2 --- Stratergies for improving bone tissue engineering --- p.11<br>Chapter 1.5 --- Signaling pathways involved in osteogenesis --- p.13<br>Chapter 1.5.1 --- ERK signaling pathway --- p.14<br>Chapter 1.5.2 --- Wnt signaling pathway --- p.15<br>Chapter 1.5.3 --- BMP signaling pathway --- p.17<br>Chapter 1.6 --- Migration of MSCs --- p.20<br>Chapter 1.7 --- Fracture healing --- p.22<br>Chapter 1.8 --- Clinical application of MSCs --- p.23<br>Chapter 1.8.1 --- BM-MSCs vs. PB-MSCs --- p.24<br>Chapter 1.8.2 --- Autologous vs. Allogeneic MSCs transplantation --- p.25<br>Chapter 1.9 --- Scope of the present study --- p.26<br>Chapter 1.9.1 --- CRBP1 --- p.26<br>Chapter 1.9.2 --- N-cadherin --- p.27<br>Chapter 1.9.3 --- SOX11 --- p.27<br>Chapter 1.10 --- Experimental scheme --- p.29<br>Chapter 2 --- p.31<br>Comparison between PB-MSCs and BM-MSCs --- p.31<br>Chapter 2.1 --- Chapter introduction --- p.32<br>Chapter 2.2 --- Materials and methods --- p.33<br>Chapter 2.2.1 --- Cell culture --- p.33<br>Chapter 2.2.2 --- Flow cytometry --- p.33<br>Chapter 2.2.3 --- Adipogenic differentiation --- p.34<br>Chapter 2.2.4 --- Osteogenic differentiation --- p.34<br>Chapter 2.2.5 --- RNA Extraction and Real-time PCR --- p.34<br>Chapter 2.3 --- Results --- p.35<br>Chapter 2.3.1 --- Morphology of PB-MSCs --- p.35<br>Chapter 2.3.2 --- Cellular surface markers of BM-MSCs and PB-MSCs --- p.36<br>Chapter 2.3.3 --- Multi-differentiation potential of BM-MSCs and PB-MSCs --- p.38<br>Chapter 2.3.4 --- Target genes expression in BM-MSCs and PB-MSCs --- p.39<br>Chapter 2.4 --- Discussion and future work --- p.40<br>Chapter 3 --- p.41<br>Role of CRBP1 in Differentiation and Migration of MSCs --- p.41<br>Chapter 3.1 --- Chapter introduction --- p.42<br>Chapter 3.2 --- Materials and methods --- p.46<br>Chapter 3.2.1 --- Chemicals --- p.46<br>Chapter 3.2.2 --- Isolation and culture of BM-MSCs --- p.46<br>Chapter 3.2.3 --- RNA Extraction and Real-time PCR --- p.47<br>Chapter 3.2.4 --- Plasmid construction, transfection, production of lentivirus and infection --- p.48<br>Chapter 3.2.5 --- Osteogenic differentiation --- p.50<br>Chapter 3.2.6 --- Adipogenic differentiation --- p.50<br>Chapter 3.2.7 --- Western blot --- p.51<br>Chapter 3.2.8 --- Immunofluorescence labeling and fluorescence microscopy --- p.52<br>Chapter 3.2.9 --- Cell migration assay --- p.52<br>Chapter 3.2.10 --- Ectopic bone formation assay --- p.52<br>Chapter 3.2.11 --- Statistical analysis --- p.53<br>Chapter 3.3 --- Results --- p.53<br>Chapter 3.3.1 --- Transducing BM-MSCs with lentivirus carrying CRBP1 or shRNAs --- p.53<br>Chapter 3.3.2 --- CRBP1 accelerates osteogenesis of BM-MSCs via enhancing ERK1/2 and β-catenin pathways --- p.56<br>Chapter 3.3.3 --- CRBP1 stabilizes β-catenin by inhibiting RXRα-induced degradation --- p.58<br>Chapter 3.3.4 --- CRBP1 inhibits adipogenesis of BM-MSCs --- p.61<br>Chapter 3.3.5 --- CRBP1 overexpression has no effect on MSCs migration potential --- p.63<br>Chapter 3.3.6 --- CRBP1 promotes ectopic bone formation in vivo --- p.64<br>Chapter 3.4 --- Discussion --- p.66<br>Chapter 3.5 --- Future work --- p.73<br>Chapter 4 --- p.74<br>Role of N-cadherin in Differentiation and Migration of MSCs --- p.74<br>Chapter 4.1 --- Chapter introduction --- p.75<br>Chapter 4.2 --- Materials and methods --- p.78<br>Chapter 4.2.1 --- Chemicals --- p.78<br>Chapter 4.2.2 --- Isolation and culture of BM-MSCs --- p.78<br>Chapter 4.2.3 --- Plasmid construction, transfection, production of lentivirus and infection --- p.79<br>Chapter 4.2.4 --- Osteogenic differentiation and ALP activity assay --- p.81<br>Chapter 4.2.5 --- Western blot --- p.81<br>Chapter 4.2.6 --- Ectopic bone formation assay --- p.82<br>Chapter 4.2.7 --- Statistical analysis --- p.82<br>Chapter 4.3 --- Results --- p.83<br>Chapter 4.3.1 --- Expression of N-cadherin during osteogenesis in MSCs --- p.83<br>Chapter 4.3.2 --- N-cadherin overexpression inhibits osteogenesis through suppressing β-catein and ERK1/2 signaling pathways --- p.84<br>Chapter 4.3.3 --- N-cadherin silencing increases osteogenesis through enhancing β-catenin and ERK1/2 signaling pathways --- p.86<br>Chapter 4.3.4 --- N-cadherin promotes migration of MSCs --- p.87<br>Chapter 4.3.5 --- Cellular surface markers of SV40-immortalized MSCs --- p.89<br>Chapter 4.3.6 --- N-cadherin inhibits ectopic bone formation in vivo --- p.89<br>Chapter 4.4 --- Discussion --- p.91<br>Chapter 4.5 --- Future work --- p.94<br>Chapter 5 --- p.96<br>Role of SOX11 in Differentiation and Migration of MSCs --- p.96<br>Chapter 5.1 --- Chapter introduction --- p.97<br>Chapter 5.2 --- Materials and methods --- p.105<br>Chapter 5.2.1 --- Plasmid construction, transfection, production of lentivirus and infection --- p.105<br>Chapter 5.2.2 --- Cell culture --- p.106<br>Chapter 5.2.3 --- Luciferase reporter gene assay --- p.106<br>Chapter 5.2.4 --- Osteogenic differentiation and ALP activity assay --- p.106<br>Chapter 5.2.5 --- Adipogenic differentiation --- p.107<br>Chapter 5.2.5 --- Chondrogenic diffferentiation --- p.107<br>Chapter 5.2.6 --- Western blot --- p.108<br>Chapter 5.2.7 --- RNA Extraction and Real-time PCR --- p.108<br>Chapter 5.2.8 --- Cell migration --- p.110<br>Chapter 5.2.9 --- Ectopic bone formation --- p.110<br>Chapter 5.2.10 --- Fracture healing model and analysis --- p.111<br>Chapter 5.2.11 --- Statistical Analysis --- p.112<br>Chapter 5.3 --- Results --- p.112<br>Chapter 5.3.1 --- SOX11 is upregulated during osteogenesis of BM-MSCs --- p.112<br>Chapter 5.3.2 --- SOX11 promotes adipogenesis in BM-MSCs --- p.113<br>Chapter 5.3.3 --- SOX11 promotes migration of BM-MSCs --- p.114<br>Chapter 5.3.4 --- SOX11 promotes osteogenesis in BM-MSCs --- p.115<br>Chapter 5.3.5 --- SOX11 promotes chondrogenesis of MSCs --- p.117<br>Chapter 5.3.6 --- Mechanisms of how SOX11 regulates differentiation and migration of MSCs --- p.118<br>Chapter 5.3.7 --- SOX11-modified MSCs promote bone fracture healing in an open femur fracture rat model --- p.122<br>Chapter 5.4 --- Discussion --- p.126<br>Chapter 5.5 --- Future work --- p.131<br>Appendix --- p.153

Meng, Fanbiao.<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 2013.<br>Includes bibliographical references (leaves 114-138).<br>Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Abstract also in Chinese.

Jia, Lin.<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 2013.<br>Includes bibliographical references (leaves 124-146).<br>Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Abstract also in Chinese.