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Upregulation of PITX2 transcription factor is associated with ovarian tumorigenesisFung, Khe Cheong, Frederic., 馮啟昌. January 2011 (has links)
published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy
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Identification of DLX1 as a FOXM1 downstream target in mediating ovarian cancer oncogenesisHui, Wing-yee, 許穎儀 January 2012 (has links)
Emerging evidences have documented that aberrant expression of FOXM1 is closely associated with human cancers. A recent comprehensive genome analysis has revealed that FOXM1 signaling is one of the major pathways involved in ovarian cancer oncogenesis. However, the regulatory network of FOXM1 in exerting the metastatic phenotypes remains unknown. Therefore, the identification of FOXM1 downstream targets will assist in understanding of its molecular mechanism in ovarian cancer oncogenesis.
In this study, by bioinformatics and a series of functional analyses, we identified DLX1 as a novel target of FOXM1. Our results clearly demonstrated that enforced expression of FOXM1 (FOXM1B and FOXM1C) could increase DLX1 in mRNA and protein levels. Conversely, depletion of FOXM1 by Thiostrepton (FOXM1 specific inhibitor) or RNAi knockdown could reduce DLX1 expression. Importantly, we demonstrated that the changes of DLX1 expression were in concomitant with the expression of a positive control gene, Cyclin-D1. Additionally, the luciferase promoter assay further showed that there are two conserved FOXM1 binding sites TFBS1 and TFBS2 which located at -61~-52bp upstream and -737~727bp upstream of the transcription factor binding sites (TSS) of DLX1 promoter respectively. In comparison of two binding sites, the more conserved binding site, TFBS1, seems have higher importance of FOXM1 binding in DLX1 transcriptional activation.
Furthermore, our study using immunohistochemical and Q-PCR analyses showed that DLX1 was frequently up-regulated in ovarian cancer samples. Noticeably, clinicopathological analysis revealed that the upregulated DLX1 was significantly associated with not only the overexpressed FOXM1 (P=0.001) but also high grade ovarian cancer (P<0.001). Previous studies have reported that DLX1 is a homeobox transcription factor controlling neuron migration and proliferation in embryogenesis. However, the oncogenic functions of DLX1 are rarely reported. In this study, we revealed that DLX1 could promote ovarian cancer cell proliferation and cell migration which are the main phenomena found in high grade tumors. To the best of our knowledge, this is the first report showing the regulation of FOXM1 on DLX1 and the metastatic functions exerted by DLX1 in ovarian cancer cells. Although ovarian cancer cells are epithelial cell type which is different from neurons, the similar cell functions derived from DLX1 reflecting that both cell types share the similar signaling pathway of DLX1. However, further investigation on the downstream network of DLX1 and the in vivo tumorigenic capacities in ovarian cancer cells are warranted.
To conclude, we have identified DLX1 as a novel target of FOXM1 and frequently up-regulated in high grade ovarian cancer. The in vitro tumorigenic assay demonstrated DLX1 could promote cell proliferation and cell migration which are the metastatic properties usually found in high grade ovarian cancer. Therefore, these data highlight the possibilities of using DLX1 as a biomarker and therapeutic target in combating ovarian cancer in the future. / published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy
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Pax6/c-Myb regulates neuronal apoptosis in a mouse model of Alzheimer's diseaseZhang, Yalun, 张亚伦 January 2011 (has links)
Alzheimer’s disease (AD) is the most frequent neurodegenerative disorder which
is characterized by impaired mental functions such as memory, language,
perception, behavior and personality, as well as cognitive skills. The molecular
mechanisms underlying this disease is still largely unknown, but numerous
evidence emerge to support a cell cycle hypothesis which implicates the
deregulation of cell cycle proteins as key mediators of neuronal dysfunction and
loss in AD brains. One of these signals in Aβ-induced neuronal death model is
Cdk/Rb/E2F pathway, where Aβ insult evokes activation of Cdk4/6, which
subsequently phosphorylates pRb protein, resulting in activation of E2F
transcription factors. However, the mechanism(s) by which Cdk/Rb/E2F mediates
neuronal death remains elusive. Therefore, the goal of this project is to
characterize the downstream events of cell cycle pathway, which include the
involvement of transcription factors c-Myb, Pax6 and Patz1 in Aβ-induced
neuronal death signaling. In this study, we showed that Pax6 is a direct target gene
for Both E2F1 and c-Myb. Both Pax6 and c-Myb are up-regulated by Aβ insults in
cultured cortical neurons. And with E2F1 silencing by siRNA, Aβ-induced Pax6
and c-Myb expression is blocked, suggesting E2F1 is responsible for their
elevation. Importantly, siRNA-mediated downregulation of either c-Myb or Pax6
protects neurons from death evoked by Aβ peptide, suggesting they are proapoptotic
proteins, delivering death signals sent from upstream E2F1. Next,
though ChIP assay, we identified two target genes for Pax6. One is Patz1, another
transcription factor that is Aβ-induced pro-apoptotic protein. The other one is
GSK3β, which is a pathogenic kinase involved in Tau protein
hyperphosphorylation and NFT formation. In conclusion, this dissertation shows
that cell cycle regulators Cdk/Rb/E2F modulate neuronal death signals by
activating downstream transcription factors c-Myb and Pax6, further upregulating
GSK3β. We provided evidence suggesting that Aβ induced neurotoxicity leads to
Tau hyperphosphorylation through a mechanism involving cell cycle activation
and subsequent activation of c-Myb/Pax6/GSK3β. In brief, in the present study,
we delineate a transcriptional cascade downstream of cell cycle pathway leads to
neuronal apoptosis as well as Tau/NFT pathology. The characterization of this
novel pathway lends support for development of new therapeutic agents and for
better experimental models for AD. Lastly, the cascade between cell cycle
activation and tauopathy in Aβ-induced neuronal death needs to be further
researched in the future. / HKU 3 Minute Thesis Award, Champion (2011) / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy
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A Sox10-GFP mutant mouse model for the study of abnormal enteric nervous system development in Hirschsprung diseaseZhang, Mei, 章梅 January 2010 (has links)
published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy
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Investigating the role of the forkhead box transcription factor FOXM1 against oxidative stress and DNA damage in human embryonic stem cellsLeung, Man-hong, 梁文康 January 2015 (has links)
abstract / Biochemistry / Master / Master of Philosophy
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Functional characterization of the B-cell lymphoma/leukemia 11A (BCL11A) transcription factorLee, Baeck-seung, 1969- 29 August 2008 (has links)
Previously a t(2;14)(p13;q32) translocation was characterized in four unusually aggressive cases of B cell chronic lymphocytic leukemia (B-CLL). A gene located near the 2p13 breakpoint, B cell lymphoma/leukemia 11A (BCL11A), was shown to overexpress 3 isoforms (BCL11A-XL, L and S). Bcl11a knockout mice are severely impaired in B cell development at the early (pro-B) stage. I have further characterized BCL11A, focusing on the most abundant and evolutionarily conserved isoform, BCL11A-XL (XL). I demonstrated that XL resides in the nuclear matrix, is modified by ubiquitination, and is destabilized by B cell antigen receptor ligation. I identified domains within XL required for its localization within nuclear paraspeckles and for its transcriptional repression. While BCL11A-XL represses model promoters in non-B cells, its biologically relevant targets in B lymphocytes were unknown. I have identified and confirmed a number of XL targets which are both up- and down-regulated by XL over-expression in B cell lines. A number of these genes have been implicated in B cell function, including the V(D)J recombination activating (RAG) genes. Both RAG1 and RAG2 transcripts were up-regulated by XL. XL binds to the RAG1 promoter and RAG enhancer (Erag) in vivo as well as in vitro. Unexpectedly, XL repressed RAG1 transcription in non-B cells, indicating that additional B cell-specific factors are required for activation. Overexpression of XL in a V(D)J recombination-competent pre-B cell line markedly induced RAG expression and VDJ recombination. IRF4 and IRF8, transcription factors previously shown to be required for early B cell development, were also induced by BCL11A-XL. I propose that the early B cell progenitor block in Bcl11a knockout mice is, at least in part, a direct result of BCL11A-XL regulation of V(D)J recombination. Further experiments are required to establish how other XL targets promote B cell lineage development and how malignant transformation such as in B-CLL may corrupt BCL11A function.
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From developing protein-protein interaction strategies to identifying gene functions: case studies for transcription factor complexes and ribosome biogenesis genes / Case studies for transcription factor complexes and ribosome biogenesis genesLi, Zhihua, doctor of cell and molecular biology 29 August 2008 (has links)
Protein-protein interactions are central to their biological functions in cells. Many approaches have been applied to study protein-protein interactions in a genomic-scale. In an attempt to develop new strategies to study protein-protein interactions, FRET by using ECFP and EYFP as the donor and receptor was evaluated for possible application in protein-protein interaction study in a high-throughput fashion. Due to the intrinsic properties of ECFP and EYFP, FRET-based protein-protein interaction assay is not suitable for large-scale studies. Instead, tandem affinity purification coupled with mass spectrometry approach proved to be a useful strategy to identify protein interacting partners. Several transcription factor complexes in yeast were successfully purified and novel components in the complexes were identified by combining a shotgun mass spectrometry approach and a differential analysis of the mass spectrometry data. In particular, a negative regulator of G1 to S phase transition during cell cycle, Whi5p, was identified to be a component of SBF complex; a regulator of nitrogen metabolism, Gln3p, was identified to be a component of Hap2/3/5 complex that regulates carbon metabolism, suggesting a crosstalk between nitrogen and carbon metabolism. Additionally, one-step purification coupled with shotgun mass spectrometry analysis was applied to simplify and improve the affinity purification approach used for protein-protein interaction studies. In order to map protein complexes in their native state, a sucrose density gradient was used to separate protein complexes in cells. The proteins within each fraction from the sucrose density gradient were analyzed and quantified with mass spectrometry to obtain the protein abundance profiles across the gradient. The known protein complexes were identified by clustering the protein abundance profiles. This method could possibly be improved to become a generic approach to mapping protein complexes. The goal of protein-protein interaction studies is to determine the protein functions. In an effort to identify ribosome biogenesis genes from a yeast gene network reconstructed from diverse large-scale interaction data sets, at least 25 new ribosome biogenesis genes were confirmed by extensive experimental validations, underscoring the value of proteinprotein interaction studies and gene interaction network.
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Ο ρόλος της καρβόξυ-τελικής επέκτασης της επικράτειας πρόσδεσης στο DNA του μεταγραφικού παράγοντα COUP-TFΖήνωνος, Ζήνων 01 December 2009 (has links)
Oι COUP-TFs (Chicken Ovalvumin Upstream Promoter – Transcription Factors)
είναι ορφανοί πυρηνικοί υποδοχείς που ανήκουν στην υπεροικογένεια των
υποδοχέων στεροειδών/θυρεοειδών ορμονών. Εμφανίζουν μεγάλη ομολογία σε όλα
τα μετάζωα και εκφράζονται κατά προτίμηση στο νευρικό σύστημα. Εκτός της
συντηρημένης αλληλουχίας τους, επίσης συντηρημένες είναι και οι θέσεις των
εσoνίων στους διάφορους οργανισμούς ενώ, μέχρι πρόσφατα, δεν είχε παρατηρηθεί
εναλλακτικό μάτισμα. Ωστόσο έχουμε δείξει ότι στα εχινόδερμα, υπάρχουν δύο
μετάγραφα, αποτέλεσμα εναλλακτικού ματίσματος ενός μικρού εξονίου 63 bps. Τα
δύο mRNAs παράγουν δύο πρωτεΐνες, η μεγαλύτερη εκ των οποίων περιέχει 21
επιπρόσθετα αμινοξέα στην κάρβοξυ-τελική επέκταση της περιοχής πρόσδεσης στο
DNA (DBD).
Πειράματα EMSA έδειξαν ότι η μικρή ισομορφή του COUP-TF, ως ομοδιμερές,
προσδένεται με διαφoρετική συγγένεια σε όλα τα στοιχεία απόκρισης που
εξετάστηκαν. Η μεγάλη ισομορφή εν αντιθέσει, δεν προσδένεται αποτελεσματικά σε
κανένα από αυτά τα στοιχεία απόκρισης, ούτε ως ομοδιμερές ούτε ως ετεροδιμερές
με τη μικρή ισομορφή. Επίσης, πειράματα ανοσοφθορισμού, έχουν δείξει ότι σε
έμβρυα αχινού στο στάδιο των 16 κυττάρων, η μεγάλη πρωτεΐνη εντοπίζεται στο
κυτταρόπλασμα, ενώ για τη μικρή πρωτεΐνη είναι γνωστή η παρουσία της στον
πυρήνα.
Ο σκοπός της εργασίας αυτής είναι η μελέτη του ρόλου της ένθεσης των 21
αμινοξέων στην μεγάλη ισομορφή, όσον αφορά στην κυτταρική διαμερισματοποίηση
και στη συγγένεια πρόσδεσης στο DNA. Στην ένθεση αυτή υπάρχουν δύο προλίνες
και τρείς θρεονίνες. Η προλίνη γενικότερα βρίσκεται σε σημεία όπου μία πρωτεΐνη
κάμπτεται. Η υπόθεσή μας είναι ότι οι προλίνες αυτές παίζουν σημαντικό ρόλο στη
στερεοδιαμόρφωση της πρωτεΐνης, που με τη σειρά της είναι υπεύθυνη για τις
αλλαγές που παρατηρούνται στη μεγάλη ισομορφή, ως προς την συγγένεια
πρόσδεσης στο DNA και την κυτταρική διαμερισματοποίηση, σε σχέση με την μικρή.
Επίσης η θρεονίνη είναι ένα αμινοξύ που μπορεί να δεκτεί φωσφορυλίωση. Γεγονότα
φωσφορυλίωσης και αποφοσφωρυλίωσης μπορεί να επηρεάζουν την ικανότητα
πρόσδεσης και την κυτταρική διαμερισματοποίηση του υποδοχέα.
2
Για να ελεχθούν οι υποθέσεις αυτές, μεταλλάχθηκαν οι δύο προλίνες και οι τρεις
θρεονίνες της μεγάλης ισομορφής σε αλανίνες, παράχθηκαν in vitro οι
μεταλλαγμένες πρωτεΐνες και προσδιορίστηκε η συγγένεια πρόσδεσης τους στο DNA
με πειράματα EMSA. Οι ίδιες μεταλλάξεις πραγματοποιήθηκαν και σε ένα
κατασκεύασμα το οποίο κωδικοποιεί για τη χιμαιρική πρωτεΐνη που αποτελείται από
τη μεγάλη ισομορφή και τη πράσινη φθορίζουσα πρωτεΐνη. Τα in vitro παραχθέντα
RNAs ενέθηκαν σε έμβρυα αχινού ώστε να μελετηθεί η ενδοκυτταρική κατανομή της
μεγάλης COUP-TF ισομορφής.
Τα αποτελέσματα δείχνουν ότι οι δύο προλίνες δεν φαίνεται να παίζουν
σημαντικό ρόλο όσον αφορά την πρόσδεση της μεγάλης COUP-TF ισομορφής ως
ομοδιμερές. Εντούτοις οι δύο αυτές προλίνες φαίνεται να επηρεάζουν την πρόσδεση
της μεγάλης ισομορφής COUP-TF ως ετεροδιμερές με τρόπο ώστε να την ενισχύει. / -
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The mitogen-activated protein kinase pathway regulates the subcellularlocalization and function of FOXM1Ma, Yam-man, Richard., 馬蔭民. January 2003 (has links)
published_or_final_version / abstract / toc / Biochemistry / Master / Master of Philosophy
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Pituitary-specific transcription factor PIT-1 in Chinese grass carp: molecular cloning, functionalcharacterization, and regulation of its transcript expression at thepituitary levelKwong, Ka-yee., 鄺嘉儀. January 2004 (has links)
published_or_final_version / abstract / toc / Zoology / Master / Master of Philosophy
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