Aim: Cisplatin is the first platinum drug that shows promising anti-tumor effect clinically. Oxaliplatin, a third-generation platinum drug that incorporates a diaminocyclohexane (DACH) structural entity, can overcome cisplatin resistance. R,R-5, a novel platinum compound that integrates the DACH entity with a demethylcantharidin (DMC) component that is derived from a traditional Chinese medicine (TCM) , can also overcome cisplatin resistance. The principal objectives of this study was to investigate in detail, the effect of these compounds at the antephase and G2 checkpoints of the cell cycle, and to establish the relationship (if any) between different structural entities with checkpoint activation. The ultimate aim of the study was to ascertain the potential for the development of novel checkpoint abrogators as anti-tumor agents. / Background: A common procedure in current cancer chemotherapy is to induce genomic stress in cancer cells, leading to irreparable DNA damage and eventually cell death. However, there are several DNA repair mechanisms in cancer cells to maintain genomic stability, which require cell cycle checkpoints to stop cell proliferation for DNA damage repair, thereby avoiding errors in cellular events like DNA replication, transcription and mitosis. Among these cell cycle checkpoints, antephase and G2 checkpoints are two gate checkpoints for mitosis. Abrogation of G2 checkpoint has been reported to give rise to synergistic cytotoxic effect with DNA damaging agents, representing a means of circumventing drug resistance in chemotherapy. / Conclusions: Acute stress to cisplatin can activate the MMR/c-Abl/MEKK1/p38MAPK pathway, leading to the activation of antephase checkpoint, and stop cells from entering mitosis immediately. DACH-containing platinum compound oxaliplatin fails to activate this antephase checkpoint. However, both cisplatin and oxaliplatin can activate the G2 checkpoint, which can be abrogated by DMC. In contrast, RR-5 can bypass both the antephase and G2 checkpoints. In summary, novel TCM-platinum compound R,R-5 can bypass mitotic DNA damage checkpoints in cancer cells and thus has the potential for further development as an anti-cancer drug. / Methods: Microarray analysis was used to detect gene transcription profiles after drug treatments. The activation of mitotic checkpoints was inspected by counting mitotic cells and utilizing flow cytometry. Using Western blotting, the activation of certain key players in the antephase and G2 checkpoint was revealed. MTT assays were performed to show the outcome of checkpoint activation. / Results: In HCT116 cells, 35 genes that facilitate G2/M transition were found to be up-regulated after R,R-5 treatment compared with oxaliplatin in the microarray analysis, implying the bypass of mitotic checkpoints by R,R-5 rather than oxaliplatin. Acute stress (2 hour) of cisplatin activated the antephase checkpoint, resulting in a rapid decrease in mitotic index and phosphorylation of histone H1, which avoided mitotic catastrophe and promoted cell survival in HeLa cells. Further experiments demonstrated that this antephase checkpoint could be abrogated by c-Abl and p38MAPK inhibitors, or siRNAs against c-Abl or MEKK1, suggesting that this checkpoint may be controlled by an MMR/c-Abl/MEKK1/p38MAPK pathway. In contrast, oxaliplatin and R,R-5 did not activate this antephase checkpoint. Moreover, after 24 hour oxaliplatin treatment in HeLa cells, the mitotic index and CDK1 activity were decreased, which could be restored by concomitant treatment with ATM/ATR inhibitor and DMC. This indicated the activation of G2 checkpoint by oxaliplatin and implied that DMC can abrogate oxaliplatin-activated G2 checkpoint by restoring CDK1 activity. Cisplatin could also activate G2 checkpoint, whereas R,R-5 apparently bypassed this G2 checkpoint. / Guan, Huaji. / Adviser: Vincent Hon Leung Lee. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 212-249). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Human multiple myeloma is an incurable hematological malignancy characterized by the proliferation of plasma cells in the bone marrow. Myeloma represents approximately 20% of all blood cancers. In this research we have explored examples of both intrinsic and acquired drug resistance in myeloma. Topoisomerases are enzymes that are critical for cell division, especially in rapidly dividing cells such as are found in cancer. Topoisomerase poisons are a common group of drugs that are used to treat cancer. Topoisomerase I and II poisons used in the treatment of multiple myeloma include topotecan, mitoxantrone, doxorubicin, and etoposide In order for topoisomerase drugs to be effective, the enzyme must be in direct contact with the DNA. In chapters one and two we examined the export of topoisomerase II alpha from the nucleus as a mechanism of drug resistance. High density cells, similar to those found in the bone marrow, export topoisomerase II alpha from the nucleus to the cytoplasm, rendering the cell drug resistant. We found that blocking nuclear export using the CRM1 inhibitor ratjadone C, or CRM1 specific siRNA, could sensitize high density cells to topoisomerase drugs. Sensitization to topoisomerase inhibitors was correlated with increased topoisomerase/DNA complexes and increased DNA strand breaks. This method of sensitizing human myeloma cells suggests a new therapeutic approach to this disease. In chapter three we examined the role of the molecular transporter ABCG2 in drug resistance in multiple myeloma. We found that ABCG2 expression in myeloma cell lines increased after exposure to topotecan or doxorubicin. Myeloma patients treated with topotecan had an increase in ABCG2 mRNA and protein expression after drug treatment and at relapse. We found that expression of ABCG2 is regulated, at least in part, by promoter methylation both in cell lines and in patient plasma cells. Demethylation of the promoter increased ABCG2 mRNA and protein expression. These findings suggest that ABCG2 is expressed and functional in human myeloma cells, regulated by promoter methylation, affected by cell density, upregulated in response to chemotherapy, and may contribute to drug resistance.

Colorectal cancer (CRC) remains a significant burden in contemporary society due to an aging population, unhealthy dietary choices and an increasingly sedentary lifestyle. While the underlying defects for many hereditary forms of CRC have been determined, many genetic and epigenetic changes promoting common sporadic CRCs have yet to be identified. The Mutated in Colorectal Cancer (MCC) gene, identified in 1991, was initially thought to be responsible for the hereditary form of CRC, familial adenomatous polyposis, before the discovery of the susceptibility gene Adenomatous Polyposis Coli (APC), which then became the focus of intense research. Recent data, however, suggests that MCC may also be important in the development of CRC. I have investigated the mechanism of MCC gene silencing, the putative structure, and multiple functions of MCC. MCC was frequently silenced by promoter hypermethylation in CRC cell lines and primary tumours. MCC methylation showed strong molecular and clinicopathological associations with hallmarks of the serrated neoplasia pathway. Furthermore, MCC methylation was more frequent in serrated precursor lesions compared with adenomas, thus occurring early during carcinogenesis. MCC is highly conserved in complex multicellular organisms. Re-introduction of MCC in CRC cell lines resulted in partial G1 to S phase, and G2/M phase cell cycle blocks, potentially by upregulating cell cycle inhibitor gene transcription and interfering with the process of mitotic checkpoints and division, respectively. Changes in MCC levels also modulated NF?B pathway signalling, the pathway required for maintaining cell viability and proliferation in colonic epithelial cells. In particular, MCC overexpression suppressed both TNF? and LPS-induced NF?B activation, decreasing both the magnitude and rate of cellular responses. Overexpression also resulted in downregulation of proteins involved in canonical NF?B pathway signalling, while increasing the transcription of non-canonical NF?B genes. Therefore, MCC may direct activation of this pathway to a specific subset of NF?B-regulated genes. These data provide a molecular basis for the role of MCC as a tumour suppressor gene in CRC. MCC may have multiple functions, regulating cell cycle progression and modulating NF?B pathway signalling, either through direct involvement in pathway signalling cascades, or by providing a scaffold on which signalling events can occur.

Though known for his fantastical writings George MacDonald is often considered to be a typical Victorian teacher of religious and moral seriousness. Approaches to MacDonald’s works normally seek to find his ‘message’ by expositing the moral, social, pedagogical, psychological or theological ‘content’ of his work. This study recasts MacDonald in the light of his shorter fairytales for the ‘childlike’ and argues that these seemingly small and insignificant works are a golden key to his artistic enterprise. This is not because of any particular ‘message’ that they carry but because of their peculiarly light mode of generating meaning and the relation of this lightness to theology. Whilst it is frequently disparaged, levity actually has strong parallels with the theological atmosphere of Christianity. Light modalities such as folly, ecstasy, play, vanity, carnival and Sabbath demonstrate that the Christian faith has greater affinities with lightness and whimsicality than its solemn defenders sometimes admit. MacDonald’s fairytales draw upon this surprising harmony between levity and faith to create environments in which readers can playfully reflect upon the nature of ultimate reality and begin to find their own place within that reality. By helping to remove the mask of ‘seriousness’ presented by things in the everyday world, fairytales engender a kind of ‘divine carelessness’ and help people to let go of the weighty cares and fears that keep them tightly bound to worldly things.

Colorectal cancer (CRC) remains a significant burden in contemporary society due to an aging population, unhealthy dietary choices and an increasingly sedentary lifestyle. While the underlying defects for many hereditary forms of CRC have been determined, many genetic and epigenetic changes promoting common sporadic CRCs have yet to be identified. The Mutated in Colorectal Cancer (MCC) gene, identified in 1991, was initially thought to be responsible for the hereditary form of CRC, familial adenomatous polyposis, before the discovery of the susceptibility gene Adenomatous Polyposis Coli (APC), which then became the focus of intense research. Recent data, however, suggests that MCC may also be important in the development of CRC. I have investigated the mechanism of MCC gene silencing, the putative structure, and multiple functions of MCC. MCC was frequently silenced by promoter hypermethylation in CRC cell lines and primary tumours. MCC methylation showed strong molecular and clinicopathological associations with hallmarks of the serrated neoplasia pathway. Furthermore, MCC methylation was more frequent in serrated precursor lesions compared with adenomas, thus occurring early during carcinogenesis. MCC is highly conserved in complex multicellular organisms. Re-introduction of MCC in CRC cell lines resulted in partial G1 to S phase, and G2/M phase cell cycle blocks, potentially by upregulating cell cycle inhibitor gene transcription and interfering with the process of mitotic checkpoints and division, respectively. Changes in MCC levels also modulated NF?B pathway signalling, the pathway required for maintaining cell viability and proliferation in colonic epithelial cells. In particular, MCC overexpression suppressed both TNF? and LPS-induced NF?B activation, decreasing both the magnitude and rate of cellular responses. Overexpression also resulted in downregulation of proteins involved in canonical NF?B pathway signalling, while increasing the transcription of non-canonical NF?B genes. Therefore, MCC may direct activation of this pathway to a specific subset of NF?B-regulated genes. These data provide a molecular basis for the role of MCC as a tumour suppressor gene in CRC. MCC may have multiple functions, regulating cell cycle progression and modulating NF?B pathway signalling, either through direct involvement in pathway signalling cascades, or by providing a scaffold on which signalling events can occur.

Generalized geometry of type B_n is the study of geometric structures in T+T^*+1, the sum of the tangent and cotangent bundles of a manifold and a trivial rank 1 bundle. The symmetries of this theory include, apart from B-fields, the novel A-fields. The relation between B_n-geometry and usual generalized geometry is stated via generalized reduction. We show that it is possible to twist T+T^*+1 by choosing a closed 2-form F and a 3-form H such that dH+F²=0. This motivates the definition of an odd exact Courant algebroid. When twisting, the differential on forms gets twisted by d+Fτ+H. We compute the cohomology of this differential, give some examples, and state its relation with T-duality when F is integral. We define B_n-generalized complex structures (B_n-gcs), which exist both in even and odd dimensional manifolds. We show that complex, symplectic, cosymplectic and normal almost contact structures are examples of B_n-gcs. A B_n-gcs is equivalent to a decomposition (T+T^*+1)_ℂ= L + L + U. We show that there is a differential operator on the exterior bundle of L+U, which turns L+U into a Lie algebroid by considering the derived bracket. We state and prove the Maurer-Cartan equation for a B_n-gcs. We then work on surfaces. By the irreducibility of the spinor representations for signature (n+1,n), there is no distinction between even and odd B_n-gcs, so the type change phenomenon already occurs on surfaces. We deal with normal forms and L+U-cohomology. We finish by defining G²₂-structures on 3-manifolds, a structure with no analogue in usual generalized geometry. We prove an analogue of the Moser argument and describe the cone of G²₂-structures in cohomology.

The purpose of this thesis is to explain new approaches to scanning and locking vulnerabilities in computer security and to design a new system to improve security of computers running the Linux operating system. The purpose of this system is to analyze remote operating systems and detect and lock down vulnerabilities by existing security standards.

Abstract Hereditary mutations in DNA damage response (DDR) genes often lead to genomic instability and ultimately tumor development. However, the molecular mechanism of how these DDR deficiencies promote genomic instability and malignancy is not well understood. Thus, the specific aim of this thesis is to identify the functional and molecular framework behind the elevated breast cancer risk observed in heterozygous PALB2 and ABRAXAS mutation carriers. The heterozygous germline alteration in PALB2 (c.1592delT) causes a haploinsufficiency phenotype in the mutation carrier cells. Due to PALB2 haploinsufficiency, elevated Cdk activity and consequently aberrant DNA replication/damage response was observed in the PALB2 mutation carrier cells. Excessive origin firing that is indicative of replication stress was also seen in the PALB2 mutation carrier cells. In addition to replication stress, PALB2 mutation carrier cells also experience G2/M checkpoint maintenance defects. The increased malignancy risk in females associated with heterozygosity for the Finnish PALB2 founder mutation is likely to be due to aberrant DNA replication, elevated genomic instability and multiple different cell cycle checkpoint defects. The heterozygous germline alteration in ABRAXAS (c.1082G>A) causes a dominant-negative phenotype in the mutation carrier cells. Decreased BRCA1 protein levels as well as reduced nuclear localization and foci formation of BRCA1 and CtIP was observed in the ABRAXAS mutation carrier cells. This causes disturbances in basal BRCA1-A complex localization, which is reflected by a restraint in error-prone DNA double-strand break (DSB) repair pathway usage, attenuated DNA damage response, deregulated G2/M checkpoint control and apoptosis. Most importantly, mutation carrier cells display a change in their transcriptional profile, which we attribute to the reduced nuclear levels of BRCA1. Thus, the Finnish ABRAXAS founder mutation acts in a dominant-negative manner on BRCA1 to promote genome destabilization in the heterozygous carrier cells. / Tiivistelmä Perinnölliset muutokset DNA-vauriovasteen geeneissä johtavat usein genomin epävakauteen ja lopulta syövän kehittymiseen. Molekyylitason mekanismeja, joilla vauriovasteen vajaatoiminta ajaa genomin epävakautta ja syöpää, ei kuitenkaan ymmärretä kunnolla. Tämän väitöskirjan tavoitteena on tunnistaa solutoiminnan ja molekyylitason vaikuttajat heterotsygoottisten PALB2- ja ABRAXAS-geenimuutosten kantajien kohonneen rintasyöpäriskin taustalla. Heterotsygoottinen ituradan suomalainen perustajamuutos PALB2-geenissä (c.1592delT) aiheuttaa haploinsuffisienssin kantajahenkilöiden soluissa. PALB2:n haploinsuffisienssin seurauksena kantajasoluissa havaittiin kohonnutta Cdk-proteiinin aktiivisuutta ja siitä johtuvaa kiihtynyttä DNA:n kahdentumista. PALB2-mutaatiota kantavissa soluissa nähtiin myös liiallista replikaation aloituskohtien käyttöä, mikä viittaa replikaatiostressiin. Replikaatiostressin lisäksi PALB2-mutaation kantajasoluilla havaittiin vaikeuksia ylläpitää solusyklin G2/M-tarkastuspisteen toimintaa. Näiden solutoiminnan poikkeavuuksien takia heterotsygoottisen PALB2 c.1592delT -mutaation kantajilla todettiin genomin epävakautta ja kohonnut syöpäriski. Heterotsygoottinen ituradan mutaatio ABRAXAS-geenissä (c.1082G>A) aiheuttaa dominantti-negatiivisen fenotyypin mutaation kantajasoluissa. ABRAXAS-mutaatiota kantavissa soluissa havaittiin BRCA1-proteiinitasojen laskua sekä BRCA1- ja CtIP-proteiinien vähentynyttä lokalisaatiota tumaan ja DNA-vauriopaikoille. Tämä aiheuttaa häiriöitä BRCA1-A-kompleksin paikallistumisessa, mikä johtaa häiriöihin virhealttiiden DNA-kaksoisjuoste¬katkoksien korjausmekanismien käytössä, DNA-vauriovasteessa, G2/M-tarkastus-pisteen säätelyssä ja ohjelmoidussa solukuolemassa. Tärkeimpänä löydöksenä havaittiin mutaation kantajasoluissa muuttunut transkriptioprofiili, joka johtunee BRCA1-proteiinitasojen laskusta tumassa. Näin ollen suomalainen ABRAXAS-perustajamutaatio toimii dominantti-negatiivisena BRCA1:n suhteen, aiheuttaen genomin epävakautta heterotsygoottisissa kantajasoluissa.

Notre travail est centré sur la démarche de découverte reposant sur des expérimentations réalisées avec Cabri-Géomètre. L'analyse d'un corpus débordant le cadre des Mathématiques clarifie la manière dont la découverte arrive ou est transmise, ainsi que le rôle de l'expérimentation dans ces processus. Elle justifie notre hypothèse de décomposition de la démarche de découverte expérimentale en macro-étapes pré- et post-conjectures elles-mêmes décomposables en micro-étapes du type exploration-interprétation..<br />L'analyse de la résolution d'une boîte noire particulière permet d'affiner notre modèle a priori de la démarche de découverte en y précisant le rôle de la figure (Duval), les niveaux de géométrie (praxéologies G1 et G2 de Parzysz) et leurs prolongements que nous développons (G1 et G2 informatiques), les cadres d'investigations (Millar) et la place de la preuve expérimentale (Johsua).<br />Les analyses des expérimentations mises en place permettent de disposer d'un modèle amélioré qui doit permettre aux enseignants d'avoir une connaissance minimale des étapes heuristiques du travail de leurs élèves, de concevoir des activités d'études et de recherches ayant des objectifs précis en liaison avec les étapes formalisées de notre modélisation et d'envisager leur possible évaluation.<br />Des analyses d'activités existantes avec notre grille montrent la validité du modèle étudié. Des propositions d'activités ont été construites pour favoriser l'apparition de telle ou telle phase de la recherche; elles montrent la viabilité de ce modèle dans la conception d'ingénieries didactiques générant une démarche conforme à la démarche postulée.

In mehrfaktoriellen 2 x 2 x 4 Untersuchungen im Zeitraum vom 7. - 28. Lebenstag und in Bilanzversuchen vom 15. - 20. Lebenstag mit männlichen Broilerküken (Cobb 500) wurden die Effekte der Versuchsfaktoren Zerkleinerung (Hammermühle vs. Walzenstuhl), thermische Behandlung (Konditionierung bei 70°C vs. Konditionierung/Expandierung 100°C) und Zusätze von Zink-Bacitracin bzw. Roxazym G2 (ohne Zusatz, mit Zink-Bacitracin 50 mg, mit Roxayzm G2 150 ppm und deren Zusatzkombination A+E) sowie die Interaktionen untersucht. Als Kriterien dienten die Parameter Futterverzehr, Lebendmassezunahme, Futteraufwand, Nährstoffansatz und -verwertung, ileale Verdaulichkeit von ausgewählten Aminosäuren, Proteinverwertung/Proteinqualität und Umsetzbarkeit der Energie. Die Versuchstiere erhielten ab dem 7. Lebenstag die entsprechenden Versuchsmischungen. Der Gehalt an XP und MEn aller Versuchsmischungen war einheitlich (XP 21,7% und MEn 12,3 MJ/kg Futter). Die Lysinversorgung wurde auf 90 % unter der optimalen Bedarfsdeckung in allen Futtermischungen limitiert. Die Auswirkungen der Versuchsfaktoren lassen sich wie folgt zusammenfassen: - Zerkleinerung : Die Zerkleinerungstechnologie mit dem Walzenstuhl übte einen signifikanten Einfluss auf den Futterverzehr (-3,5 %) und Futteraufwand (-2,8 %) gegenüber der Zerkleinerung mit der Hammermühle aus. Die Nährstoffverwertung (XP und Energie) zeigten durch Walzenstuhl-Zerkleinerung tendenzielle Verbesserungen. Die ileale Lysinverdaulichkeit blieb unverändert, die ileale Verdaulichkeit von Threonin und Met+Cys wurde signifikant erhöht. Die Walzenstuhl-Zerkleinerung führte zu einer besseren Futterstruktur und zu einer höheren Nährstoffdichte in den Pellets. Das wird deutlich durch die höhere N-Aufnahme bzw. N-Bilanz sowie durch gesteigerte N-Verwertungsparameter und einen erhöhten Gehalt an N-korrigierter umsetzbarer Energie (MEn). - Thermische Behandlung : Durch erhöhte Hitzeapplikation mit dem Expander konnten in der vorliegenden Arbeit hinsichtlich der Leistungsparameter, Nährstoffansatz und -verwertung keine Unterschiede gegenüber der Konditionierung festgestellt werden. Die Expandierung führte zu einer signifikant erhöhten ilealen Lysinverdaulichkeit, die durch die gemessene Lysinwirksamkeit im Bilanzversuch jedoch nicht widergespiegelt wurde. Auch signifikant niedrigere N-Bilanz und physiologische Proteinnutzwerte (PNu) sowie die tendenzielle Verringerung der N-Verdaulichkeit und des Gehaltes an umsetzbarer Energie deuten auf eine negative Wirkung der intensiveren thermischen Behandlung durch Expandieren hin. Hierzu sind weitere klärende Untersuchungen notwendig. - Futterzusätze: Durch die alleinige Supplementierung mit dem Antibiotikum Zink-Bacitracin oder NSP-spaltenden Enzym Roxazym G2 bzw. deren Kombination reagierten Mastleistung und Futterverwertung signifikant positiv. Während der Effekt der Enzymzulagen bei Nährstoffverwertung und ilealer Verdaulichkeit ausgewählter Aminosäure signifikant höher gegenüber der unsupplementierten Gruppe war, blieb ein Effekt von Zink-Bacitracin hinsichtlich dieser Parameter aus. Der Effekt der Zusatzkombination war bei Mastleistung, Nährstoffansatz und -verwertung und bei der ilealen Verdaulichkeit der ausgewählten Aminosäuren gegenüber der Kontrolle oder dem alleinigen Zusatz signifikant höher. Das deutet auf einen synergistischen Effekt der gleichzeitigen Applikation der beiden Additive hin. Die N-Verwertung einschließlich des Gehalts an N-korrigierter scheinbar umsetzbarer Energie lag nach alleiniger Applikation von Zink-Bacitracin unerwartet signifikant niedriger gegenüber den anderen Zusätzen bzw. tendenziell gegenüber der Kontrolle. Die Gehalte an scheinbar umsetzbarer Energie (AMEn) waren deutlich durch den Enzymzusatz allein oder in Kombination mit Zink-Bacitracin erhöht. -Interaktionen: Die Abhängigkeit der Versuchsfaktoren voneinander im Mastversuch war nicht stark ausgeprägt. Die Zerkleinerung in Verbindung mit anschließender thermischer Behandlung führte zur Beeinflussung der Futterverzehrsdaten. Danach verbesserten die Verfahrenskombinationen Hammermühle x Konditionierung oder Walzenstuhl x Expandierung bedingt durch einen erhöhten Futterverzehr die Lebendmassezunahme und den Nährstoffansatz signifikant. Hinsichtlich der ilealen Aminosäurenverdaulichkeit zeigten die Futterzusätze eine Abhängigkeit von der Behandlung bzw. Zerkleinerung und Behandlung. Die Enzymzulage allein oder in Kombination mit Zink-Bacitracin zeigte stärkere Effektivität in Verbindung mit der thermischen Behandlung durch Expandieren.