Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-04-03T19:41:30Z No. of bitstreams: 1 RafaelVianaSales_DISSERT.pdf: 3545507 bytes, checksum: 4ef61dd2d94d0034feabb461fc8913f1 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-04-05T19:14:13Z (GMT) No. of bitstreams: 1 RafaelVianaSales_DISSERT.pdf: 3545507 bytes, checksum: 4ef61dd2d94d0034feabb461fc8913f1 (MD5) / Made available in DSpace on 2017-04-05T19:14:13Z (GMT). No. of bitstreams: 1 RafaelVianaSales_DISSERT.pdf: 3545507 bytes, checksum: 4ef61dd2d94d0034feabb461fc8913f1 (MD5) Previous issue date: 2015-12-21 / A emiss?o de compostos sulfurados provenientes da queima de combust?veis derivados do refino do petr?leo, como ?leo diesel e gasolina, ? respons?vel por ocasionar danos ambientais, ? sa?de humana e aumento de custos industriais. Neste trabalho, foram estudados processos adsortivos de compostos sulfurados do diesel proveniente da Refinaria Potiguar Clara Camar?o, RPCC, com elevado teor de enxofre, utilizando s?licas mesoporosas do tipo MCM-41, que foram obtidas por m?todo hidrot?rmico, a partir de duas s?licas amorfas, s?lica MP1, novo material avaliado neste trabalho e uma s?lica comercial, utilizada para compara??o. Para os adsorventes mesoporosos MCM-41, sintetizados, foi avaliada a capacidade de adsor??o de enxofre em colunas de leito fixo e, posteriormente, os mesmos foram modificados, p?s-situ, com c?tions met?licos por impregna??o ?mida e dispers?o f?sica. Diferentes composi??es qu?micas dos compostos met?licos foram testadas, com a utiliza??o de ?xidos e sais, para a obten??o de s?tios ativos e avalia??o da complexa??o, entre o enxofre dos compostos org?nicos e os c?tions met?licos inseridos nos adsorventes, favorecendo a adsor??o. Os adsorventes sintetizados, puros e modificados, foram caracterizados atrav?s das t?cnicas de difratometria de raios X - DRX, espectroscopia de infravermelho com transformada de Fourier - FTIR, an?lises termogravim?tricas - TG/DTG, an?lises texturais pelo m?todo de BET/BJH e microscopia eletr?nica de varredura ? MEV com espectroscopia de energia dispersiva (EDS). Um planejamento fatorial do tipo 23, foi aplicado para o estudo do processo de adsor??o onde blends dos c?tions met?licos foram testados na adsor??o dos compostos sulfurados existentes. O monitoramento do teor de enxofre no diesel, durante o processo de adsor??o, ocorreu por t?cnica de Espectrometria de Fluoresc?ncia na Regi?o do Ultravioleta (FUV). A cin?tica e o equil?brio de adsor??o foram avaliados, onde isotermas de equil?brio foram obtidas para a avalia??o da capacidade m?xima de adsor??o de compostos sulfurados, nos sistemas adsorventes desenvolvidos neste trabalho. Foram obtidas capacidades m?ximas de adsor??o, em mg.g-1, de 16,64 e 14,98, respectivamente, para os adsorventes testados: 2AgS/MCM-41(M) e 1NiS1AgS/MCM-41(M). Os dados experimentais foram ajustados aos modelos de equil?brio de Langmuir e Freundlich. A remo??o de enxofre presente no diesel foi significante, atingindo o m?ximo de adsor??o de 94,9%, em rela??o ? concentra??o inicial deste contaminante. Atrav?s do estudo estat?stico, constatou-se que os blends preparados a partir dos adsorventes modificados por sais apresentaram uma capacidade de adsor??o maior em rela??o aos preparados por impregna??o de ?xidos met?licos, o que pode contribuir para a redu??o de custos industriais, uma vez que o m?todo de impregna??o ?mida dispensa uma segunda calcina??o. / The emission of sulfur compounds from the burning of oil products, such as diesel and gasoline, is responsible for causing environmental damage to human health and increase industrial costs. In this work, adsorptive processes of sulfur compounds from the diesel provided by the refinery Potiguar Clara Camar?o, RPCC, with high sulfur content, were studied using MCM-41 type of mesoporous silica which were obtained by hydrothermal method, from two amorphous silicas, MP1 silica, a new material evaluated in this work and a commercial silica, used for comparison. For MCM-41 mesoporous adsorbents, synthesized, it was evaluated the capability of sulfur adsorption in fixed bed columns, and later they were modified post-situ with metal cations by wet impregnation and physical dispersion. Different chemical compositions of metal compounds were tested with the use of oxides and salts, to obtain active sites and evaluation of complexing, between the sulfur of organic compounds and the metal cations inserted on the adsorbent, favoring adsorption. The synthesized adsorbents, pure and modified, were characterized by the techniques of X-ray diffraction - XRD, Fourier transform infrared spectroscopy - FTIR, thermogravimetric analysis - TG / DTG, textural analysis by the method of BET / BJH and scanning electron microscopy - SEM with energy dispersive spectroscopy (EDS). A 23 factorial plan was applied to study the adsorption process which metal cations blends were tested in the adsorption of existing sulfur compounds. The sulfur content monitoring in diesel fuel during the adsorption process occurred by ultraviolet fluorescence Spectrometry (UVF). The adsorption kinetics and equilibrium were evaluated, where equilibrium isotherms were obtained for the evaluation of the maximum adsorption capacity of sulfur compounds in adsorbent systems developed in this work. Maximum adsorption capacities were obtained ,at mg.g-1, 16,64 and 14,98, respectively, for the tested adsorbent: 2AgS/MCM-41(M) e 1NiS1AgS/MCM-41(M. The experimental data were fit to a Langmuir balance models and Freundlich. The removal of sulfur present in the diesel was significant, ranging from 54.3% to 88.9% compared to the initial concentration of contaminant. By statistical analysis it was found that blends prepared from the adsorbent modified by salts showed a higher adsorption capacity than the ones prepared by impregnation of metal oxides, which may contribute to the reduction of industrial costs, since the wet impregnation method dispenses a second calcination.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / In this work the mesoporous material MCM-41 was functionalized with 3-aminopropyltrimetoxysilane (APS) for several purposes such as adsorption of the remazol red dye, immobilization of lanthanide ions (Eu+3, Tb+3 or Gd+3), coordination with the 2,6-pyridinedicarboxylic acid (dpa) and immobilization of the complex [RuCl2(PPh3)2(Meo-bipy)]. The structural and textural properties of the MCM-41 and NH2-MCM-41 were characterized by absorption spectroscopy in the infrared region, thermal analysis, X-ray diffraction and nitrogen adsorption-dessorption. The adsorption capacity of NH2-MCM-41 was studied with Remazol Red dye. The following parameters were studied in the adsorption process: pH, temperature, adsorbent dosage and initial concentration. The desorption process was studied in a NaOH solution which different concentrations. The Freundlich isotherm model was found to be fit with the equilibrium isotherm data. Kinetics of adsorption follows the modified Avrami rate equation. The NH2-MCM-41 adsorbed 99,1 % of the dye in 360 minutes at 25 ºC. Data from X-ray diffractograms of the NH2-MCM-41-Ln(dpa) (Ln = Eu+3, Tb+3 or Gd+3) material indicated that the materials showed a hexagonal structure with a low degree of ordering. The insertion of the lanthanide ions was indicated by the shift of the amine group of APTS in the spectra of NH2-MCM-41-Ln. The coordination of dpa to NH2-MCM-41-Ln (Ln = Eu+3, Tb+3 or Gd+3) materials was evidenced by the shift of the bands of COH and COO-. The elemental analysis indicated the coordination in the ratio 1:3 (metal:dpa). The nitrogen adsorption analysis shows that with changes in MCM-41, the surface area, pore volume and diameter of the material decreased indicating the immobilization of ions lanthanides and dpa within the pores of the mesoporous material. The evaluation of the triplet level of the ligand, from the spectra of the NH2-MCM-41-Gd(dpa)3 shows that their position favors the energy transfer of metal to ligand for NH2-MCM-41-Eu(dpa)3 material, however the NH2-MCM-41-Tb(dpa)3 material this process is complicated due to the triplet level of ligand is below the issuing level of the Tb+3 ion. Data from X-ray diffractograms of NH2-MCM-41-[RuCl(PPh3)2(Meo-bipy)] showed a hexagonal structure with a low degree of ordering. In addition, the adsorption spectra in the infrared region of the immobilized complex have a displacement in the band 1612 cm-1 characteristic of the ruthenium complex. The thermogravimetric analysis showed that the complex immobilized on the functionalized MCM-41 present high thermal stability compared to free complex. The nitrogen adsorption analysis showed that surface area, pore volume and diameter of the NH2-MCM-41-[RuCl(PPh3)2(Meo-bipy)] are smaller than for the NH2-MCM-41, indicating the immobilization the complex within the pores of the NH2-MCM-41. Catalytic tests for reduction of ketones were carried out with the [RuCl2(PPh3)2(Meo-bipy)] complex. For acetophenone and sulcatone, [RuCl2(PPh3)2(Meo-bipy)] proved to be a good catalyst with 90.50 % and 33.95 % conversion, respectively. / Neste trabalho, o material mesoporoso MCM-41 foi funcionalizado com 3-aminopropilmetoxisilano (APTS) para diversas finalidades como, adsorção do corante vermelho de remazol, imobilização de íons lantanídeos (Eu+3, Tb+3 ou Gd+3), coordenação com o ácido 2,6-piridina-dicarboxílico (dpa) e imobilização do complexo [RuCl2(PPh3)2(Meo-bipy)]. As propriedades estruturais e texturais do MCM-41 e do NH2-MCM-41 foram caracterizados por espectroscopia de absorção na região do infravermelho, análise térmica, difratometria de raios-X e adsorção-dessorção de nitrogênio. A capacidade de adsorção do corante vermelho de remazol foi avaliada com o NH2-MCM-41. Os seguintes parâmetros foram estudados: pH, temperatura, dose do adsorvente e concentração do corante. O processo de dessorção foi examinado em solução aquosa de NaOH em diferentes concentrações. O modelo de isoterma de Freudlich obteve maior adequação ao processo de adsorção e a cinética de adsorção seguiu o modelo modificado de Avrami. O NH2-MCM-41 adsorveu 99,1 % do corante em 360 minutos a 25 ºC. Os dados dos difratogramas de raios-X dos materiais NH2-MCM-41-Ln(dpa) (Ln = Eu+3, Tb+3 ou Gd+3), indicaram que estes materiais apresentaram estruturas hexagonais com baixo grau de ordenação. A inserção dos íons lantanídeos foi indicada pelo deslocamento da banda do grupo amina do APTS nos espectros do NH2-MCM-41-Ln. A coordenação do dpa aos materiais NH2-MCM-41-Ln (Ln = Eu+3, Tb+3 ou Gd+3) foi evidenciada através do deslocamento das bandas do COH e do COO-. A análise elementar indicou a coordenação na proporção 1:3 (metal: dpa). A análise da adsorção de nitrogênio revela que com as modificações no MCM-41, a área superficial, diâmetro e volume de poros dos materiais diminuíram indicando a imobilização dos íons lantanídeos e do dpa no interior dos poros do material mesoporoso. A avaliação do nível tripleto do ligante, a partir do espectro do NH2-MCM-41-Gd(dpa)3, demonstra que sua posição favorece a transferência de energia ligante-metal para o material NH2-MCM-41-Eu(dpa)3, entretanto, para o material NH2-MCM-41-Tb(dpa)3 este processo é dificultado devido o nível tripleto do ligante está abaixo do nível emissor do íon Tb+3. Os dados de difratogramas de raios-X do NH2-MCM-41-[RuCl(PPh3)2(Meo-bipy)] evidenciaram uma estrutura hexagonal com baixo grau de ordenação. Além disso, o espectro de absorção na região do infravermelho do complexo imobilizado apresenta um deslocamento na banda em 1612 cm-1 característica do complexo de rutênio. As análises termogravimétricas demonstram que os complexos imobilizados no MCM-41 funcionalizado apresentam elevada estabilidade térmica comparada com a do complexo livre. Os dados da adsorção de nitrogênio demonstraram que a área superficial, diâmetro e volume de poros do NH2-MCM-41-[RuCl(PPh3)2(Meo-bipy)] são menores que para o NH2-MCM-41, indicando a eficiência no processo de imobilização do complexo no interior dos poros do NH2-MCM-41. Testes catalíticos para redução de cetonas foram realizados com o complexo [RuCl2(PPh3)2(Meo-bipy)]. Para a acetofenona e sulcatona, o [RuCl2(PPh3)2(Meo-bipy)] mostrou-se ser um bom catalisador com 90,50 % e 33,95 % de conversão, respectivamente.

La tendance actuelle vers l’encapsulation d’un assemblage comportant plusieurs puces favorise l’incorporation d’un procédé de réusinage compatible avec l’enlèvement et le remplacement d’une puce défectueuse. La vérification électrique est un procédé inhérent au réusinage qui identifie facilement les puces défectueuses sans en compromettre leur intégrité. Malheureusement, l’aspect qui optimise l’intégrité mécanique et électrique des dispositifs, soit l’encapsulant (underfill), rend les étapes subséquentes d’enlèvement des puces extrêmement difficile. Il devient alors souhaitable d’évaluer le comportement de certaines puces sans underfill qui sont sujettes aux conditions de vérifications électriques souhaitées afin de déterminer s’il existe une zone de test favorable à la qualité et la fiabilité du produit. Ceci est d’autant plus vrai en considérant le peu de données publiées concernant un chargement en compression qu’une puce sans underfill et sans capot peut supporter. Ce projet de recherche présente une étude du comportement des puces renversées sans capot, utilisant un alliage SAC, qui est sans plomb, pour les interconnexions, face à une charge compressive appliquée directement. Les puces sont assemblées sur un substrat organique sans underfill dans une configuration à plusieurs puces. Pour différentes tailles de puces, une série de compressions ont été observées afin de couvrir une vaste étendue de forces potentiellement utilisées pour assurer le contact électrique du laminé non planaire. Les chargements ont été faits à température ambiante et élevée ainsi qu’à angle normal et incliné. La caractérisation par l’analyse des courbes de chargement, la coplanarité, la mesure des hauteurs des interconnections et la tomographie par rayon X a démontré qu’un angle normal ne provoquait qu’une légère déformation qui était relativement indépendante de la température ou de la force dans l’étendu testé. Bien que les données de fluage semblent entrer dans le cadre des modèles précédemment publiés et un nouveau modèle plus juste a été créé avec l’aide de test de nanoindentation. L’inclinaison du chargement a démontré produire des déformations significatives qui, durant un test électrique typique de 20 minutes, étaient similaire pour un faible ou un grand chargement. Par contre, ces déformations avaient tendance à être plus importantes pour une température élevée et pour une petite taille de puce. De telles déformations ont démontrés induire une recristallisation des grains en une taille plus fine sans retrouver leur forme après un recuit. De plus, les déformations sévères ont démontrées une présence de fissurations à température ambiante et d’espace réduit à température élevée pouvant nuire à l’intégrité de l’assemblage. Un cyclage thermique de 1000 cycles n’a pas induit de défaillance électrique pour le nombre d’échantillons testés, mais des tests avec un plus grand échantillonnage sont recommandés. En somme, cette étude conclue qu’une vérification électrique dans des conditions normales d’une puce sans capot ne va pas endommager l’intégrité des interconnections sans plomb. Toutefois, un contrôle des procédés robustes est nécessaire afin d’éviter des conditions anormales résultant en une inclinaison de la charge qui pourrait compromettre l’intégrité des assemblages.

The CMG (Cdc45, MCM, GINS) helicase is required for cellular proliferation and functions to unwind double-stranded DNA to allow the replication machinery to duplicate the genome. Cancer cells mismanage helicase activation through a variety of mechanisms, leading to the potential for the development of novel anti-cancer treatments. Mammalian cells load an excess of MCM complexes that act as reserves for new replication origins to be created when replication forks stall due to stress conditions, such as drug treatment. Targeting the helicase through inhibition of the MCM complex has sensitized cancer cells to drugs that inhibit DNA replication, such as aphidicolin and hydroxyurea. However, these drugs are not used in the clinical management of cancer. We hypothesized that the effectiveness of the clinically relevant drugs gemcitabine and 5-FU against pancreatic cancer cells, and oxaliplatin and etoposide against colorectal cells, could be increased through co-suppression of the MCM complex. The oncogene c-Myc also leads to the mismanagement of CMG helicases in part due to a non-transcriptional role in overactivating replication origins and causing DNA damage. We sought to elucidate the mechanism by which Myc causes overactivation of CMG helicases. Herein we demonstrate that co-suppression of reserve MCM complexes in pancreatic or colorectal cancer cell lines treated with clinically applicable chemotherapeutic compounds causes significant loss of proliferative capacity compared with cells containing the full complement of reserve MCMs. This is in part due to an inability to recover DNA replication following drug exposure, leading to an increase in apoptosis. Targeting of Myc to genomic sites induced large-scale decondensation of higher order chromatin that was required for CMG helicase assembly and activation at reserve MCM complexes. The physiological mediators of Myc, GCN5 and Tip60, are required for the chromatin unfolding and Cdc45 recruitment. We conclude that depletion of the reserve MCM complexes causes chemosensitization of multiple human tumor cell types to several chemotherapeutic drugs used in the clinical management of human cancer. This argues for the development and use of anti-MCM drugs in combination with chemotherapeutic compounds, which has the potential to increase the therapeutic index of existing clinical compounds. We have also identified a previously unknown role for Myc in normal cell cycle progression whereby DNA replication initiation is regulated through the assembly and activation of CMG helicases on Myc-mediated open chromatin regions. Our results also provide new mechanistic insight into Myc oncogenic transformation in which overstimulation of DNA replication could result in genomic instability and provide an explanation for Myc driven oncogenic transformation.

Tumorigenesis is a multifaceted set of events consisting of the deregulation of several cell-autonomous and tissue microenvironmental processes that ultimately leads to the acquisition of malignant disease. Transforming growth factor beta (TGFß) and its family members are regulatory cytokines that function to ensure proper organismal development and the maintenance of homeostasis by controlling cellular differentiation, proliferation, adhesion, and survival, as well as by modulating components of the cellular microenvironment and immune system. The pleiotropic control by TGFß of these cell intrinsic and extrinsic factors is intimately linked to the prevention of tumor formation, the specifics of which are dependent on the various cellular and/or molecular signaling contexts that exist for TGFß. The diverse roles and the various levels of signal control for TGFß lend themselves to certain characteristics that are more advantageous for cancers to usurp in order to promote tumorigenesis, while other anti-tumorigenic roles for TGFß are more beneficial to tumor development if they are circumvented or disabled. Transforming growth factor ß1 (TGF-ß1) exerts its anti-tumor effects in large part by potently inhibiting cell cycle progression at any point in G1 phase to control the proliferation of a variety of cell lineages. Loss of sensitivity to TGF-ß1-induced cell cycle arrest is a crucial event during early tumorigenesis. Indeed, cancer cells of almost all tumor types display insensitivity to TGF-ß1 inhibition. As such, the pursuit of the molecular details underlying the TGF-ß1 growth arrest pathway is important for our understanding of cell cycle regulation, and significantly, how disruption of these mechanisms contributes to TGF-ß1 insensitivity and tumorigenesis. TGF-ß1 inhibition of the cell cycle in G1 phase has been shown to involve two main transcriptionally based molecular events, including the induction of cyclin-dependent kinase (CDK) inhibitors and the suppression of the c-Myc protein. Both mechanisms contribute to the maintenance of the retinoblastoma (Rb) protein in its hypophosphorylated and antiproliferative form, thus preventing progression through the cell cycle. However, this type of regulation does not offer answers to all of the questions regarding TGF-ß1 arrest. While these transcriptional mechanisms provide explanations for TGF-ß1 arrest throughout most of G1, inhibition late in G1 by TGF-ß1 however, does not require any acute regulation of transcription. In addition, the chance to utilize canonical TGF-ß1 arrest mechanisms at this time has already passed (i.e. Rb is already hyperphosphorylated by late-G1). Previous work from our group shows instead that late-G1 TGF-ß1 cell cycle arrest requires an intact direct interaction between the N-terminus of Rb (RbN) and the C terminus of Mcm7, a subunit of the Cdc45-MCM-GINS (CMG) replicative helicase. Our studies show that TGF-ß1 exposure in late-G1 prevents the disassociation of Rb with fully assembled helicases, which remain inactive. In addition, it was found that early-G1 treatment with TGF-ß1 also targets CMG components, namely MCM protein accumulation (and therefore hexamer formation) in G1 is blocked. However, the residue(s) of RbN involved as well as the molecular mechanisms Rb utilizes for late-G1 TGF-ß1 arrest are not described, nor is it evident from this work if TGF-ß1 affects other genes involved in CMG assembly and/or activation. In the following study we explore these unanswered questions for TGF-ß1 growth arrest as a means to understand novel aspects of cell cycle regulation that must be abrogated during tumorigenesis. Our hypothesis is that CMG helicase control on some level is critical for all TGF-ß1-induced inhibition of cell cycle progression throughout the entire G1 phase. In Chapter 2 herein we have investigated the details and mechanistic implications of the Rb/RbN inhibitory-interaction with the CMG helicase that is required for late-G1 TGF-ß1 arrest. We show that N-terminal exons of Rb that are lost in partially penetrant hereditary retinoblastomas inhibit DNA replication and elongation using a bipartite mechanism. Specifically, Rb exon 7 is necessary and sufficient to inhibit CMG helicase activation, while an independent loop domain within RbN that forms a projection blocks DNA polymerase α (Pol-α) and Ctf4 recruitment without affecting polymerases δ and ε or the CMG helicase. Individual disruption of exon 7 or the projection in RbN or Rb, as occurs in inherited cancers, partially impairs the ability of Rb/RbN to inhibit DNA replication and block G1-S cell cycle transit. Importantly, their combined loss abolishes these functions of Rb. Thus, TGF-ß1 cell cycle arrest in late-G1 requires the growth suppressive role of Rb in which replicative complexes are blocked directly via independent and additive N-terminal domains. TGF-ß1-induced arrest in late-G1 also requires the presence of Smad3 and Smad4, suggesting that a novel transcription-independent role may exist for Smad signaling proteins in blocking cell cycle transit directly in Rb-CMG inhibitory complexes. TGF-ß1 is thought to require a functional Rb protein to inhibit the cell cycle at any point in G1 phase. Intriguingly, while cells lacking Rb (and the inhibitory N-terminal domains) lose sensitivity to TGF-ß1 arrest in late-G1, these same cells remain sensitive to TGF-ß1 inhibition in early-G1. This Rb-independent TGF-ß1 growth arrest also occurs in the absence of c-Myc and MCM suppression, as well as without CyclinE-Cdk2 inhibition, but requires Smad3 and Smad4 respectively. Here (Chapter 3) we have identified the mechanism by which TGF-ß1 achieves Smad-dependent G1 arrest in the absence of these common mediators. TGF-ß1 inhibits the assembly of CMG replicative helicases by suppressing the recruitment of the MCM complex to chromatin. Accordingly, the entire heterohexamer fails to load onto DNA. Cdc6 phosphorylation in its amino terminus is known to be required for Cdt1-dependent loading of the MCM complex. We show that in Rb-lacking cells early-G1 TGF-ß1 treatment blocks the phosphorylation of Cdc6 at serine 54, without affecting total Cdc6 protein levels, to prevent MCM heterohexamer formation on DNA. Consistent with TGF-ß1 signals targeting this recruitment and loading step, Cdt1 overexpression promotes S-phase entry in the presence of TGF-ß1, circumventing the need for Cdc6 phosphorylation. Importantly, Cdt1 requires an intact C-terminal MCM-binding domain in order to overcome this TGF-ß1-induced cell cycle arrest mechanism. These data indicate that early-G1 TGF-ß1 arrest can occur by perturbing Cdc6 phosphorylation to block Cdt1-mediated MCM recruitment and loading, leading to inhibition of CMG assembly and S-phase entry despite the lack of Rb and normal c-Myc and CyclinE-Cdk2 activities. We conclude that the main event governing TGF-ß1-induced cell cycle arrest at any point in G1 is the inhibition of the assembly and/or activation of the replicative CMG helicase. However, TGF-ß1 growth arrest has a temporal dependence on the presence of the Rb protein. In normal cells containing Rb, the accumulation of MCM subunit proteins is blocked by TGF-ß1 in early-G1 and accordingly MCM heterohexamers are unable to form. However, if cells are allowed to transit to late-G1 when MCM complexes have already assembled on origins, but before functional CMG helicases have formed at G1-S, exposure to TGF-ß1 signaling prevents CMG activation via interactions with critical inhibitory domains within RbN. Cells lacking Rb (and these residues) are not sensitive to TGF-ß1 in late-G1. Surprisingly, these cells remain sensitive to TGF-ß1 early in G1 phase despite a lack of c-Myc/MCM protein suppression and CyclinE-Cdk2 inhibition. In these cells the recruitment and loading of the MCM complex is blocked to facilitate a TGF-ß1-mediated G1 arrest. It is only when this mechanism is overcome by Cdt1 overexpression that TGF-ß1 is unable to elicit cell cycle arrest in these cells. These data provide molecular explanations for studies reporting instances of TGF-ß1 arrest without canonical effectors, such as Rb, c-Myc loss, or CDK inhibitors. Additionally, this work argues for the development of novel cancer therapeutics targeting CMG helicase assembly or activation, the regulation of which is likely lost in a variety of TGF-ß1-insensitive and/or Rb-deficient malignancies. Indeed, reintroduction of these tumor suppressive pathways has shown efficacy in blocking growth of tumors or cancer cells lacking the same mechanisms. Our studies of Rb/RbN inhibition of DNA replication also provide proof of principle for this type of therapy, as well as the framework for how the CMG might be targeted by exploring further and perhaps mimicking Rb exon7-mediated CMG inhibition biochemically.

In der vorliegenden Arbeit wird die Herstellung und Charakterisierung von partiell porösen Materialien auf der Grundlage von Ionenaustausch und Phasenseparation in Alkaliborosilicatgläsern beschrieben. Ausgehend von einem homogenen Natriumborosilicatglas wird durch den Austausch von Natrium gegen Lithium das Glas teilweise in die Mischungslücke überführt und so zur Phasenseparation befähigt. Es werden zwei möglich Prozessrouten beschrieben, bei der einerseits die Prozesse Ionenaustausch und Phasenseparation getrennt voneinander und andererseits parallel ablaufen. Sowohl der Ionenaustausch als auch die Phasenseparartion werden gesondert untersucht und die wesentlichen Einflussfaktoren Temperatur und Dauer der Prozesse auf die resultierende Porenstruktur und Porenweite diskutiert. Ein besonderes Augenmerk wird auf die Extraktion der phasenseparierten Schichten gelegt und der Einfluss der Extraktionsbedingungen auf die Stabilität der Materialien untersucht. Es wird eine Verfahrensprozedur entwickelt, mit der es möglich ist sowohl die Porenweite als auch die erzielbare Dicke der porösen Schicht unabhängig voneinander einzustellen. Die Untersuchung des Ionenaustauschs erfolgt gravimetrisch, mit ICP-OES und mittels REM-EDX. Die Charakterisierung der porösen Schichten erfolgt mit Stickstofftieftemperaturadsorption, Quecksilberporosimetrie, REM und Positronenlebensdauerspektroskopie. Mit den untersuchten Bedingungen können mesoporöse Schichten (10-50 nm Porenweite) mit einer ungeordneten Durchdringungsstruktur erzeugt werden, wobei die Schichtdicke im Bereich zwischen 1 und 60 mm variabel ist. In den anwendungsorientierten Untersuchungen wird das entwickelte Verfahrensprinzip auf die Herstellung von Core-Shell-Kugeln erfolgreich übertragen. Weiterhin wird gezeigt, dass durch eine pseudomorphe Transformation zu MCM-41 die Oberfläche der porösen Schicht deutlich vergrößert werden kann und auf diesem Wege geordnete Porensysteme als Schicht sowie Poren kleiner 10 nm zugänglich sind. Der Nachweis der MCM-41 Strukturen erfolgt über Röntgenkleinwinkelstreuung.

No description available.

The adsorption of cytochrome c onto mesoporous silica (MCM-41) was investigated in this study. MCM-41 was synthesized and characterized by different methods. The pore size of MCM-41 was calculated from each method and all were in agreement with each other. Result from SAXS method showed a well ordered 2D hexagonal structure of MCM- 41. To investigate the effect of pH on adsorption process, different buffers were used with various pH in the range from 3.8 to 10.7. It was observed that the maximum adsorption occurs at pH near the isoelectric point of cytochrome c. The surface charges of cytochrome c and MCM-41 play an essential role for the process of adsorption. Desorption of cytochrome c from MCM-41 was investigated as well. Pure water and buffers with pH 7.1 and 10.7 were used to study the desorption. The result shows that desorption of cytochrome c from MCM-41 takes place at a pH above its isoelectric point.

No description available.

No description available.