Rost- und säurebeständige Stähle, wie z.B. der Austenit 1.4404, zeichnen sich durch eine hervorragende Korrosionsbeständigkeit aus. Der Einsatz dieser Stähle war aber aufgrund ihrer schlechten Verschleißeigenschaften bisher eher eingeschränkt. Um diesen Anforderungen gerecht zu werden, ist es notwendig, eine Oberflächenmodifizierung vorzunehmen. Dies kann besonders mit Nitrier- und Carburierverfahren bzw. einer Kombination aus beiden erreicht werden, wobei das im Mischkristall gelöste Chrom nicht zur Ausscheidung als Chromnitrid bzw. Chromcarbid gebracht werden darf. Dazu wurde bereits Mitte der 80-er Jahre das Verfahren der so genannten Tieftemperaturnitrierung entwickelt. Mit Hilfe dieses Verfahrens ist es möglich, auf rost- und säurebeständigen Stählen eine Schicht mit hohem Verschleißwiderstand zu erzeugen, ohne dass es zum Verlust der Korrosionsbeständigkeit kommt. Die werkstoffwissenschaftliche Forschung hat mit dieser überwiegend technologischen Entwicklung nicht Schritt gehalten. Verallgemeinerungsfähige Aussagen über das Korrosionsverhalten dieser verschleißbeständigen Randschichten in Abhängigkeit von ihrer Zusammensetzung und Struktur sind gegenwärtig noch nicht möglich. Die Zielstellung der im Rahmen dieser Arbeit durchzuführenden Untersuchungen besteht deshalb in der Ermittlung vertiefter Kenntnisse über den Zusammenhang zwischen den Behandlungsbedingungen, der Struktur und der chemischen Zusammensetzung der dabei entstehenden Randschichten und ihrem Korrosionsverhalten.

The saturation of the near surface layers of metals with different elements is a powerful tool to change their surface properties. In this work, structure and transport changes induced by incorporation of large amounts of nitrogen at moderate temperatures (∼370−430°C) in austenitic stainless steel are investigated. The structural study of the plasma nitrided ASS has been carried out using a combination of global (X-ray diffraction, nuclear reaction analysis) and local probe techniques (Mossbauer, X-ray absorption near edge structure, extended X-ray absorption fine structure spectroscopies). It reveals that nitriding at moderate temperatures (∼400°C) results in the nitrided layer with Fe, Cr and Ni being in different local chemical environments: Cr in the CrN-like state, Fe in the Fe4N-like state, Ni in the metallic state. The results demonstrate that the incorporation of interstitial nitrogen destabilizes homogeneous distribution of the ASS constituents, which leads to the segregation of the elements into small zones rich in Cr and Ni and provide strong evidence that the decomposition is of a spinodal nature. These experimental findings contradict the widely accepted view that the phase formed during nitriding at moderate temperatures is a homogeneous supersaturated nitrogen solid solution. The nitrogen atomic transport study has been carried out by using ion beam nitriding of single-crystalline stainless steel, and the issues of the influence of the crystalline orientation, nitriding temperature, ion flux and ion energy are addressed. The diffusion coefficients have been extracted from the fitting of the nitrogen depth profiles by using the trapping-detrapping model. It is shown that the crystalline orientation plays a significant role in nitrogen diffusion: the penetration depth is largest, intermediate and lowest for the (001), (110) and (111) orientation, respectively. The pre-exponential factor D0 varies by two orders of magnitude depending on the orientation, while the activation energy E is similar (∼1.1 eV) for the (111) and (110) orientations and higher for the (001) orientation (∼1.4 eV). It is found that the nitrogen ion energy and the flux have the effect on the nitrogen transport in the bulk with higher energies (or fluxes) showing larger diffusion coefficients. The ion energy effect is more pronounced for the (001) than for the (111) orientation, while the flux effect is similar in both orientations. In addition, the diffusivity during post-nitriding thermal annealing without ion irradiation is found to be independent of the crystalline orientation. The observed radiation enhanced diffusion and anisotropy are discussed on the basis of nitrogen incorporation induced changes in the matrix structure (ASS decomposition and formation heterogeneous structure), ion bombardment induced effects (defects, localized vibrations) and correlated diffusion.

Rost- und säurebeständige Stähle, wie z.B. der Austenit 1.4404, zeichnen sich durch eine hervorragende Korrosionsbeständigkeit aus. Der Einsatz dieser Stähle war aber aufgrund ihrer schlechten Verschleißeigenschaften bisher eher eingeschränkt. Um diesen Anforderungen gerecht zu werden, ist es notwendig, eine Oberflächenmodifizierung vorzunehmen. Dies kann besonders mit Nitrier- und Carburierverfahren bzw. einer Kombination aus beiden erreicht werden, wobei das im Mischkristall gelöste Chrom nicht zur Ausscheidung als Chromnitrid bzw. Chromcarbid gebracht werden darf. Dazu wurde bereits Mitte der 80-er Jahre das Verfahren der so genannten Tieftemperaturnitrierung entwickelt. Mit Hilfe dieses Verfahrens ist es möglich, auf rost- und säurebeständigen Stählen eine Schicht mit hohem Verschleißwiderstand zu erzeugen, ohne dass es zum Verlust der Korrosionsbeständigkeit kommt. Die werkstoffwissenschaftliche Forschung hat mit dieser überwiegend technologischen Entwicklung nicht Schritt gehalten. Verallgemeinerungsfähige Aussagen über das Korrosionsverhalten dieser verschleißbeständigen Randschichten in Abhängigkeit von ihrer Zusammensetzung und Struktur sind gegenwärtig noch nicht möglich. Die Zielstellung der im Rahmen dieser Arbeit durchzuführenden Untersuchungen besteht deshalb in der Ermittlung vertiefter Kenntnisse über den Zusammenhang zwischen den Behandlungsbedingungen, der Struktur und der chemischen Zusammensetzung der dabei entstehenden Randschichten und ihrem Korrosionsverhalten.

The essential cell cycle kinase Dbf4/Cdc7 (DDK) triggers DNA replication through phosphorylation of the Mcm2-7 helicase at replication origins. Prior work has implicated various Mcm2-7 subunits as targets of DDK, however it is not well understood which specific subunits mediate the docking of the DDK complex. Through yeast two-hybrid and co-immunoprecipitation analyses, we found that Dbf4 and Cdc7 interact with distinct subunits of the Mcm2-7 helicase complex. Dbf4 showed the strongest interaction with Mcm2 while Cdc7 associated with Mcm4 and Mcm5. Dissection of the N-terminal region of Mcm2 revealed two regions that mediate the interaction with Dbf4, whereas in Mcm4, a region near the N-terminus has been previously identified by another group as the DDK docking domain. Mutant forms of Mcm2 (Mcm2ΔDDD) or Mcm4 (Mcm4ΔDDD) lacking the DDK docking domain were expressed in cells and resulted in modest growth and replication defects. Combining the two mutations resulted in synthetic lethality, suggesting a redundant mechanism of Mcm2 and Mcm4 in targeting the DDK complex to Mcm rings. Furthermore, growth inhibition could be induced in a Mcm4ΔDDD background by overexpressing Mcm2 to titrate Dbf4 from Mcm rings. These growth defects were exacerbated in the presence of genotoxic agents such as hydroxyurea and methyl methanesulfonate, suggesting that DDK-Mcm interactions may play a role in stabilizing replication forks under S-phase checkpoint conditions. Regions of Cdc7 were examined for their interaction with Mcm4 and Dbf4. Results have shown that the N-terminal amino acid region 55-124 and the C-terminal region 453-507 of Cdc7 are likely target regions for Dbf4-binding. Several conserved residues were identified within the N-terminal 55-124 Cdc7 region that interface with conserved residues within motif-C of Dbf4. Conserved residues were identified within the DDD domain of Mcm2 and mutating these residues resulted in a decreased interaction with Dbf4. Lastly, bioinformatics analysis has revealed potential conserved residues within the Mcm4DDD region, which may play a role in binding to Cdc7. This research is significant because these factors, which are conserved in all eukaryotes studied to date, should give further insight as to how DNA replication is triggered and how it is affected when cells are exposed to DNA damaging or replication compromising agents. This research also has implications in cancer genetics, as prior studies have shown elevated DDK and Mcm protein levels in tumour cell lines and melanomas, with Cdc7 showing great promise as a cancer therapeutic target. Such knowledge will further enhance our understanding of the DNA replication process and the roles of cell cycle proteins involved, under both normal and checkpoint conditions.

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 83-112).

Cellular checkpoints are typically considered to both facilitate the ordered execution of the cell cycle and to act as a barrier to oncogene driven cell cycles and the transmission of unresolved genetic lesions from one phase to the next. Furthermore, these mechanisms are also believed to underpin the responses of cells, both in normal and cancerous tissues, to those therapies that either directly or indirectly generate DNA damage. In recent studies however, it has become clear these checkpoints permit the passage of significant genomic aberrations into subsequent cell cycle phases and even descendant cells, and that heterogeneous responses are apparent amongst genetically identical cells. The consequences of this checkpoint ‘negligence’ remain relatively uncharacterised despite the importance of checkpoints in current models for how genomic instability is avoided in the face of ubiquitous DNA damage. Unresolved DNA damage is presumably inherited by subsequent cell cycle phases and descendant cells yet characterisation of the consequences of this has been relatively limited to date. I therefore utilised microscopy-based lineage tracing of cells expressing genetically encoded fluorescent sensors, particularly the Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) probes (Sakaue-Sawano et al., 2008), with semi-automated image analysis to characterise the response of single cells and their descendants to DNA lesions across multiple cell cycle generations. This approach, complemented by generational tracing by flow cytometry, permitted me to characterise the timing of cell fate determination in treated and descendant cells, the non-genetic heterogeneity in checkpoint responses and overall lineage behaviour, correlations between cells (similarly to Sandler et al., 2015) and cell cycle timing dependencies in the response to DNA damaging agents. With these single cell analytical approaches I show that the consequences of DNA damage on descendant cell fate is dramatic, suggesting checkpoint mechanisms may have consequences and even cooperate across phases and generations. U2OS cell lineages traced for three generations following the induction of DNA damage in the form of strand breaks showed greatly induced cell death in the daughters and granddaughters of DNA damaged cells, termed delayed death. Furthermore, lineage behaviour was characterised as highly heterogeneous in when and whether cell death occurred. Complementary flow cytometric approaches validated the findings in U2OS cells and suggested HeLa cells may show similar behaviour. These findings indicate that checkpoint models need to incorporate multigenerational behaviour in order to better describe the response of cells to DNA damage. Understanding the processes governing cell fate determination in descendant cells will impact upon our understanding of the development of genomic instability during carcinogenesis and how DNA-damaging chemotherapeutics drive cells to ‘quit’ the cell cycle.

The cell division cycles of differentiating cells are coordinated so as to generate sufficient numbers of mature cells. The cell cycle may also regulate the process of differentiation, in ways that are not well understood. We previously discovered that during erythropoiesis, the cell cycle is synchronized with a specific developmental switch, where erythroid progenitors known as colony-forming-unit-erythroid (CFU-e) transition from a self-renewal state to a state of erythroid terminal differentiation (ETD). This switch takes place during a single cell cycle S phase and is dependent on S-phase progression. My work shows that this S phase is unusual, in that it is shorter than S phase in preceding cycles, as a result of a global increase in replication fork speed. I found that the CDK inhibitor, p57KIP2, negatively regulates replication fork speed in self-renewing CFU-e, and its down-regulation at the switch to ETD results in S-phase shortening. p57KIP2-mediated inhibition of CDK2 is essential for CFU-e self-renewal. It exerts this effect by reducing replication stress and also reducing the probability of transition from CFU-e to ETD, promoting CFU-e self-renewal instead. CDK2 inhibiting drugs that mimic the action of p57KIP2 stimulate erythropoiesis both in vitro and in vivo, through expansion of the CFU-e pool. In addition to p57KIP2, E2f4 also regulates S-phase shortening and the efficiency of the CFU-e to ETD transition. Overall, my work shows that S-phase speed regulates a key erythroid cell fate decision, and suggests a possible translational application of CDK2 inhibiting drugs in the stimulation of erythropoiesis.

Integrity of the human genome is frequently threatened by endogenous and exogenous DNA damaging reagents that may lead to genome instability and cancer. Cells have evolved multiple mechanisms to repair DNA damage or to eliminate the damaged cells beyond repair and to prevent diverse diseases. Among these are ataxia telangiectasia and Rad3-related (ATR)-mediated DNA damage checkpoint and nucleotide excision repair (NER) that are the major pathways by which cells handle ultraviolet C (UV-C)- or other exogenous genotoxin-induced bulky DNA damage. However, it is unclear how these 2 pathways may be coordinated. In this study we show that ATR physically interacts with NER factor xeroderma pigmentosum group A (XPA) where an ATR phosphorylation site on serine 196 is located. Phosphorylation of XPA on serine 196 is required for repair of UV-induced DNA damage. In addition, a K188A point mutation of XPA that disrupts the ATR-XPA interaction inhibits the UV-induced XPA phosphorylation and DNA repair. Moreover, we show that depletion of p53, a downstream checkpoint of ATR, and inhibition of p53 transcriptional activities reduced the UV-induced XPA import. Furthermore, we found that the ATR-directed XPA nuclear import happens primarily in the S phase of the cell cycle. In effort to determine the mechanism involved in the XPA nuclear import, we found that, in addition to the nuclear localization signal (NLS) of XPA, importin-α4 is required for the UV-induced XPA nuclear import in an ATR-dependent manner. These data suggest that NER could be regulated by the ATR-dependent checkpoint via modulation of XPA phosphorylation and nuclear import. In a separate study we show that, upon UV damage, cytoplasmic ATR translocates to mitochondria, blocks the recruitment of proapoptotic Bcl-2–associated X (Bax) protein to mitochondria and prevents the loss of mitochondrial membrane potential (ΔΨ) and apoptosis. Bax-depletion reduces the effect of ATR on ΔΨ. Remarkably, the cytoplasmic ATR exhibits no checkpoint kinase activity, a hallmark function of nuclear ATR. Silencing of ATR’s kinase activity failed to affect Bax relocalization to mitochondria. These results reveal a novel checkpoint-independent antiapoptotic function of ATR at mitochondria in the cellular response to DNA damage.

No description available.

Thermochemische Randschichthärteverfahren ermöglichen eine ausscheidungsfreie Einlagerung von Kohlenstoff bzw. Stickstoff innerhalb des austenitischen Mischkristalls. Im Zusammenhang mit einer Randschichtbehandlung thermisch gespritzter Schichtsysteme stellen die charakteristischen Strukturmerkmale eine bislang weitgehend unerforschte Einflussgröße für die Beurteilung von Diffusionsprozessen dar. Bei der Verarbeitung von randschichtgehärtetem Pulver durch Verfahren des thermischen Spritzens ist die Phasenstabilität des Spritzzusatzwerkstoffs von übergeordneter Bedeutung. Die beiden Möglichkeiten einer Verfahrenskombination werden für hochgeschwindigkeitsflamm- und atmosphärisch plasmagespritzte Schichtsysteme des Werkstoffs EN 1.4404 durch systematische Prozess- und Parametervariation eingehend betrachtet. Für die einzelnen Schichtsysteme werden Einflussfaktoren struktur- und prozessspezifisch sowie in Abhängigkeit vom Anreicherungsmedium erfasst und im Kontext der Massivwerkstoffreferenz eingeordnet. Die daraus abgeleiteten allgemeingültigen Aussagen zu verfahrenstechnischen Wechselwirkungseffekten ermöglichen eine anwendungsorientierte Verfahrensauswahl bzw. Entwicklungsstrategie. / Thermochemical surface hardening enables a precipitation-free solvation of carbon or nitrogen on interstices of the austenitic crystal lattice. However, the interplay of the diffusion mechanisms with the structural properties of thermal spray coatings has not yet been understood. Thermal spraying of surface-hardened powders is a further opportunity, where the phase stability of the feedstock material is of crucial importance. A process and parameter study is conducted on high velocity oxy-fuel and atmospheric plasma spraying of AISI 316L considering both basic concepts. Structural and process-specific influence factors are examined for the different coating systems in comparison to the bulk material reference. Correlation effects are determined allowing for an application-oriented process selection or development strategy.