Indiana University-Purdue University Indianapolis (IUPUI) / Articular cartilage (AC) is a strong but flexible connective tissue that covers and protects the end of the long bones. Although cartilage has excellent friction and wear properties that allow smooth joint function during daily activities, these properties are not fully understood. Many material properties of cartilage are anisotropic and vary with anatomic location and the composition of the tissue, but whether this is also true for cartilage friction and wear has not been previously determined. Furthermore, cartilage disease and injury are major health concerns that affect millions of people, but there are few available treatments to prevent the progression of cartilage degeneration. Collagen crosslinking may be a potential treatment to reduce cartilage wear and slow or prevent the progression of cartilage disease. The objectives of this thesis were to investigate the relationships between the friction/wear characteristics of cartilage and the orientation of the preferred fiber direction, the anatomic location of the tissue, the composition of the tissue, and exogenous photochemical crosslinking. In the superficial zone, AC has preferential fiber direction which leads to anisotropic material behavior. Therefore, we hypothesized that AC will show anisotropic behavior between longitudinal and transverse direction in an accelerated, in vitro wear test on bovine cartilage in terms of friction and wear. This hypothesis was proven by the quantification of glycosaminoglycans released from the tissue during the wear test, which showed that more glycosaminoglycans were released when the wear direction was transverse to the direction of the fibers. However, the hydroxyproline released from the tissue during the wear test was not significantly different between the two directions, nor was the coefficient of friction. The material properties of AC can also vary with anatomic location, perhaps due to differences in how the tissue is loaded in vivo. We hypothesized that cartilage from a higher load bearing site will give better wear resistance than cartilage from lower load bearing regions. However, no differences in friction or wear were observed between the different anatomic locations on the bovine femoral condyles. The concentration of collagen, glycosaminoglycans, cells and water in the tissue was also quantified, but no significant differences in tissue composition were found among the locations that were tested. Although wear did not vary with anatomic location, variation in the wear measurements were relatively high. One potential source of variation is the composition of the cartilage. To determine whether cartilage composition influences friction and wear, a correlation analysis was conducted. An accelerated, in vitro wear test was conducted on cartilage from bovine femoral condyles, and the tissue adjacent to the wear test specimens was analyzed for collagen, glycosaminoglycan, cell, and water content. Because wear occurs on the cartilage surface, the superficial zone of the cartilage might play an important role in wear test. Therefore, composition of the adjacent cartilage was determined in both the superficial zone and the full thickness of the tissue. A significant negative correlation was found between wear and collagen content in the full thickness of the tissue, and between the initial coefficient of friction and the collagen content in the superficial zone. This correlation suggests that variation in the collagen content in the full thickness of the cartilage partially explains differences in amount of wear between specimens. The wear resistance of cartilage can be improved with exogenous crosslinking agents, but the use of photochemical crosslinking to improve wear resistance is not well understood. Two photochemical crosslinking protocols were analyzed to improve the wear resistance of the cartilage by using chloro-aluminum phthalocyanine tetrasulfonic acid (CASPc) and 670nm laser light. The cartilage treated with the two crosslinking protocols had lower wear than the non-treated group without changing the friction properties of the cartilage.

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One of the major classes of polymer matrix resins under consideration for structural composite applications in the infrastructure and construction industries is the dimethacrylate matrix resin. An investigation of the relationships between the chemical structures and properties of these dimethacrylate/styrene networks has been conducted. Oligomer number average molecular weights of the polyhydroxyether ranging from 700 to 1200g/mole were studied with systematically varied styrene concentrations to assess the effects of crosslink density and chemical composition on glass transition temperatures, toughness, tensile properties and matrix strain. Network densities have been estimated from measurements of the rubbery moduli at Tg + 40°C. Within this rather small range in vinyl ester molecular weight, toughness of the resultant networks improved tremendously as the vinyl ester oligomer Mn was increased from 700g/mole to 1200g/mole due to improvements in the resistance to crack propagation. As styrene concentration was increased along all series' of materials, brittleness increased even though the molecular weight between crosslinks increased. This was attributed to the inherent relative brittleness of the polystyrene chemical structure relative to the polyhydroxyether component. This may also be related to the reactivity ratios dictating styrene and vinyl ester sequence length and warrants further investigation. As expected, the volume contraction upon cure also decreased significantly as styrene was decreased, and thus residual cure stresses may be reduced in fiber-reinforced composites. Vickers microhardness values decreased for each of the series when molecular weight increased and styrene content decreased. Two different cure procedures were compared to assess the effects of conversion on the physical and mechanical properties. All mechanical properties investigated (i.e. fracture toughness, tensile strength, and microhardness) were dependent on the cure procedure. Materials cured at 140°C were harder, more brittle, had lower elongations and higher rubbery moduli than those cured at 25°C followed by a 93°C postcure. A maximum in the degree of conversion occurred with increasing polymerization temperature and can be explained by the competition between the chemical reaction and molecular mobility. The overall shrinkage per moles of vinyl groups converted was the same when the materials were cured at 25°C or 140°C. However, in the room temperature cured samples, there was essentially no further densification of the network during postcure, regardless of the postcure temperature. A mono-methacrylate analogue of the dimethacrylate terminated poly(hydroxyether) oligomer was synthesized and copolymerized with styrene to study the effects of chain transfer during elevated temperature reactions. / Ph. D.

DNA crosslinking agents exhibit a variety of DNA lesions, such as monoadducts, DNA-DNA interstrand or intrastrand crosslinks or DNA-protein crosslinks. Agents that produce interstrand crosslinks (ICLs) exist naturally and are widely used in chemotherapy. Therefore, it is important to understand how the lesions induced by these agents are repaired. In bacteria, the repair is mainly dependent on nucleotide excision repair (NER) together with homologous recombination (HR) or translesion synthesis (TLS). In human cells, it is not clear how these lesions are repaired, and it is believed to be a more complicated process in which NER does not play as important a role as in prokaryotes. Here, we investigated the repair mechanisms mainly after treatment with psoralen but also with acetaldehyde, cisplatin and mitomycin C in some studies. As expected from studies on plasmids and in bacteria, we used new techniques to confirm that various ICL-inducing agents block replication fork elongation in mammalian cells. We also found that the replication fork was unable to bypass these lesions. We confirmed that ERCC1/XPF and the HR proteins BRCA2 and XRCC2/3 are vital for protection against ICL treatments. These proteins were also found to be equally important for the repair of monoadducts. To better understand ICL repair in mammalian cells, we developed a method to study the induction and unhooking of ICL in human fibroblasts. We found that ICLs were repaired and that 50% of the induced ICLs were unhooked within 3 hours following exposure. Additionally, we determined that XPA, but not XPE, is involved in ICL unhooking, although not affecting lethality. A step in ICL repair is the formation of double-strand breaks (DSBs), and we identified a replication-dependent formation of DSBs following ICL treatment. Furthermore, ERCC1/XPF was not necessary for DSB formation. The repair of these DSBs was performed by HR and involved ERCC1/XPF. Additionally, we were able to quantify the ICL unhooking in human fibroblasts and found that they can unhook ~2500 ICL/h. We also determined that a dose of approximately 400 ICL/cell is lethal to 50% of the cells, indicating that ICL unhooking is not the most critical step during the repair process. / DNA-skadande ämnen är vanligt i cancerbehandling, då snabbt växande celler, såsom cancerceller är betydligt känsligare än normala celler för DNA skador. En grupp av ämnen som vanligen används i cancerbehandling är korsbindare av DNA. Dessa ämnen kommer reagera två gånger med DNA och skapa två bindningar mitt emot varandra. DNA strängen, som består av två delar, måste kunna separeras och kopieras (replikation) på ett tillförlitligt sätt för att cellerna ska kunna dela sig och bli flera. DNA strängen måste också kunna dela sig och bli avläst rätt för att nya proteiner ska kunna bildas (transkription). När korsbindarna har bundit till DNA strängarna, hindrar detta deras separation och därigenom förhindras även avläsningen och kopieringen.  För att göra undersökningarna av DNA korsbindande ämnen ännu lite svårare, så ger korsbindare flera olika typer av skador. Dels kan det bli flera olika typer av korsbindningar, både mellan två DNA-strängar (ICL) vilket är den farligaste och mest svårreparerade typen, men det kan också ske inom samma DNA-sträng (intrastrand crosslink) eller mellan en DNA-sträng och ett protein (DNA-protein crosslink). Korsbindare kan även bilda enbindningsskador (monoaddukt), vilket innebär den bara binder en gång till DNA. För att cellen ska kunna överleva, så måste den reparera skadorna och ta bort korsbindningen eller monoaddukten. Hur detta sker i människor är inte helt klarlagt men det verkar som det sker i flera steg. Till att börja med klipps DNA sönder i ena strängen på båda sidorna om korsbindningen, detta gör att den kvarvarande delen av korsbindningen kan böjas bort. Därefter kommer cellen att skapa nytt DNA för att fylla mellanrummet som bildats. Cellen använder sig av den andra DNA strängen som mall för att sätta in rätt DNA baser, men i fallet med korsbindande ämnen så är även den strängen skadad och därför finns det en stor risk för att fel DNA baser sätts in och då uppstår mutationer. Nästa steg är att klippa den kvarvarande delen av korsbindningen, även denna gång skapas ett mellanrum som måste fyllas med nya baser. Den första artikeln i avhandlingen handlar om att försöka reda ut om det är ICLen eller monoaddukten som är orsak till olika effekter som påträffas efter behandling med korsbindande ämnen. Det vi fann var att även om det bara var från ICLs som vi kunde mäta en effekt på replikationen, så fick vi nästan lika stark effekt från monoaddukterna, som från ICL, för en av de vanligast använda markörerna (kännetecknen) för båda DNA strängarna var brutna på samma ställe (dubbelstränsbrott). Detta berodde dock inte på att även monoaddukterna skapade dubbelsträngsbrott, utan på att markören vi använde var ospecifik. Vi fann även att även om ICLs har mycket större effekt än monoaddukten på cellens överlevnad m.m., så kan man inte bortse ifrån effekten av monoaddukten och att den troligen har en betydande roll för de korsbindande ämnen som endast ger en liten del ICLs. I artikel två har vi utvecklat en ny metod, som gör det möjligt att mäta hur många ICLs som bildas vid en viss dos av de korsbindande ämnen vi undersöker. Vi kan även mäta hur fort ICLerna kan repareras i mänskliga celler med hjälp av metoden. Tack vare en kombination av våra mätningar och med hjälp av datorsimuleringar, kunde vi räkna ut hur många ICLs som bildades per dos för tre vanliga korsbindare. Vi kunde även visa att 50 % av ICLen har påbörjat reparationen och kommit så långt att de var bortklippta från ena stängen inom 3 timmar efter behandlingen. I artikel tre undersöker vi vilka proteiner som är inblandade i den tidiga delen av ICL reparationen, alltså fram till och med att celler klipper ut korsbindningen på båda sidorna om skadan i ena strängen. Här visar vi att celler som är defekta i reparationsprotein kallat XPA, har en betydligt långsammare borttagning av ICLer än vad båda normala celler och celler defekta i reparationsprotein XPE har. Vi visar även att detta inte påverkar cellens replikationshastighet, eller har någon effekt på cellens överlevnad. Den fjärde artikeln handlar om acetaldehyd, som bildas när alkohol förbränns i kroppen. Acetaldehyd har föreslagits bilda ICL och därför undersökte vi vilka effekter den har på cellerna. Vi visar i den här artikeln att det krävs nysyntes av DNA för att acetaldehyd ska leda till dubbelsträngsbrott. Celler kan reparera dessa dubbelsträngsbrott med hjälp av reparationssystem, som kallas homolog rekombination, men att reparationen ibland blir felaktig. I den femte och sista artikeln i avhandligen undersöker vi ett av de vanligast föreslagna proteinen för att sköta klippningen av DNA (ERCC1/XPF) och hur den är inblandad i reparationen av korsbindningar. Vi kan här visa att även det krosbindande ämnet mitomycin C bromsar replikationshastigheter och att ERCC1/XPF är nödvändigt för att kunna fullfölja homolog rekombination av ICLs. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Submitted. Paper 2: Manuscript. Paper 3: Manuscript. Paper 4: Submitted.</p>

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Budding yeast Slx4 binds to the structure-specific DNA repair nucleases Slx1 and Rad1XPF-Rad10ERCC1, and it was reported that Slx4 is essential for DNA flap cleavage by Rad1XPF-Rad10ERCC1 during certain types of DNA repair in yeast. At the outset of this thesis, bioinformatic analyses identified the uncharacterised protein BTBD12 in higher eukaryotes as a putative orthologue of yeast Slx4. In the first results chapter of this thesis, I describe the identification of BTBD12-interacting proteins, including XPF-ERCC1 and SLX1. These findings led me to refer to BTBD12 as human SLX4. I found that SLX4 binds to another structure-specific nuclease MUS81-EME1, and other proteins involved in telomere maintenance and cell cycle progression. The remainder of this chapter describes detailed biochemical analysis of the nuclease activities associated with the SLX4 complex isolated from human cells. Work from this lab and others revealed that depletion of SLX4 from human cells using siRNAs causes defects in the repair of DNA interstrand crosslinks (ICLs). Inherited mutations in humans that reduce the efficiency of ICL repair cause Fanconi anaemia (FA). The cellular sensitivity of SLX4 depleted cells to ICLs prompted me to investigate SLX4 as a candidate FA gene. Dr. Johan de Winter (VU University Medical Center, Amsterdam) and Dr. Detlev Schindler (University of Wurzburg) had identified several patients with unclassified FA that was not caused by mutations in the FA genes known at the time. In the second results I describe characterisation of SLX4, and the SLX4 holo-complex, in cells from some of these FA patients who had bi-allelic SLX4 mutations. In three of the patients SLX4 was expressed at normal levels but was missing part of the first, and all of the second, UBZ-type putative ubiquitin-binding domain. This prompted me to investigate the function of the SLX4 UBZ domains. I found that the first, but not the second, UBZ domain of SLX4 binds to ubiquitin in vitro and targets SLX4 to sites of DNA damage in vivo. Furthermore, the first but not the second SLX4 UBZ domain appears to be required for ICL repair, demonstrating the important of correctly localising SLX4 for DNA repair. In the final chapter, I present preliminary data which suggests that SLX4 is regulated in an unusual manner in during S-phase of the cell cycle, and that SLX4 interacts with the PLK1 kinase in a phosphorylation-dependent manner.

Kazunori Yoshikiyo, Katja Kratz, Kouji Hirota, Kana Nishihara, Minoru Takata, Hitoshi Kurumizaka, Satoshi Horimoto, Shunichi Takeda, and Josef Jiricny "KIAA1018/FAN1 nuclease protects cells against genomic instability induced by interstrand cross-linking agents" PNAS 2010 107 (50) 21553-21557; published ahead of print November 29, 2010, doi:10.1073/pnas.1011081107 / 京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12772号 / 論医博第2063号 / 新制||医||1000(附属図書館) / 30755 / (主査)教授 小松 賢志, 教授 小川 誠司, 教授 松本 智裕 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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