Magister Scientiae - MSc / The aim of this study is to investigate the repair bond strength of composite resin following micromechanical and chemical means of retention in improving the repair of composite resin specimens.

This research explores the application of expert systems to the maintenance of boilers, are as follows: and its main objectives [1] To explore the application of expert systems in the maintenance of boilers. [2] To simplify the task of boiler maintenance with the help of expert systems. [3] To improve the skills of boiler maintenance workers through the knowledge 'captured ' or 'preserved' by expert systems. [4] To minimise errors which may cost time, money, effort and possible risk of injury to boiler maintenance personnel. [5] To develop a prototype expert system which can advise and assist boiler maintenance personnel. [6) To contribute to research in the application of expert systems in the field of engineering.

The mainstay of treatment for injuries to the anterior cruciate ligament (ACL) is with a tendon graft harvested from elsewhere in the knee. This procedure, known as ACL reconstruction (ACLR), has excellent reported outcomes in terms of restoring the gross stability of the knee. However there are reported rates of graft failure in the pediatric population.1 ACL reconstruction also does not provide full protection from developing premature osteoarthritis, which is often seen in patients after ACL injury. A new method must be found in order to find a solution to these adverse outcomes from the current standard treatment of ACL injuries. The Bridge-enhanced anterior cruciate ligament repair (BEAR) technique is a new innovation on primary repair of a torn ACL. It avoids the requirement for autograft harvesting and use of an allograft by combining a primary suture repair with an extraceullular matrix scaffold (the BEAR scaffold). This scaffold is placed between the two torn ends of the ACL to help facilitate natural healing of the ligament in order to restore the intrinsic functions of the ACL to protect the knee from trauma and instability. This ability to repair the native ACL maintains the proper anatomy of its insertion points, avoids donor site morbidity, and helps to prevent early osteoarthritis by maintaining innate proprioceptive functions of the ACL. Our study will prove the BRIDGE repair to be a promising step in the direction of preserving the native ACL.

Indiana University-Purdue University Indianapolis (IUPUI) / Metnase is a novel bifunctional protein that contains a SET domain and a transposase domain. Metnase contains sequence-specific DNA binding activity and sequence non-specific DNA cleavage activity, as well as enhances genomic integration of exogenous DNA. Although Metnase can bind specifically to DNA sequences containing a core Terminal Inverted Repeat sequence, this does not explain how the protein could function at sites of DNA damage. Through immunoprecipitation and gel shift assays, I have identified the Pso4 protein as a binding partner of Metnase both in vitro and in vivo. Pso4 is essential for cell survival in yeast, and cells containing a mutation in Pso4 show increased sensitivity to DNA cross-linking agents. In addition, the protein has sequence-independent DNA binding activity, favoring double-stranded DNA over single-stranded DNA. I demonstrated that the two proteins form a 1:1 stochiometric complex, and once formed, Metnase can localize to DNA damage foci as shown by knockdown of Pso4 protein using in vivo immunofluorescence. In conclusion, this shows that Metnase plays an indispensable role in DNA end joining, possibly through its cleavage activity and association with DNA Ligase IV.

Indiana University-Purdue University Indianapolis (IUPUI) / Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa. Toxoplasma infection is a serious threat to immunocompromised individuals such as AIDS patients and organ transplant recipients. Side effects associated with current drug treatment calls for identification of new drug targets. DNA repair is essential for cell viability and proliferation. In addition to reactive oxygen species produced as a byproduct of their own metabolism, intracellular parasites also have to manage oxidative stress generated as a defense mechanism by the host immune response. Most of the oxidative DNA damage is repaired through the base excision repair (BER) pathway, of which, the apurinic /apyrimidinic (AP) endonucleases are the rate limiting enzymes. Toxoplasma possesses two different AP endonucleases. The first, TgAPE, is a magnesium-dependent homologue of the human APE1 (hAPE1), but considerably divergent from hAPE1. The second, TgAPN, is a magnesium-independent homologue of yeast (Saccharomyces cerevisiae) APN1 and is not present in mammals. We have expressed and purified recombinant versions of TgAPE and TgAPN in E. coli and shown AP endonuclease activity. Our data shows that TgAPN is the more abundant AP endonuclease and confers protection against a DNA damaging agent when over-expressed in Toxoplasma tachyzoites. We also generated TgAPN knockdown Toxoplasma tachyzoites to establish that TgAPN is important for parasite protection against DNA damage. We have also identified pharmacological inhibitors of TgAPN in a high-throughput screen. The lead compound inhibits Toxoplasma replication at concentrations that do not have overt toxicity to the host cells. The importance of TgAPN in parasite physiology and the fact that humans lack APN1 makes TgAPN a promising candidate for drug development to treat toxoplasmosis.

No description available.

We have used a non-replicating recombinant adenovirus, AdCA17𝘭𝘢𝘤𝘡, which expresses the β-galactosidase (β-gal) reporter gene under the control of the human cytomegalovirus immediate early (HCMV-IE) promoter, to examine host cell reactivation (HCR) of a UVC-damaged reporter gene in human fibroblasts. Since β-gal expression in mammalian cells is expected to occur following transcription of a lesion-free 𝘭𝘢𝘤𝘡 reporter gene, the removal of UV-induced DNA lesions and subsequent β-gal expression is a measure of the DNA repair capacity of the infected cell. In the present work, we have examined HCR and UV-enhanced HCR of the UV-damaged reporter construct, AdCA17𝘭𝘢𝘤𝘡, in normal and nucleotide excision repair (NER) deficient fibroblasts. Xeroderma pigmentosum (XP) group C and E fibroblasts are deficient in the global genome pathway (GGR) of NER, while Cockayne syndrome (CS) fibroblasts are deficient in the transcription-coupled repair pathway (TCR) of NER and XP group A fibroblasts are deficient in both TCR and GGR. HCR of the UV-damaged reporter gene activity was significantly reduced in XP-A, XP-C, XP-E and CS-B cell strains relative to that in normal human fibroblasts, indicating that both TCR and GGR contribute to expression of the UV-damaged reporter gene in human cells. Pre-treatment of human fibroblasts with UVC (12 J/m²) immediately prior to infection results in enhanced HCR of the UV-damaged reporter gene. UV-enhanced HCR of the UV-damaged reporter gene was detected in normal fibroblasts at 12 hr post-infection and in CS-B, XP-C and XP-E fibroblasts, but not XP-A fibroblasts at 24 and 40 hr post-infection. These results indicate that both TCR and GGR are UV-inducible in human cells. We have examined the colony survival of various human cells following exposure to UVC and UVA radiation. HaCAT, an immortalized human keratinocyte cell line, demonstrated increased resistance to UVC exposme compared to glioma (U373, T98G) and colon carcinoma (HT29) cell lines, while exhibiting increased sensitivity to UVA radiation and H₂O₂ treatment. This increased sensitivity towards both UVA and H₂O₂ treatment suggests that HaCAT cells have a reduced capacity to repair oxidative DNA damage. In addition, we show that p53-null keratinocytes (HPV-G) have reduced clonogenic survival compared to p53-mutant keratinocytes (HaCAT) following H₂O₂ treatment, consistent with an involvement of p53 in the survival of keratinocytes following H₂O₂ treatment. We also examined whether the interaction of H₂O₂ with growth media resulted in chemical by-products that were toxic to cells. Even though the H₂O₂ levels in the media were reduced following 24 hr incubation, similar clonogenic survival curves of HaCAT cells were observed following treatment of cells with immediately-prepared or 24hr-incubated H₂O₂-containing media solutions. Thus, extended incubation (24 hr) of H₂O₂ with media did not alter its cytotoxicity towards HaCAT cells, indicating that no detectable levels of toxic chemical by-products were produced. Ultraviolet irradiation of cells can have both cytotoxic and/or mutagenic consequences, resulting in increased levels of cell death or the induction of a state of genomic instability that persists for several cell generations after irradiation. However, the extent of the effects induced in non-irradiated cells by UV -irradiated cells has not been fully investigated. Using the medium transfer technique, we have tried to address whether UV irradiation of cells can induce biological effects in non-irradiated cells. Medium obtained from UVA, but not UVC, irradiation of various human cell lines in phosphate-buffered saline (PBS) solution was capable of reducing the relative clonogenic survival and colony size of non-irradiated HaCAT cells. In the absence of cells, UVA-irradiation of PBS reduced the clonogenic survival, but not the colony size, of various non-irradiated human cells. These results indicate a cytotoxicity of UVA-irradiated PBS towards non-irradiated cells. Hydrogen peroxide, a reactive oxygen species (ROS) generated following UV A irradiation, was measured following UV A treatment (or mock-treatment) of PBS (in the presence or absence of cells) either immediately or 24 hr post-irradiation. Hydrogen peroxide levels increased immediately following UV A irradiation, suggesting that it may contribute to the reduced survival of non-irradiated human cells. However, human glioma T98G and U373 cells produced elevated H₂O₂ levels in mock-irradiated conditions at 24 hr post-incubation, while demonstrating different sensitivities towards treatment with medium containing UVA-irradiated PBS. These results suggest that UVA-induced H₂O₂ is not responsible for the reduction in clonogenic survival of non-irradiated human cells. We also examined colony number and size at different times following the addition of media containing UVA-irradiated PBS and media from UVA-irradiated cell cultures. Increasing the time before scoring for colonies resulted in an increased clonogenic survival and a decrease in relative colony size for HaCAT cells. These preliminary results suggest that the relative survival and relative colony size of non-irradiated cells are inter-related, and indicate that treating non-irradiated HaCAT cells with medium from UVA-irradiated cells or medium containing UVA-irradiated PBS reduced the growth rate of HaCAT colonies. Therefore, in order to properly evaluate clonogenic survival, a time-dependent examination of relative survival and relative colony size should be conducted in order to address whether the treatment results in an inhibition of cell growth and/or true cell killing. / Thesis / Master of Science (MS)

The stability of DNA is a critical factor for several diseases, the most prevalent of which is cancer. Several neurodegenerative and accelerated aging diseases are also characterized by genomic instability. The number and complexity of DNA repair pathways that human cells possess underscores the importance of genomic stability. These pathways ensure that damaged DNA is repaired and that a cells complement of DNA remains stable upon cell division. How one particular type of DNA alteration, a DNA loop, is processed in human cells was the focus of this study. We have employed an in vitro system to study defined DNA loop substrates by human nuclear extracts. The influence of either a 5 or 3 nick, the range of loop sizes processed, and the role of DNA mismatch repair, DNA nucleotide excision repair, and the Werner Syndrome helicase proteins were variables tested. The results indicate tha t DNA loops containing between 5 to 12 nucleotides are processed in a strand - specific manner when either a 5 or 3 nick is present , with repair being targeted solely to the nicked strand . This repair occurs by both mismatch repair dependent and independent pathways. The processing of DNA loops containing 30 nucleotides in length is directed either by a 5 nick, or by the loop itself, but not by a 3 nick. The nick independent pathway results solely in loop removal. The large loop pathway is independent of mismatch repair, nucleotide excision repair, and the WRN helicase/exonuclease protein. Both of the 5 nick directed pathways occur by excision that initiates at the pre- existing nick and proceeds towards the loop along the shortest path between the nick and loop. DNA resynthesis occurs using either DNA polymerase , , or and also initiates at the pre-existing 5 nick. The 3 nick directed intermediate loop repair pathway proceeds in a similar fashion, likely after a nick is made 5 to the loop region on the strand that contained the pre-existing nick. DNA synthesis inhibition has only a minor affect on the nick independent loop removal pathway as only a short tract of DNA surrounding the loop site is processed. In total, the results point to at least 3 novel pathways that process DNA loops that likely contribute to total genomic stability.

Thesis. 1977. M.Arch.--Massachusetts Institute of Technology. Dept. of Architecture. / MICROFICHE COPY AVAILABLE IN ARCHIVES AND ROTCH. / Bibliography : leaf 58. / by Marcia K. Hnatowich. / M.Arch.

Abstract Given that most utilities are located beneath public right of ways, it is difficult to perform repairs to the utility without significantly disturbing the existing roadway. Currently there are several standard orders of procedure that deal with small-scale repairs on asphaltic surfaces. This study investigates the use of Grade D Aggregate as a backfill during a utility repair versus the condition of the repair. Five East Tennessee utilities provided a total of 60 utility repair locations over three years of age; 30 of which incorporated Grade D Aggregate and 30 incorporated #57 Stone. The Tennessee Department of Transportation specifies a smoothness criterion of the roadway to regulate any deviation of the surface of the roadway greater than ¼ inch over a 12-foot span. The parameters measured during this study include smoothness, condition of the asphalt topcoat, adjacent stress cracking, depth of repair and disturbed surface area. Multiple linear regression and analysis of variance tests were used to analyze the results. Results suggest that there is no difference between using and not using Grade D Aggregate except with failures of one inch or greater. The results also suggest that there is little to no relationship between roadway characteristics and the performance of the repair except with failures one inch or greater. There is a correlation between slope and failures that had a deviation of one inch or greater. The results suggest that Grade D Aggregate performs better when significant failures occur. Recommendations include implementation of a cutback area, development and implementation of installation guidelines within the municipality, and implementation of a maintenance program that will address the repair cut failures in a timely manner.