DNA interstrand crosslinks (ICLs) are a potent type of DNA damage that arise as a consequence of normal cell metabolism. By covalently linking opposing strands of the double helix, ICLs block essential DNA transactions such as replication, transcription, and recombination. If unrepaired, or incorrectly repaired, ICLs can lead to gross genome instability and cell death. This cytotoxicity has been exploited in the clinic, where ICL inducing drugs are among the oldest and most widely prescribed anti-cancer therapies. However, acquired resistance is a significant limitation of these drugs, and the mechanism by which this occurs remains largely elusive. In order to develop more effective ICL-based therapies, it is imperative to first fully elucidate how healthy cells respond to and repair ICLs. Moreover, better understanding ICL repair mechanisms is necessary to fully unravel the complex DNA repair networks that govern genomic integrity, and understand the physiology of diseases such as Fanconi Anemia, which result from the inability to efficiently repair ICL lesions. Multiple mechanisms of ICL repair exist, and repair pathway choice is primarily determined by the phase of the cell cycle. In proliferating cells, the ICL repair occurs during S-phase, and in a process termed “replication coupled repair” (RCR). In contrast, slowly or non-dividing cells rely on an alternative modality of repair called “replication independent repair” (RIR). RIR is critical for homeostasis and survival in quiescent healthy cells that (for example, neurons) and in cycling cells deficient for replication coupled repair proteins (i.e. Fanconi Anemia cells). Despite its importance, little is known about RIR. This is due, in part, to the fact that ICL repair has been primarily studied in systems, such as cultured cells, that favor RCR and are therefore bias against RIR. More recently, non-replicating Xenopus cell-free extracts has emerged as a powerful system to study RIR. This system faithfully recapitulates RIR and has been instrumental in identifying DNA polymerase kappa (Pol κ) and the eukaryotic sliding clamp, proliferating cell nuclear antigen (PCNA), as two critical RIR factors. However, other important RIR factors are yet to be identified. ICL repair is unique among DNA repair pathways as it harnesses proteins from diverse DNA repair pathways including, Base Excision Repair (BER), Nucleotide Excision Repair (NER), Mismatch Repair (MMR), and Double Strand Break Repair (DSBR). Chapter 1 provides an overview of these pathways including the types of DNA damage that each pathway responds to, key steps of the repair process, and the corresponding proteins that are involved. This chapter provides context for the rest of the thesis in which I explore the contribution of multiple DNA repair proteins on the repair of ICL lesions. In Chapter 2, I detail our studies assessing the contribution of the MMR machinery to RIR. We show that the mismatch repair sensor, MutS complex (MSH2-MSH6), is critical for ICL recognition, and the stepwise recruitment of other MMR proteins including MutL (MLH1-PMS2) and EXO1. In this chapter, I also investigate how ICL structure influences repair. I find that more distorting ICLs use an MMR-dependent ICL repair mechanism, while less distorting ICLs are repaired MMR-independently (see also Appendix A), or not repaired at all. Appendix B further explores the contribution of the MMR pathway on ICL repair in mammalian cells. Finally, in Appendix C and D we provide further evidence that RIR is fundamentally distinct from replication coupled ICL repair, as depletion of key RCR proteins from our extracts yields no phenotype. I summarize all of these findings in Chapter 3, and discuss their implications to the DNA repair field as well as the clinic, where crosslinker drugs remain a mainstay in the treatment of cancer.

Thesis (M.S.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 1982. / MICROFICHE COPY AVAILABLE IN ARCHIVES AND ENGINEERING / Includes bibliographical references. / by Elizabeth Hsiu-Yun Chen. / M.S.

The reactions of carboxylic acids with organoaluminium reagents were studied, which led to the formation of novel aluminium compounds. The reactions of orthofunctionalised derivatives of benzoic acid with trivalent aluminium organyls AlR3, led to the formation of different Al-based molecular clusters, depending on the nature of R, the reaction stoichiometry and the character of the benzoic acid derivative. The obtained compounds were characterised in the solid state by X-ray diffraction methods and two main motifs were observed. When the acid and AlR3 reacted in a one-to-two stoichiometry the obtained products, [iBu4Al2(μ-O2CC6H4-2-μ- O)]2, [(Me2Al)2(μ-O2CC6H4-2-μ-NH)]2, [(iBu2Al)2(μ-O2CC6H4-2-μ-NH)]2, [(Me2Al)2(μ- O2CC6H4-2-μ-NMe)]2 and [(iBu2Al)2(μ-O2CC6H4-2-μ-NMe)]2, consisted of a central distorted 12-membered macrocycle, formed by two [Al-O-C-O-Al-X] units (X= O,N) and was found to be dimeric. The reaction between anthranilic acid derivatives and AlR3 could also take place in a one-to-one ratio. For anthranilic acid and Nmethylanthranilic acid the obtained crystals only allowed a qualitative analysis and showed the structure of the products, [MeAl(μ-O2CC6H4-2-μ-NH)]4, [iBuAl(μ-O2CC6H4- 2-μ-NMe)]4 to be tetrameric and each consisting of a distorted 16-membered ring formed by four [O-C-O-Al] units. With the reaction of N-phenylanthranilic acid it was possible to isolate a structural analogous product [iBuAl(μ-O2CC6H4-2-μ-NPh)]4 which could be fully characterised by x-ray crystallography and NMR spectroscopy. Where the quantity and quality of the obtained product was sufficient, the solution behaviour of the compounds was elucidated by multinuclear and multidimensional NMR spectroscopic techniques. The 27Al NMR showed that the aforementioned aggregates are maintained in solution, which for the 12-membered [Al-O-C-O-Al-N] macrocycle of [(iBu2Al)2(μ-O2CC6H4-2-μ-NH)]2 was confirmed by a NOESY spectrum. The second part of this project focused on the preliminary studies towards the application of aluminium compounds in the crosslinking of guar and carboxymethyl hydroxypropyl guar, which are common additives in hydraulic fracturing. Different commercially available aluminium compounds were tested for their general ability to crosslink the aforementioned polysaccharides, yielding promising results for aluminium lactate, aluminium acetylacetonate and aluminium isopropoxide. For the system comprising aluminium lactate in combination with CMHPG, rheological studies were carried out to determine the viscosity, the viscoelasticity, the shear recovery and the stability towards high temperatures. These sought to evaluate the crosslinking properties of the aluminium additive and to optimise the required conditions of the different system components. Finally, it was possible to obtain first proof-of-concept data suggesting that synthetically obtained aluminium compounds such as [Me2Al(μ- O2CPh)]2 and Al[MeC(CH2O)3]2(AlMe2)3 can be employed for the crosslinking of guar and CMHPG.

Parkinson’s Disease (PD) is a progressively debilitating neurodegenerative disease that affects the central nervous system and leads to severe difficulties with body movements. PD occurs due to the selective degeneration of neurons in the region of the brain known as the substantia nigra pars compacta. To date PD remains an incurable disease. Currently prescribed drugs provide only symptomatic relief to patients. Furthermore, they have considerable side effects and are often ineffective in the later stages of the disease or need to be used in combination in order to be effective. The role of neuroprotectants as a preventative measure in PD therapy is consequently receiving a great deal of attention and is being subjected to extensive research. This study sought to develop a novel prolonged-release drug delivery device for providing site-specific administration of newly researched neuroprotective agents. Nicotine was employed as the model neuroprotectant to incorporate in a novel reinforced crosslinked multiple-unit multi-polymeric drug delivery device. The study was the first of its kind to develop and employ the alkaloid for this purpose in a formulated delivery system. The device was intended to be one that provided zero-order prolonged release of the drug over a period of 1-2 months. The device was formulated such that its design was in keeping with the potential for implantation into the substantia nigra pars compacta to provide site-specific drug delivery. In order to do so, polymers, with biocompatible and bioerodable characteristics were selected to incorporate the drug within a reinforced crosslinked matrix. The study elucidated the mechanism of crosslinking of ionotropically crosslinked alginate spheres (gelispheres) with a variety of crosslinking agents through an evaluation of physicomechanical properties of the crosslinked system. The presence of barium in the crosslinked matrices generated densely networked gelispheres which retained their robustness following exposure to hydrating media and displayed promising potential for 4 entrapping drug molecules and retarding their release. The system was reinforced employing hydroxyethylcellulose (HEC) and polyacrylic acid (PAA). A Design of Experiments approach employing a Plackett-Burman screening design enabled optimisation of the proposed device in terms of reinforcing polymers (HEC and PAA) and crosslinking agents (barium and calcium). In order to further attenuate drug release rate the optimised crosslinked gelispheres were exposed to dilute hydrochloric acid (HCl) which significantly decreased gelisphere matrix swelling and erosion following exposure to simulated cerebrospinal fluid (CSF). These gelispheres were thereafter incorporated into a compressed release-rate modulating polymeric discs. Zero-order drug release was observed for a period of 50 days in simulated CSF when the optimised gelispheres were incorporated into compressed poly(lactic-co-glycolic) acid (PLGA) discs. Alternative approaches to modify drug release kinetics were also evaluated including the use of PLGA coatings on compressed hydroxypropylmethylcellulose-polyethylene oxide (HPMC-PEO) discs incorporating gelispheres and the use of crosslinked alginate-pectinate gelispheres as carrier systems to deliver PLGA-PLA (polylactic acid) microparticles incorporating drug. A Box-Behnken statistical design was employed to formulate and optimise the drug carrying PLGA-PLA microparticles. In both of the abovementioned cases we obtained sustained zeroorder drug release.

Diabetes is a debilitating disease affecting millions of people worldwide. The transplantation of insulin-producing, pancreatic islet cells has been an extensively explored approach for the treatment of Type 1 Diabetes. However, the need for a multi-donor source, the strong host immune responses, and a life-long immunosuppressive therapy regimen limits the widespread applicability of islet transplantation. Encapsulation of islet cells within a semi-permeable biomaterial as a means to mask transplanted cells from the host has been shown to be a viable option for the protection of islets upon transplantation. Recent advancements, incorporating additional knowledge of biomaterials, have revitalized the field of islet encapsulation. This thesis work focused on both micro- and nano-scale encapsulation techniques. Initially, a novel, covalently linked alginate-poly(ethylene glycol) (PEG), termed XAlginate-PEG, microcapsule was evaluated, and was shown to exhibit superior stability over traditional ionically bound alginate microcapsules. The XAlginate-PEG capsules exhibited a 5-fold decrease in osmotic swelling than traditional alginate microcapsules, and remained completely intact upon chelation of ionic interactions. In addition, in vitro study of the novel polymer matrix showed high compatibility with mouse insulinoma cell lines, rat and human islets. Furthermore, no disruption in islet function was observed upon encapsulation. The second study of this thesis work focused on the nano-scale encapsulation of islets with a single layer PEG coating. A PEG polymer was grafted directly on the collagen matrix of the islet capsule to form a stable amide bond. PEGylation of the islet cells was shown to camouflage inflammatory agents, such as tissue factor (TF), present on the surface of the islet, while maintaining islet morphology and function. In summary, PEG dampened coagulation cascade activation, and concealed activated factor X (afX) generation under pro-inflammatory culture conditions. The present findings contribute to the field of cellular encapsulation, both in the fabrication of novel encapsulation techniques and the evaluation of nano-scale coatings. The future potential of this research includes the attenuation of immune responses to transplanted cells, elimination of continuous immunosuppression, and provide flexibility in cell source. Furthermore, the platforms evaluated in this thesis are generalized for all cell types, thereby permitting translation of techniques to alternative cellular therapies.

Tissue-engineering (TE) is one of the most innovative approaches for tackling many diseases and body parts that need to be replaced, by developing artificial tissues and organs. For this, tissue scaffolds play an important role in various TE applications. A tissue scaffold is a 3D (3D) structure with interconnected pore networks and used to facilitate cell growth and transport of nutrients and wastes while degrading gradually itself. Many fabrication techniques have been developed recently for incorporating living cells into the scaffold fabrication process and among them; dispensing-based rapid prototyping techniques have been drawn considerable attention due to its fast and efficient material processing. This research is aimed at conducting a preliminary study on the dispensing-based biofabrication of 3D cell-encapsulated alginate hydrogel scaffolds. Dispensing-based polymer deposition system was used to fabricate 3D porous hydrogel scaffolds. Sodium alginate was chosen and used as a scaffolding biomaterial. The influences of fabrication process parameters were studied. With knowledge and information gained from this study, 3D hydrogel scaffolds were successfully fabricated. Calcium chloride was employed as crosslinker in order to form hydrogels from alginate solution. The mechanical properties of formed hydrogels were characterized and examined by means of compressive tests. The influences of reagent concentrations, gelation time, and gelation type were studied. A post-fabrication treatment was used and characterized in terms of strengthening the hydrogels formed. In addition, the influence of calcium ions used as crosslinker on cell viability and proliferation during and after the dispensing fabrication process was examined and so was the influence of concentration of calcium solutions and exposing time in both media and alginate hydrogel. The study also showed that the density of encapsulated cells could affect the viscosity of alginate solution. In summary, this thesis presents a preliminary study on the dispensing-based biofabrication of 3D cell-encapsulated alginate hydrogel scaffolds. The results obtained regarding the influence of various factors on the cell viability and scaffold fabrication would form the basis and rational to continue research on fabricating 3D cell-encapsulated scaffolds for specific applications.

The 3D structures of proteins may provide important clues to their functions and roles in complex biological pathways. Traditional methods such as X-ray crystallography and NMR are not feasible for all proteins, while theoretical models are typically not validated by experimental data. This project investigates the use of chemical crosslinkers as an experimental means of validating these models. Five target proteins were successfully purified from yeast whole cell extract: Transketolase (TKL1), inorganic pyrophosphatase (IPP1), amidotransferase/cyclase HIS7, phosphoglycerate kinase (PGK1) and enolase (ENO1). These TAP-tagged target proteins from yeast <em>Saccharomyces cerevisiae</em> allowed the protein to be isolated in two affinity purification steps. Subsequent structural analysis used the homobifunctional chemical crosslinker BS³ to join pairs of lysine residues on the surface of the purified protein via a flexible spacer arm. Mass spectrometry (MS) analysis of the crosslinked protein generated a set of mass values for crosslinked and non-crosslinked peptides, which was used to identify surface lysine residues in close proximity. The Automatic Spectrum Assignment Program was used to assign sequence information to the crosslinked peptides. This data provided inter-residue distance constraints that can be used to validate or refute theoretical protein structure models generated by structure prediction software such as SWISS-MODEL and RAPTOR. This approach was able to validate the structure models for four of the target proteins, TKL1, IPP1, HIS7 and ENO1. It also successfully selected the correct models for TKL1 and IPP1 from a protein model library and provided weak support for the HIS7, PGK1 and ENO1 models.

The 3D structures of proteins may provide important clues to their functions and roles in complex biological pathways. Traditional methods such as X-ray crystallography and NMR are not feasible for all proteins, while theoretical models are typically not validated by experimental data. This project investigates the use of chemical crosslinkers as an experimental means of validating these models. Five target proteins were successfully purified from yeast whole cell extract: Transketolase (TKL1), inorganic pyrophosphatase (IPP1), amidotransferase/cyclase HIS7, phosphoglycerate kinase (PGK1) and enolase (ENO1). These TAP-tagged target proteins from yeast <em>Saccharomyces cerevisiae</em> allowed the protein to be isolated in two affinity purification steps. Subsequent structural analysis used the homobifunctional chemical crosslinker BS³ to join pairs of lysine residues on the surface of the purified protein via a flexible spacer arm. Mass spectrometry (MS) analysis of the crosslinked protein generated a set of mass values for crosslinked and non-crosslinked peptides, which was used to identify surface lysine residues in close proximity. The Automatic Spectrum Assignment Program was used to assign sequence information to the crosslinked peptides. This data provided inter-residue distance constraints that can be used to validate or refute theoretical protein structure models generated by structure prediction software such as SWISS-MODEL and RAPTOR. This approach was able to validate the structure models for four of the target proteins, TKL1, IPP1, HIS7 and ENO1. It also successfully selected the correct models for TKL1 and IPP1 from a protein model library and provided weak support for the HIS7, PGK1 and ENO1 models.

Tissue-engineering (TE) is one of the most innovative approaches for tackling many diseases and body parts that need to be replaced, by developing artificial tissues and organs. For this, tissue scaffolds play an important role in various TE applications. A tissue scaffold is a 3D (3D) structure with interconnected pore networks and used to facilitate cell growth and transport of nutrients and wastes while degrading gradually itself. Many fabrication techniques have been developed recently for incorporating living cells into the scaffold fabrication process and among them; dispensing-based rapid prototyping techniques have been drawn considerable attention due to its fast and efficient material processing. This research is aimed at conducting a preliminary study on the dispensing-based biofabrication of 3D cell-encapsulated alginate hydrogel scaffolds. Dispensing-based polymer deposition system was used to fabricate 3D porous hydrogel scaffolds. Sodium alginate was chosen and used as a scaffolding biomaterial. The influences of fabrication process parameters were studied. With knowledge and information gained from this study, 3D hydrogel scaffolds were successfully fabricated. Calcium chloride was employed as crosslinker in order to form hydrogels from alginate solution. The mechanical properties of formed hydrogels were characterized and examined by means of compressive tests. The influences of reagent concentrations, gelation time, and gelation type were studied. A post-fabrication treatment was used and characterized in terms of strengthening the hydrogels formed. In addition, the influence of calcium ions used as crosslinker on cell viability and proliferation during and after the dispensing fabrication process was examined and so was the influence of concentration of calcium solutions and exposing time in both media and alginate hydrogel. The study also showed that the density of encapsulated cells could affect the viscosity of alginate solution. In summary, this thesis presents a preliminary study on the dispensing-based biofabrication of 3D cell-encapsulated alginate hydrogel scaffolds. The results obtained regarding the influence of various factors on the cell viability and scaffold fabrication would form the basis and rational to continue research on fabricating 3D cell-encapsulated scaffolds for specific applications.

This study investigates the changes due to crosslinking treatment in stiffness, permeability, and glycosaminoglycan (GAG) content of bovine intervertebral discs. The objective of this study was to determine the mechanical and biochemical effects of crosslinking treatment on lumbar bovine tissue. Previous studies have found that crosslinking can increase stiffness and permeability in the intervertebral disc. These changes have not yet been investigated by confined compression, stress-relaxation tests of young bovine tissue. Eleven lumbar motion segments were harvested from calf spines and soaked in a saline solution or one of four crosslinking treatments (genipin, methylglyoxal, proanthrocyanidin, and EDC). Five mm diameter samples were removed from the midannulus region at anterior / anterior-lateral locations, confined in a saline bath, swelled to equilibrium, and tested in confined compression stress-relaxation to 15% strain in 5% increments. Radial samples were also harvested, treated with saline solution and EDC, and tested in the same manner. The aggregate modulus and hydraulic permeability were calculated using the nonlinear biphasic theory. Swelling pressure was calculated as the load at swelling equilibrium. GAG content was measured using the dimethylmethylene blue assay. Differences with P value < 0.05 were considered significant. In the axial orientation, all crosslinking treatments except methyglyoxal at least doubled the aggregate modulus relative to soaked controls (P less than 0.05). Genipin treatment resulted in 78% lower axial permeability, proanthrocyanidin (PA) 50% lower, and EDC treatment 84% lower relative to soaked controls (P &lt; 0.05). GAG content measured in the methyglyoxal treatment group was 25% lower than in soaked control group. Genipin (G), proanthrocyanidin (PA), and EDC treatment increased the swelling pressure by at least 65% (P less than 0.05). In the radial orientation, EDC treatment increased the stiffness by 75%, and did not significantly affect the permeability or swelling pressure. Some crosslinking treatments proved effective in increasing the stiffness and swelling pressure of the disc. The increased swelling pressure in G, PA, and EDC treatment groups relative to soaked controls suggests reduced GAG leaching during soaking treatment, further confirmed by the reduction in permeability in these groups.