Substantial savings in operations can be achieved by reducing the amount of wastewater that must be pumped and treated. Utilities have long dealt with the infiltration and inflow (I and I) issues in their system by televising their pipes and identifying leak points, but this primarily addresses only the infiltration part of “I and I.” Inflow, which creates hydraulic issues during rain events, leads to sanitary sewer overflows and can subject the utility to fines from regulatory agencies. As a result, dealing with the inflow portion of I and I is needed. The goal of this thesis is to differentiate inflow and infiltration from baseflow and to determine the effectiveness of different methods used to reduce inflow and infiltration in sanitary sewer lines. An analysis was conducted on the benefits and cost effectiveness of different inflow/infiltration approaches (slip-lining sewer lines, stormwater manhole inserts, replacing sewer lines, smoke testing, etc.) and cost savings municipalities can expect to receive from each. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2015. / FAU Electronic Theses and Dissertations Collection

Proliferating cell nuclear antigen (PCNA) is a sliding clamp that acts as a central co-ordinator for mismatch repair as well as DNA replication. Loss of Elg1, the major subunit of the PCNA unloader complex, causes over-accumulation of PCNA on DNA and also increases mutation rate, but it has been unclear if the two effects are linked. In this study, I showed that timely control of PCNA retention on DNA by Elg1 replication factor C-like complex (Elg1-RLC) ensures correct mismatch repair. Although premature unloading of PCNA generally increases mutation rate, PCNA mutants PCNA-R14E and PCNA-D150E that spontaneously fall off DNA attenuate the mutator phenotype of elg1Δ. In contrast, PCNA-D21K that accumulates on DNA due to enhanced electrostatic PCNA-DNA interactions exacerbates the elg1Δ mutator phenotype. Next, I addressed how accumulation of PCNA on DNA increases mutation rate. Epistasis analysis suggests that PCNA over-accumulation on DNA predominantly prevents the Msh2-Msh6-dependent and Exo1-independent mismatch repair pathways. In elg1Δ, over-retained PCNA hyper-recruits the Msh2-Msh6 mismatch recognition complex through its PCNA-interacting peptide motif, causing accumulation of mismatch repair intermediates. The results suggest that PCNA retention controlled by the Elg1-RLC is critical for efficient mismatch repair: PCNA needs to be on DNA long enough to enable mismatch repair, but if it is retained too long it interferes with downstream repair steps.

Unlike the peripheral nervous system (PNS), adult neurons in the central nervous system (CNS) have limited regenerative capacity after injury. One interesting phenomenon observed nearly four decades ago was that lesion of a peripheral nerve can significantly enhance the regenerative capacity of the central axons of the corresponding dorsal root ganglion (DRG) neurons, termed a ‘conditioning lesion’, but the underlying mechanism is still not fully understood. Since ATP is released after nerve injury and extracellular ATP has a broad range of biological activities, we postulated that ATP might be the injury signalling molecule that triggers the regenerative machinery in the injured neurons. If that were the case, injection of ATP into a peripheral nerve should be able to mimic the effect of a conditioning lesion. To test this theory, we injected ATP into a peripheral (sciatic) nerve after a dorsal column transection and found that ATP injection did promote the regeneration of injured axons into the lesion cavity. We also found that ATP injection activated transcription factor STAT3 and increased the expression of growth associated protein 43 (GAP43) in the corresponding DRG neurons. ATP injection increased the concentrations of ciliary neurotrophic factor and interleukin-6 in sciatic nerve and DRG. These results indicate that intraneural injection of ATP can mimic conditioning lesion to a certain degree. Most interestingly, we found that a second injection of ATP one week after the first one markedly boosted the effects of the first injection as many more axons grew into or across the lesion compared with double saline injection or ATP plus saline injection. Double ATP injection is also more effective in sustaining the expression of phospho- STAT3 and GAP43. Immunohistochemical analysis showed ATP injection caused little Wallerian degeneration at the injection site. Behavioural tests showed no long-term adverse effects to the injected sciatic nerve. In order to explore the underlying mechanism of ATP induced elevation of the regeneration state of DRG neurons and look for more potent purinoceptor agonists to stimulate axonal regeneration, we first tried to identify the expression of purinoceptor subtypes in sciatic nerves using quantitative PCR and immunohistochemistry. We found that mRNAs for all the four P1 and fourteen P2 purinoceptor subtypes were expressed in the sciatic nerve, DRG or dissociated Schwann cells at various levels. Immunohistochemical analysis showed that purinoceptor subtypes are expressed by different types of cells. Due to the expression of nearly all purinoceptor subtypes in the sciatic nerve, it will be a big challenge to identify the receptor subtype(s) responsible for ATP induced axonal regeneration. We have set up a compartmented co-culture system to test various agonists/antagonists of purinoceptors. Taken together, we have shown that intraneural ATP injection can mimic conditioning lesion in promoting sensory axonal regeneration. Identification of the receptor subtype(s) and other molecules involved in the enhanced regeneration capacity of injured neurons may lead to the development of therapeutic agents to effectively promote the axonal regeneration of both peripheral and central neurons.

Melanoma is one of the most common cancers in the world. For primary melanoma, early diagnosis and surgical excision are effective treatments but, despite the new targeted therapies and immunotherapies, there is still a need for more effective treatment options to improve overall survival for patients with metastatic melanoma. Chemotherapy with genotoxic agents remains the main approach for most cancers, but DNA repair pathways in cancer cells reduce their effectiveness. So disruption of key DNA repair pathways, such as nucleotide excision repair (NER), could be an effective option to combine with chemotherapy for melanoma. The structure-specific endonuclease ERCC1-XPF, which heterodimerises through the C-terminal helix-hairpin-helix (HhH)2 domains of both proteins, is essential for NER. The aim of my project was to determine the mechanism involved in regulating the level of the ERCC1-XPF heterodimer with a view to disrupting NER activity. The project started by determining the ERCC1 and XPF response in six melanoma cell lines to the chemotherapeutic cisplatin at the mRNA and protein levels. Although the mRNA and protein levels of both ERCC1 and XPF increased, there was variable consistency between the cell lines, raising the possibility that post translational modification may play an important role in the regulation of ERCC1- XPF activity. We chose to focus on ubiquitination, because it can affect a protein’s activity at both expression and activation levels and several examples of ubiquitinated DNA repair proteins were known. In the pilot study we found that ERCC1 was accumulated after proteasome inhibitor treatment and decreased by treatment with a translation inhibitor in two melanoma cell lines, suggesting that ERCC1 may be ubiquitinated. By cotransfecting His-tagged ubiquitin and non-tagged ERCC1 constructs into melanoma cells and performing an ubiquitin assay, we found that ERCC1 was degraded by the proteasome system through polyubiquitination or multiple monoubiquitination. To determine the nature of the ubiquitination type, we mutated each of the seven Lys residues on ubiquitin and carried out additional assays with ubiquitin single and combination mutants, and discovered that Lys33 was most likely involved in the proteasome dependent degradation of ERCC1. By immunoprecipitation with an antibody to linear ubiquitin from melanoma cell extracts containing a ubiquitin construct with all seven Lys residues mutated to Arg, we found that the N-Met of ubiquitin was also most likely involved in ERCC1 ubiquitination. To determine which Lys of ERCC1 is used by ubiquitin, we did another series of in vivo ubiquitin assays with full length and truncated ERCC1 constructs and found that the key amino acid is most likely within the C-terminal XPF binding domain of ERCC1. By cotransfecting the full length ERCC1 and ERCC1 truncation constructs together with full length XPF, we showed that the ubiquitination of ERCC1 was not an artefact resulting from overexpression of ERCC1 alone and that the stability of XPF was dependent on the overexpression and stability of ERCC1. We then made single lysine and lysine combination mutants in the XPF binding domain of ERCC1 and found that none of the lysines were essential for ubiquitination of ERCC1, indicating that a non- lysine amino acid might be used for ubiquitination. However, using a transfection-based NER assay in ERCC1-deficient cells, we found that ubiquitination of Lys 295 could be involved in regulating the DNA repair activity of ERCC1-XPF. The in vivo ubiquitin assay result after cotransfection of ERCC1 and XPF, which showed that XPF was dependent on the presence of ERCC1 for stability, but not vice versa, was inconsistent with previous published data suggesting that heterodimerization was essential for the stability of both proteins. Instead we hypothesised that homodimerization of ERCC1 might be another mechanism to keep ERCC1 stable and obtained evidence for this at the overexpression level by immunoprecipitation following cotransfection of Myc-tagged ERCC1 and Flag-tagged ERCC1 or ERCC1 truncations, which was supported at the endogenous expression level by size exclusion chromatography on melanoma cell extracts to identify ERCC1 in different molecular weight fractions. In the previous in vivo ubiquitin assay, we found that levels of transfected full length ERCC1 and XPF were dramatically reduced by cotransfection with the Flag-tagged ERCC1 (220-297) construct that just contains the XPF binding domain of ERCC1. This led to another hypothesis, that the ERCC1 (220-297) peptide can decrease endogenous levels of ERCC1 and XPF and so be a potential drug in combination with cisplatin chemotherapy. This hypothesis was verified in stable transgenic cell lines expressing ERCC1 (220-297) which showed reduced levels of ERCC1 and XPF and of NER and increased sensitivity to cisplatin and UV irradiation. Based on the above results and supporting bioinformatics analysis we have made the following conclusions: ERCC1 is regulated by the ubiquitin-proteasome degradation pathway through linkages most likely involving Lys33 and N-Met; the XPF binding domain is most likely the key domain for ERCC1 ubiquitination; XPF stability is dependent on the presence of ERCC1 and seems affected by ERCC1 ubiquitination; ERCC1 seems to be ubiquitinated in a non-conventional lysine-independent manner and ubiquitination of Lys 295 might be involved in the regulation of the DNA repair activity of ERCC1- XPF; homodimerization is most likely a novel mechanism to keep ERCC1 stable; the ERCC1 (220-297) peptide can destabilise both ERCC1 and XPF and could be a potential drug in combination with genotoxic therapies.

This study was conducted at the University of Massachusetts Medical Center in the Volkert laboratory. Human alkyl adenine DNA glycosylase (hAAG) is a DNA repair enzyme that repairs alkylated DNA bases. hAAG was cloned in 1991 and a second isoform was classified in 1994. The difference between the two isoforms of hAAG is an alternate spliced first exon. Both isoforms of the hAAG gene were present in the Volkert laboratory collection, however the second isoform (hAAG-2) was phenotypically different than the first and became the first focus of this study. Using the improperly functioning isoform as a template, and constructing a 5' primer with the identical upstream sequence as the functioning isoform (hAAG-1), a phenotypically similar gene was constructed by PCR. The new isoform (hAAG-2) was cloned into an expression vector and its activity as a DNA repair agent was studied. A second version of hAAG-2 was also constructed, incorporating a histidine tag for protein purification and identification purposes. Efforts included using the ability of hAAG to complement glycosylase deficient alkA tagA E. coli double mutant strains to assess and to compare the ability of the two isoforms of hAAG and to determine if the histidine tag affected function. The ability of hAAG to rescue cells from exposure to a variety of DNA damaging agents was studied by inducing each isoform and analyzing the sensitivity of the cells to increased doses of DNA damaging agents. Both hAAG-1 and hAAG-2 were able to restore the wild type resistance of the alkA and tag genes when exposed to the alkylating agents MNNG and MMS. In order to study the ability of hAAG to repair alkyl lesions larger than methyl groups, it was necessary to inactivate the uvrA dependent nucleotide excision repair gene. In E. coli, methyl lesions are repaired primarily by glycosylases, while nucleotide excision repairs bulky lesions. Thus, in order to detect hAAG activity on these types of damage, it was necessary to inactivate the bacterial uvrA gene. Each isoform of hAAG was transformed into a triple mutant strain deficient in alkA tagA and uvrA, then exposed to CNU, BCNU, and Mitomycin C. Each of these DNA damaging agent caused increased toxicity in the presence of hAAG. hAAG-1 expressed in the alkA tag double mutant strain was exposed to Mitomycin C and showed greater resistance than hAAG-1 expressed in the alkA tag uvrA triple mutant. In fact, in the nucleotide excision proficient strain, expression increased Mitomycin C resistance above that seen in the control, suggesting that glycosylase activity may function in a partnership with nucleotide excision repair and that the two isoforms of hAAG have subtle differences. An ompT protease knockout host strain was constructed using P1-transduction and used to examine protein products. hAAG-2 was inserted into the pBlueScript plasmid so that the gene could be regulated by the T7 promoter for use beyond the scope of this thesis. A protein synthesis time course assay was conducted to determine the expression levels of hAAG-1 and hAAG-2 when induced by IPTG. Immunoblot detection of the histidine tag was used to measure expression levels of each isoform.

With the majority of bridges in the country aging, over capacity and costly to rehabilitate or replace, it is essential that engineers refine their inspection and evaluation techniques. Over the past 130 years the information gathering techniques and methods used by engineers to inspect bridges have changed little. All of the available methods rely on one technique, visual inspection. In addition, over the past 40 years individual bridge inspectors have gone from being information gathers to being solely responsible for the condition rating of bridges they inspect. The reliance on the visual abilities of a single individual to determine the health of a particular bridge has led to inconsistent and sometimes erroneous results. In an effort to provide bridge inspectors and engineers with more reliable inspection and evaluation techniques, this thesis will detail the case for development of a new inspection tool, and the assembly and use of one new tool called Fringe Interferometry

Digitized by Kansas Correctional Industries

Typescript (photocopy). / Digitized by Kansas Correctional Industries

CRISPR/Cas9 is the gene editing tool of choice in basic research and poised to become one in clinical context. However, current studies on the topic suffer from a number of shortcomings. Mutagenesis is often assessed using bulk methods, which means rare events go undetected, unresolved or are discarded as potential sequencing errors. Many of the genotyping methods rely on short-range PCR, which excludes larger structural variants. Other methods, such as FISH, do not provide basepair resolution, making the genotype assessment imprecise. Furthermore, it is not well understood how Cas9 delivery format influences the dynamics of indel introduction. Finally, many studies of on-target activity were conducted in cancerous cell lines, which do not accurately model the mutagenesis of normal cells in the therapeutic context. In my thesis, I have investigated on-target lesions induced by Cas9 complexed with single gRNAs and no exogenous template. I have followed the time dynamics of Cas9-induced small indels as a function of reagent delivery methods, established an assay for quantification of Cas9-induced genomic lesions that are not small indels ("complex lesions") and used this assay to isolate and genotype complex lesions, many of which would be missed by standard methods. I found that DNA breaks introduced by single guide RNAs frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and cross-over events were identified. Frequent and extensive DNA damage in mitotically active cells caused by CRISPR/Cas9 editing may have pathogenic consequences.

Typescript (photocopy). / Digitized by Kansas Correctional Industries / Department: Family Economics.