The genomic integrity of a cell is constantly being pressured by both intrinsic and extrinsic forces. Cell cycle checkpoints exist to protect the cells by arresting cell cycle progression in response to DNA damage or replication stress. It has been shown that the interaction between the checkpoint proteins Rad9A and TopBP1 is a crucial upstream event required for the ATR-dependent checkpoint response to DNA damage, which can be activated throughout different points in the cell cycle. The Bimolecular Fluorescence Complementation (BiFC) technique has recently emerged as a simple and effective tool for analyzing protein-protein interactions in live cell cultures. By fusing complementary fragments of fluorescent proteins to proteins of interest, one can visualize protein-protein interactions through the formation of a mature fluorophore from these fragments. In the current work, the BiFC assay system was employed to study the interaction between TopBP1 and Rad9A; the human homologue of fission yeast Rad9. BiFC vectors expressing TopBP1, Rad9A, and the Rad9A-S387 mutant were constructed and optimized for transfection in HeLa cells. It was shown that the BiFC fusion protein of Rad9A lacked phosphorylation on its constitutive S387 site, although it retained its upstream damage dependent S272 phosphorylation after IR treatment. BiFC signals could be detected in cells containing the BiFC fusion proteins of Rad9A and TopBP1 using confocal microscopy and flow cytometry techniques. However, the signals could not be distinguished from that of the negative control samples. Our results suggest a possibility that our BiFC fusion proteins of interest interact in a non-specific manner, although further characterization is required to confirm this. The BiFC assay employed in this project must be further optimized to effectively study the interaction between Rad9A and TopBP1, as well as other checkpoint proteins. However, this study has given us great insight into the implementation of this new BiFC technique for studying protein interactions in the context of cell cycle proteins, and the knowledge gained from this study will be invaluable for future work. / Thesis (Master, Biochemistry) -- Queen's University, 2009-12-17 12:42:06.717

Next Generation 9-1-1 (NG 9-1-1) will revolutionize how the public accesses emergency services and will alter the technological landscape within which existing public safety agencies operate. A lack of systematic methodologies exists for quality control of the required geospatial data layers for NG 9-1-1 systems. The primary objective of this study was to develop and systematize a highly accurate NG 9-1-1 GIS database for Counties of Southern Illinois (CSI). The project goals included mapping relevant geospatial data layers required by and based on NENA standard data formats; conducting data quality control and standardization; and providing standardized spatial datasets for NG 9-1-1 to relevant stakeholders. The approach was developed using a conceptual model for error and uncertainty analysis of the GIS-based NG 9-1-1 system. This included the identification of various sources of input uncertainties often associated with spatial data layers; modeling the accumulation and propagation of errors; analyzing their impact on the quality of the spatial data layers; and correcting the errors. Modeling uncertainty propagation focused on positional errors and was conducted through a simulation procedure. The results showed that the original spatial datasets possessed a large account of uncertainties, especially location errors of railroads and roads. The errors had different sources, including input map errors, the use of different map projection and coordinate systems, a lack of topological structures, etc. In addition, they varied from county to county. From the error propagation simulation, it was also found that the location errors measured as root mean square error (RMSE) fluctuated when the perturbed distance of the ground control points (GCP) was less than 15 m. After that, the RMSE increased as the perturbed distance of GCPs increased. This relationship was significantly linear. In addition, the location errors from railroads were larger than those from roads.

Dispatchers and calltakers who work at 9-1-1 call centers are confronted with memories of emergencies they must address at work even though they are not physically present at the event. The language they use to talk about their work thus always references a potentially traumatic experience processed second-hand. These telecommunicators use personal messaging through the dispatch platform, verbal communication, and texting in cellphones to tell stories about their work and manage emergency response. Often two to three mediums are used in order to communicate different aspects of the same narrative. Through storytelling, dispatchers manage an environment influenced by social hierarchies, workplace command structures, gender dynamics, and the emotional stress of the calls they must process. The fragmented experiences of dispatchers are reflected in the disjointed methods and narrative structures of their storytelling. This study offers an approach to multi-modal communication and presents an analysis of an occupational folk group not previously studied by folklorists. / 2015-07-11

Replication protein A (RPA) is a eukaryotic single-stranded DNA-binding protein consisting of three subunits of 70-, 32-, and 14-kDa (RPA70, RPA32, RPA14, respectively). It is a protein essential for most cellular DNA metabolic pathways. Checkpoint proteins Rad9, Rad1, and Hus1 form a clamp-like complex which plays a central role in the DNA damage-induced checkpoint response. In this report, we presented the evidence that Rad9-Rad1-Hus1 (9-1-1) complex directly interacted with RPA in human cells, and this interaction was mediated by the binding of Rad9 protein to both RPA70 and RPA32 subunits. In addition, the cellular interaction of 9-1-1 with RPA or hyperphosphorylated RPA was stimulated by UV irradiation or camptothecin treatment in a dose-dependent manner. Such treatments also resulted in the colocalization of the nuclear foci formed with the two complexes. Consistently, knockdown of the RPA expression in cells by the small interference RNA (siRNA) blocked the DNA damage-dependent chromatin association of 9-1-1, and also inhibited the 9-1-1 complex formation. Taken together, our results suggest that 9-1-1 and RPA complexes collaboratively function in DNA damage responses, and that the RPA may serve as a regulator for the activity of 9-1-1 complex in the cellular checkpoint network.

L'analyse quantitative a été réalisée en cotutelle avec Rémi Boivin et Pierre Tremblay et publiée dans la Revue de Criminologie: Boivin, R., Lamige, C,. Tremblay, P. (2009) La police devrait-elle cibler les taudis malfamés? Criminologie, (42)1, 225-266. / Les modèles de police de résolution de problèmes et de police communautaire sont souvent présentés en opposition l’un de l’autre. Ce mémoire présente l’évaluation d’une intervention policière réunissant des éléments des modèles mentionnés précédemment et ayant pour objectif de mettre fin au foyer de désordre causé par la présence d’un immeuble de location de chambres. Les analyses reposent à la fois sur une évaluation qualitative et quantitative des interventions communautaires et policières réalisées dans cet édifice surnommé le « Motel ». Les résultats des entretiens réalisés auprès d’organismes communautaires, de policiers et de citoyens du quartier ont permis de mieux comprendre les étapes de cette opération policière et communautaire. Ils ont également permis de connaître le mode de fonctionnement relatif au rachat d’un immeuble par une administration municipale de même que d’en mesurer l’impact d’un point de vue préventif. Les résultats obtenus ont également permis de saisir l’importance des maisons de chambres pour certains locataires « démunis » de ce quartier, mais aussi la réputation qui en découle. La méthode de gestion de ce genre d’immeubles possède une étroite corrélation avec le type de locataires présents. Une analyse de terrain effectuée trois ans après l’opération a rendu possible l’identification du type de problèmes dont souffrent la majorité des locataires de maisons de chambres à gestion privée. Dans ce cas-ci, il s’agit de problèmes reliés à la santé mentale. Les résultats de l’évaluation quantitative ont quant à eux révélé qu’un régime de patrouille intensive favorise la stimulation de la fréquence des appels logés au 9-1-1 par les citoyens et que la stratégie de profiler un taudis de mauvaise réputation pour faire diminuer les désordres dans l’ensemble du quartier n’a pas été, dans le site observé, particulièrement concluante. / Models of problem-oriented policing and community policing are often shown in opposition to each other. This thesis presents an evaluation of a police crackdown, combining elements of the above mentioned models and aimed at ending disorder outbreaks caused by tenants of a troublesome flophouse. Analyses are based on both qualitative and quantitative evaluations of community and police interventions conducted in a building known as the "Motel". Results obtained through discussions with community organizations, police and neighbourhood residents have lead to better understanding the phases of this police and community operation. They also provided the operation mode on property buyback by a municipal administration and assessed its impact by means of a preventive perspective. The results also helped to acknowledge the importance of flophouses for a number of helpless tenants in this district, but also the reputation that follows. The management process for this category of building is in close correlation with the type of tenants who live there. A field analysis, conducted three years after the operation, has made it possible to identify the type of problems afflicting the majority of tenants living in flophouses that are privately managed. In this case, the problems are linked to mental health. Results of the quantitative evaluation have demonstrated that intensive patrol surveillance increases residents’ propensity to place 9-1-1 calls and the strategy to reduce disorder in the general neighbourhood by targeting notorious slums has not been particularly conclusive on the site observed.

Les modèles de police de résolution de problèmes et de police communautaire sont souvent présentés en opposition l’un de l’autre. Ce mémoire présente l’évaluation d’une intervention policière réunissant des éléments des modèles mentionnés précédemment et ayant pour objectif de mettre fin au foyer de désordre causé par la présence d’un immeuble de location de chambres. Les analyses reposent à la fois sur une évaluation qualitative et quantitative des interventions communautaires et policières réalisées dans cet édifice surnommé le « Motel ». Les résultats des entretiens réalisés auprès d’organismes communautaires, de policiers et de citoyens du quartier ont permis de mieux comprendre les étapes de cette opération policière et communautaire. Ils ont également permis de connaître le mode de fonctionnement relatif au rachat d’un immeuble par une administration municipale de même que d’en mesurer l’impact d’un point de vue préventif. Les résultats obtenus ont également permis de saisir l’importance des maisons de chambres pour certains locataires « démunis » de ce quartier, mais aussi la réputation qui en découle. La méthode de gestion de ce genre d’immeubles possède une étroite corrélation avec le type de locataires présents. Une analyse de terrain effectuée trois ans après l’opération a rendu possible l’identification du type de problèmes dont souffrent la majorité des locataires de maisons de chambres à gestion privée. Dans ce cas-ci, il s’agit de problèmes reliés à la santé mentale. Les résultats de l’évaluation quantitative ont quant à eux révélé qu’un régime de patrouille intensive favorise la stimulation de la fréquence des appels logés au 9-1-1 par les citoyens et que la stratégie de profiler un taudis de mauvaise réputation pour faire diminuer les désordres dans l’ensemble du quartier n’a pas été, dans le site observé, particulièrement concluante. / Models of problem-oriented policing and community policing are often shown in opposition to each other. This thesis presents an evaluation of a police crackdown, combining elements of the above mentioned models and aimed at ending disorder outbreaks caused by tenants of a troublesome flophouse. Analyses are based on both qualitative and quantitative evaluations of community and police interventions conducted in a building known as the "Motel". Results obtained through discussions with community organizations, police and neighbourhood residents have lead to better understanding the phases of this police and community operation. They also provided the operation mode on property buyback by a municipal administration and assessed its impact by means of a preventive perspective. The results also helped to acknowledge the importance of flophouses for a number of helpless tenants in this district, but also the reputation that follows. The management process for this category of building is in close correlation with the type of tenants who live there. A field analysis, conducted three years after the operation, has made it possible to identify the type of problems afflicting the majority of tenants living in flophouses that are privately managed. In this case, the problems are linked to mental health. Results of the quantitative evaluation have demonstrated that intensive patrol surveillance increases residents’ propensity to place 9-1-1 calls and the strategy to reduce disorder in the general neighbourhood by targeting notorious slums has not been particularly conclusive on the site observed. / L'analyse quantitative a été réalisée en cotutelle avec Rémi Boivin et Pierre Tremblay et publiée dans la Revue de Criminologie: Boivin, R., Lamige, C,. Tremblay, P. (2009) La police devrait-elle cibler les taudis malfamés? Criminologie, (42)1, 225-266.

DNA is subject to attack by DNA damaging agents from both environmental and endogenous sources. In response to DNA damage, living organisms enhance expression of many related genes to facilitate DNA repair and survival. The SOS response is a well-understood prokaryotic regulatory cascade that controls the expression of more than 30 genes in response to DNA damage. However, in eukaryotic organisms from simple budding yeast to human, such a regulatory network has not been reported.<p>Previous research in our laboratory found that among DNA repair mutants of <i>Saccharomyces cerevisiae</i>, only rad6 and rad18 defective in the post-replication repair pathway significantly affected DNA damage induction of several genes examined. Rad6 and Rad18 form a ubiquitin conjugation-ligase complex and are required for the cellular tolerance to damaged DNA. Since the Rad6-Rad18 complex binds to single-stranded DNA, it may act as a DNA damage sensor required for the activation of DNA damage-induced transcription. We performed microarray analysis and found that the induction of up to 379 genes, including those involved in DNA repair, control of replication and transcription, regulation of the cell cycle and cell metabolism, are compromised in the rad6 and rad18 mutants. Although Rad6/Rad18 monoubiquitinates proliferating cell nuclear antigen (PCNA) following DNA damage to initiate a damage tolerance response, PCNA ubiquitination is not required for DNA damage induction. In budding yeast, cell-cycle checkpoints are involved in the control of DNA damage induction of gene expression through phosphorylation of a protein kinase Rad53 by two pathways represented by Rad24 and Sgs1. The Rad6-Rad18 complex appears to function in the Rad24 pathway and parallel to Sgs1. We further demonstrated that the Rad17 subunit of the 9-1-1 complex is subject to Rad6/Rad18- and DNA damage-dependent mono-ubiquitination and that the Rad17-Lys197 residue with flanking sequences homologous to Lys164 of PCNA is absolutely required for the DNA damage induction by Rad6-Rad18. Hence, by ubiquitinating two DNA clamps, PCNA and 9-1-1, the Rad6-Rad18 complex plays a central role in the cellular response to DNA damage by coordinating translesion synthesis, error-free bypass, homologous recombination, as well as transcriptional regulation, reminiscent of roles of RecA in <i>E. coli</i> cells.<p>Several individual genes have also been examined in this study to elucidate the regulatory mechanisms acting on specific DNA damage-inducible genes. In the microarray analysis, DDI2 and DDI3, two identical genes located in duplicated chromosomal regions, were identified due to the highest induction ratio (122-fold) after MMS treatment. Interestingly, DDI2/DDI3 can only be highly induced by SN2-type alkylating agents. Promoter deletion analysis mapped the putative upstream acting sequence (UASDDI2) responsible for 40% of basal expression and 90% of induced expression by MMS.<p>The CRT10 gene was identified through screening of the yeast deletion library for hydroxyurea (HU) resistance. CRT10 encodes a putative 957 amino acid, 110 kDa protein with a leucine repeat and a WD40 repeat near the N-terminus. Deletion of CRT10 resulted in an enhanced resistance to HU reminiscent of the inactivation of two other ribonucleotide reductase (Rnr) suppressors, CRT1 and SML1, which regulate Rnr activity at transcriptional and translational levels, respectively. Epistasis analysis indicates that CRT10 belongs to the CRT1 pathway but not the SML1 pathway. Indeed, deletion of CRT10 enhanced the survival of the mec1 null mutant and increased basal level and DNA damage-induced expression of RNR2 and RNR3, suggesting that Crt10 regulates RNR genes at the transcriptional level. Furthermore, the dun1 mutation is epistatic to crt10 with respect to both HU sensitivity and RNR gene expression. Interestingly, the expression of CRT10 itself is induced by DNA damaging agents and this induction requires DUN1, suggesting that CRT10 plays a role in cellular response to DNA damage and replication blocks. The CRT10 function appears to be achieved by positive regulation of the CRT1 transcript level, indicating that CRT10 is a component of the regulatory circuit.

Cell-cycle checkpoint proteins help maintain genomic integrity by sensing damaged DNA and initiating DNA repair or apoptosis. Checkpoint protein activation to cell-cycle damaging agents can involve post-translational modifications and these alterations provide a means to determine whether DNA in a cell is damaged or not. Steinmoeller et al. (2009) showed that checkpoint proteins are suitable biomarkers for detecting genotoxins in Oncorhynchus mykiss (rainbow trout). In this project, two evolutionarily conserved checkpoint proteins, Rad1 and Hus1, have been cloned from rainbow trout and antibodies against these proteins were developed. This is the first time that either Rad1 or Hus1 has been characterized in rainbow trout. For rtRad1, it was determined that the open-reading frame was 840bp, which encodes 279aa with a predicted protein size of 31kDa. The rtRad1 amino-acid sequence is highly conserved and contains conserved exonuclease and leucine zipper domains. RT-PCR was used to identify alternatively spliced variants of rtRad1 and it appears that these variants encode different sized Rad1 proteins that are tissue and cell-line specific. A Rad1 splice variant that encodes an 18kDa protein appears to be abundant only in heart tissue and in the RTgill-W1 and RTbrain-W1 cell-lines. A genotoxicity study was completed where RTgill-W1 and RTbrain-W1 cells were treated with bleomycin, which induces double-stranded DNA breaks. In RTgill-W1, levels of an 18kDa Rad1 protein increased in a dose-dependent manner while in RTbrain-W1 the Rad1 levels remained the same. It appears that this 18kDa Rad1 protein may be directly involved in maintaining genomic integrity and shows potential to be used as a genotoxicity biomarker. This is the first time that an isoform of Rad1 has shown to be modified in the presence of a damaging agent. Both Rad1 and Hus1 need to be further characterized to determine their usefulness as genotoxicity biomarkers.

DNA is subject to attack by DNA damaging agents from both environmental and endogenous sources. In response to DNA damage, living organisms enhance expression of many related genes to facilitate DNA repair and survival. The SOS response is a well-understood prokaryotic regulatory cascade that controls the expression of more than 30 genes in response to DNA damage. However, in eukaryotic organisms from simple budding yeast to human, such a regulatory network has not been reported.<p>Previous research in our laboratory found that among DNA repair mutants of <i>Saccharomyces cerevisiae</i>, only rad6 and rad18 defective in the post-replication repair pathway significantly affected DNA damage induction of several genes examined. Rad6 and Rad18 form a ubiquitin conjugation-ligase complex and are required for the cellular tolerance to damaged DNA. Since the Rad6-Rad18 complex binds to single-stranded DNA, it may act as a DNA damage sensor required for the activation of DNA damage-induced transcription. We performed microarray analysis and found that the induction of up to 379 genes, including those involved in DNA repair, control of replication and transcription, regulation of the cell cycle and cell metabolism, are compromised in the rad6 and rad18 mutants. Although Rad6/Rad18 monoubiquitinates proliferating cell nuclear antigen (PCNA) following DNA damage to initiate a damage tolerance response, PCNA ubiquitination is not required for DNA damage induction. In budding yeast, cell-cycle checkpoints are involved in the control of DNA damage induction of gene expression through phosphorylation of a protein kinase Rad53 by two pathways represented by Rad24 and Sgs1. The Rad6-Rad18 complex appears to function in the Rad24 pathway and parallel to Sgs1. We further demonstrated that the Rad17 subunit of the 9-1-1 complex is subject to Rad6/Rad18- and DNA damage-dependent mono-ubiquitination and that the Rad17-Lys197 residue with flanking sequences homologous to Lys164 of PCNA is absolutely required for the DNA damage induction by Rad6-Rad18. Hence, by ubiquitinating two DNA clamps, PCNA and 9-1-1, the Rad6-Rad18 complex plays a central role in the cellular response to DNA damage by coordinating translesion synthesis, error-free bypass, homologous recombination, as well as transcriptional regulation, reminiscent of roles of RecA in <i>E. coli</i> cells.<p>Several individual genes have also been examined in this study to elucidate the regulatory mechanisms acting on specific DNA damage-inducible genes. In the microarray analysis, DDI2 and DDI3, two identical genes located in duplicated chromosomal regions, were identified due to the highest induction ratio (122-fold) after MMS treatment. Interestingly, DDI2/DDI3 can only be highly induced by SN2-type alkylating agents. Promoter deletion analysis mapped the putative upstream acting sequence (UASDDI2) responsible for 40% of basal expression and 90% of induced expression by MMS.<p>The CRT10 gene was identified through screening of the yeast deletion library for hydroxyurea (HU) resistance. CRT10 encodes a putative 957 amino acid, 110 kDa protein with a leucine repeat and a WD40 repeat near the N-terminus. Deletion of CRT10 resulted in an enhanced resistance to HU reminiscent of the inactivation of two other ribonucleotide reductase (Rnr) suppressors, CRT1 and SML1, which regulate Rnr activity at transcriptional and translational levels, respectively. Epistasis analysis indicates that CRT10 belongs to the CRT1 pathway but not the SML1 pathway. Indeed, deletion of CRT10 enhanced the survival of the mec1 null mutant and increased basal level and DNA damage-induced expression of RNR2 and RNR3, suggesting that Crt10 regulates RNR genes at the transcriptional level. Furthermore, the dun1 mutation is epistatic to crt10 with respect to both HU sensitivity and RNR gene expression. Interestingly, the expression of CRT10 itself is induced by DNA damaging agents and this induction requires DUN1, suggesting that CRT10 plays a role in cellular response to DNA damage and replication blocks. The CRT10 function appears to be achieved by positive regulation of the CRT1 transcript level, indicating that CRT10 is a component of the regulatory circuit.

Cell-cycle checkpoint proteins help maintain genomic integrity by sensing damaged DNA and initiating DNA repair or apoptosis. Checkpoint protein activation to cell-cycle damaging agents can involve post-translational modifications and these alterations provide a means to determine whether DNA in a cell is damaged or not. Steinmoeller et al. (2009) showed that checkpoint proteins are suitable biomarkers for detecting genotoxins in Oncorhynchus mykiss (rainbow trout). In this project, two evolutionarily conserved checkpoint proteins, Rad1 and Hus1, have been cloned from rainbow trout and antibodies against these proteins were developed. This is the first time that either Rad1 or Hus1 has been characterized in rainbow trout. For rtRad1, it was determined that the open-reading frame was 840bp, which encodes 279aa with a predicted protein size of 31kDa. The rtRad1 amino-acid sequence is highly conserved and contains conserved exonuclease and leucine zipper domains. RT-PCR was used to identify alternatively spliced variants of rtRad1 and it appears that these variants encode different sized Rad1 proteins that are tissue and cell-line specific. A Rad1 splice variant that encodes an 18kDa protein appears to be abundant only in heart tissue and in the RTgill-W1 and RTbrain-W1 cell-lines. A genotoxicity study was completed where RTgill-W1 and RTbrain-W1 cells were treated with bleomycin, which induces double-stranded DNA breaks. In RTgill-W1, levels of an 18kDa Rad1 protein increased in a dose-dependent manner while in RTbrain-W1 the Rad1 levels remained the same. It appears that this 18kDa Rad1 protein may be directly involved in maintaining genomic integrity and shows potential to be used as a genotoxicity biomarker. This is the first time that an isoform of Rad1 has shown to be modified in the presence of a damaging agent. Both Rad1 and Hus1 need to be further characterized to determine their usefulness as genotoxicity biomarkers.