As aderências intra-abdominais são complicações relativamente comuns das cirurgias abdominais dos equinos após uma intervenção cirúrgica, podendo resultar em processos obstrutivos, dor abdominal recorrente e vôlvulos. Os avanços técnicos e farmacológicos são direcionados a minimizar o trauma e a inflamação peritoneal, aumentar o processo de fibrinólise, promover movimentação intestinal e separar tecidos potencialmente adesiogênicos durante a fase inicial de cicatrização. No presente trabalho foram utilizados 10 equinos, machos, SRD, entre 4 e 15 anos e peso entre 300 e 400kg. Incluíram-se no experimento os animais que não apresentaram alterações no exame físico e laboratorial, prévios. A membrana biológica utilizada era proveniente do banco de membranas biológicas do HOVET da FMVZ-USP. O protocolo anestésico consistiu de sedação com associação de xilazina 10% (0,7mg/kg) e tartarato de butorfanol (0,04mg/kg). A laparotomia foi realizada pelo flanco esquerdo com os animais em posição quadrupedal. No ponto médio de sua exteriorização (flexura pélvica) foi induzida lesão por abrasão em superfície serosa, com dimensão de 7x7 cm. Após a indução das lesões, nos animais do grupo 1 (cinco animais), a área lesionada foi recoberta com membrana biológica. Nos animais do grupo 2 (cinco animais), o segmento intestinal exteriorizado, após a criação das lesões, foi lavado com solução fisiológica e reposicionado na cavidade abdominal. Em ambos os grupos foi realizada a sutura de um ponto simples no centro da lesão utilizando-se fio de nylon 2-0, com o objetivo de referendar a lesão criada. As suturas dos planos musculares e subcutâneo foram realizadas com sutura contínua com fio absorvível Poliglactina 910 n0 2 e n0 00 respectivamente, e a pele foi suturada de maneira contínua utilizando-se nylon n0 0. A avaliação pós-operatória consistiu de avaliação física diária e coleta de material para análise laboratorial (sangue, soro e líquido peritoneal) semanalmente, durante quatro semanas. Decorridos 30 dias após o procedimento cirúrgico, os animais foram submetidos à nova laparotomia a fim de verificar a presença de aderências intra-abdominais, aspecto macroscópico das lesões e do implante, e realização de biópsia intestinal. A coleta do material para a realização dos exames laboratoriais foi realizada nos momentos: M0- período pré-operatório imediato, M7- 7 dias de pós-operatório, M14- 14 dias de pós-operatório, M21- 21 dias de pós-operatório, M28- 28 dias de pós-operatório. A laparotomia realizada em posição mais ventral e seguindo a técnica de grade possibilitou menor trauma cirúrgico, mas esta abordagem ofereceu ao cirurgião menor campo operatório, determinando alguma dificuldade de exteriorização do segmento intestinal. A evolução clínica dos animais foi satisfatória apresentando vantagem quando comparada a citação da literatura. Durante a reavaliação cirúrgica verificou-se presença de tecido cicatricial fibroso entre os planos musculares que determinou pequena dificuldade para o acesso da cavidade abdominal. Não foram observadas aderências intra-abdominais em ambos os grupos. O aspecto macroscópico das lesões no grupo experimental caracterizou-se por extensa área de hiperemia com cobertura de tecido seroso; e no grupo controle, verificou-se pequeno tecido cicatricial recobrindo o material de sutura. A realização das biópsias intestinais foi executada com facilidade bem como sua sutura. A avaliação histológica demonstrou em ambos os grupos intensa infiltração eosinofílica, presença de colágeno em fase de maturação além de evidências de intensa neovascularização. A partir da análise dos valores laboratoriais observaram-se diferenças estatísticas significativas nos valores sanguíneos de bilirrubinas, hematócrito, monócito, uréia; e no líquido peritoneal estas diferenças foram manifestadas em proteína, densidade e número de leucócitos segmentados. Conclui-se que a abrasão focal de serosa de cólon maior como único fator promotor de aderências intra-abdominais fibrosas foi insuficiente. Além disso, a utilização de pericárdio equino como proteção de áreas de lesão intestinal, mesmo não tendo sido comprovada sua eficácia na minimização das aderências intra-abdominais, apresenta-se viável e possível, pois não foram observadas diferenças estatisticamente significativas (hematológicas, bioquímicas ou do líquido peritoneal) na comparação entre os grupos, ou alterações que determinasse efeitos deletérios aos animais. / The intra-abdominal adhesions are relatively common complications of abdominal surgery in horses and they can result in obstructive processes, abdominal pain and recurrent volvulus. The technical and pharmacological advances are aimed to minimize trauma and peritoneal inflammation, increase the fibrinolysis process, promote intestinal peristalsis and separate potentially adhesiogenic tissues during the initial phase of healing. In this study were used 10 horses, male, mixed breed, between 4 and 15 years old and weighing between 300 and 400kg. The experiment included animals that showed no changes in physical and laboratorial examination. The biological membrane was from the bank of biological membranes of the HOVET-FMVZ-USP. The anesthetic protocol consisted of sedation with a combination of ketamine 10% (0.7 mg / kg) and butorphanol (0.04 mg / kg). The laparotomy was performed by the left flank and the animal was kept in standing position. At the midpoint of the pelvic flexure was induced the injury by abrasion in serous surface, with a size of 7x7 cm. After the lesion induction, the group 1 (five animals) had the injured area covered with a biological membrane. The group 2 (five animals) had the intestinal segment exteriorized, and after the lesion creation it was washed with saline and repositioned in the abdominal cavity. In both groups was performed suturing a single point in the center of the lesion using 2-0 nylon, in order to endorse the lesion created. The sutures of the muscle layers and subcutaneous tissue were performed with continuous suture with Polyglactin 910 n0 2 and n0 00 respectively, and the skin was sutured continuously using nylon n0 0. The postoperative evaluation consisted of daily physical assessment and collection of material for laboratory analysis (blood, serum and peritoneal fluid) weekly for four weeks. After 30 days, the animals underwent another laparotomy to verify the presence of intra-abdominal adhesions, macroscopic aspect of the lesions and implant, and intestinal biopsy. The collection of material for the completion of laboratory tests was performed at moments M0- pre-operative, M7- 7 days post-operatively, M14- 14 days post-operatively, M21- 21 days post-operatively, M28- 28 days post-operatively. The laparotomy was performed in ventral position following the technique of grid that´s promoted less surgical trauma, but this approach offered to surgeon less surgical field, causing some difficulty for exteriorization of the intestinal segment. The clinical outcome was satisfactory showing advantage when compared literature. During the surgical reassessment verified the presence of fibrous scar tissue between the muscle layers that determined some trouble to access the abdominal cavity. There were no intra-abdominal adhesions in both groups. The macroscopic aspect of lesions in the experimental group was characterized by extensive area of redness covered serous tissue, and the control group there was little scar tissue overlying the suture material. The intestinal biopsies and suture were performed easily. The histological evaluation showed in both groups intense eosinophilic infiltration, presence of collagen in the maturation stage in addition to evidence of intense neovascularization. The laboratory results showed statistically significant differences in blood levels of bilirubin, hematocrit, monocyte, urea, and into peritoneal fluid, these differences were manifested in protein density and leukocytes. Concluded that the focal serosal abrasion of the large colon as the single factor promoting intra-abdominal fibrous adhesions was insufficient. Furthermore, the use of equine pericardium as protecting areas of intestinal injury, even without prove their effectiveness in minimizing intra-abdominal adhesions, it appears feasible and possible, because there were no statistically difference (hematological, biochemical or peritoneal fluid) between the groups, or changes that would determine deleterious effects to the animals.

Obstacles such as DNA damage can block the progression of DNA replication forks. This is a major source of genome instability that can lead to cell transformation or death. The budding yeast MMS1 and MMS22 genes were identified in a screen for mutants that were hypersensitive to DNA alkylation that blocks replisome progression. I set out to investigate the cellular roles of these genes and found that cells lacking MMS1 or MMS22 are hypersensitive to a wide variety of genotoxins that stall or block replication forks, and are severely defective in their ability to recover from DNA alkylation damage. Homologous recombination (HR) is an important mechanism for the rescue of stalled or blocked replication forks and for the repair of double-strand breaks (DSBs). Strikingly, MMS1 and MMS22 are required for HR induced by replication stress but not by DSBs, and the underlying mechanisms were explored.I next identified the uncharacterized protein C6ORF167 (MMS22L) as a putative human Mms22 orthologue. MMS22L interacts with NF?BIL2/TONSL, the histone chaperone ASF1 and subunits of the MCM replicative helicase. MMS22L colocalizes with TONSL at perturbed replication forks and at sites of DNA damage. MMS22L and TONSL are important for the repair of collapsed replication forks as depletion of MMS22L or TONSL from human cells causes DNA damage during S–phase and hypersensitivity to agents that cause fork collapse. These defects are consistent with the observations that MMS22L and TONSL are required for the efficient loading of the RAD51 recombinase onto resected DNA ends and for efficient HR. These data indicate that MMS22L and TONSL are novel regulators of genome stability that enable efficient HR.

As aderências intra-abdominais são complicações relativamente comuns das cirurgias abdominais dos equinos após uma intervenção cirúrgica, podendo resultar em processos obstrutivos, dor abdominal recorrente e vôlvulos. Os avanços técnicos e farmacológicos são direcionados a minimizar o trauma e a inflamação peritoneal, aumentar o processo de fibrinólise, promover movimentação intestinal e separar tecidos potencialmente adesiogênicos durante a fase inicial de cicatrização. No presente trabalho foram utilizados 10 equinos, machos, SRD, entre 4 e 15 anos e peso entre 300 e 400kg. Incluíram-se no experimento os animais que não apresentaram alterações no exame físico e laboratorial, prévios. A membrana biológica utilizada era proveniente do banco de membranas biológicas do HOVET da FMVZ-USP. O protocolo anestésico consistiu de sedação com associação de xilazina 10% (0,7mg/kg) e tartarato de butorfanol (0,04mg/kg). A laparotomia foi realizada pelo flanco esquerdo com os animais em posição quadrupedal. No ponto médio de sua exteriorização (flexura pélvica) foi induzida lesão por abrasão em superfície serosa, com dimensão de 7x7 cm. Após a indução das lesões, nos animais do grupo 1 (cinco animais), a área lesionada foi recoberta com membrana biológica. Nos animais do grupo 2 (cinco animais), o segmento intestinal exteriorizado, após a criação das lesões, foi lavado com solução fisiológica e reposicionado na cavidade abdominal. Em ambos os grupos foi realizada a sutura de um ponto simples no centro da lesão utilizando-se fio de nylon 2-0, com o objetivo de referendar a lesão criada. As suturas dos planos musculares e subcutâneo foram realizadas com sutura contínua com fio absorvível Poliglactina 910 n0 2 e n0 00 respectivamente, e a pele foi suturada de maneira contínua utilizando-se nylon n0 0. A avaliação pós-operatória consistiu de avaliação física diária e coleta de material para análise laboratorial (sangue, soro e líquido peritoneal) semanalmente, durante quatro semanas. Decorridos 30 dias após o procedimento cirúrgico, os animais foram submetidos à nova laparotomia a fim de verificar a presença de aderências intra-abdominais, aspecto macroscópico das lesões e do implante, e realização de biópsia intestinal. A coleta do material para a realização dos exames laboratoriais foi realizada nos momentos: M0- período pré-operatório imediato, M7- 7 dias de pós-operatório, M14- 14 dias de pós-operatório, M21- 21 dias de pós-operatório, M28- 28 dias de pós-operatório. A laparotomia realizada em posição mais ventral e seguindo a técnica de grade possibilitou menor trauma cirúrgico, mas esta abordagem ofereceu ao cirurgião menor campo operatório, determinando alguma dificuldade de exteriorização do segmento intestinal. A evolução clínica dos animais foi satisfatória apresentando vantagem quando comparada a citação da literatura. Durante a reavaliação cirúrgica verificou-se presença de tecido cicatricial fibroso entre os planos musculares que determinou pequena dificuldade para o acesso da cavidade abdominal. Não foram observadas aderências intra-abdominais em ambos os grupos. O aspecto macroscópico das lesões no grupo experimental caracterizou-se por extensa área de hiperemia com cobertura de tecido seroso; e no grupo controle, verificou-se pequeno tecido cicatricial recobrindo o material de sutura. A realização das biópsias intestinais foi executada com facilidade bem como sua sutura. A avaliação histológica demonstrou em ambos os grupos intensa infiltração eosinofílica, presença de colágeno em fase de maturação além de evidências de intensa neovascularização. A partir da análise dos valores laboratoriais observaram-se diferenças estatísticas significativas nos valores sanguíneos de bilirrubinas, hematócrito, monócito, uréia; e no líquido peritoneal estas diferenças foram manifestadas em proteína, densidade e número de leucócitos segmentados. Conclui-se que a abrasão focal de serosa de cólon maior como único fator promotor de aderências intra-abdominais fibrosas foi insuficiente. Além disso, a utilização de pericárdio equino como proteção de áreas de lesão intestinal, mesmo não tendo sido comprovada sua eficácia na minimização das aderências intra-abdominais, apresenta-se viável e possível, pois não foram observadas diferenças estatisticamente significativas (hematológicas, bioquímicas ou do líquido peritoneal) na comparação entre os grupos, ou alterações que determinasse efeitos deletérios aos animais. / The intra-abdominal adhesions are relatively common complications of abdominal surgery in horses and they can result in obstructive processes, abdominal pain and recurrent volvulus. The technical and pharmacological advances are aimed to minimize trauma and peritoneal inflammation, increase the fibrinolysis process, promote intestinal peristalsis and separate potentially adhesiogenic tissues during the initial phase of healing. In this study were used 10 horses, male, mixed breed, between 4 and 15 years old and weighing between 300 and 400kg. The experiment included animals that showed no changes in physical and laboratorial examination. The biological membrane was from the bank of biological membranes of the HOVET-FMVZ-USP. The anesthetic protocol consisted of sedation with a combination of ketamine 10% (0.7 mg / kg) and butorphanol (0.04 mg / kg). The laparotomy was performed by the left flank and the animal was kept in standing position. At the midpoint of the pelvic flexure was induced the injury by abrasion in serous surface, with a size of 7x7 cm. After the lesion induction, the group 1 (five animals) had the injured area covered with a biological membrane. The group 2 (five animals) had the intestinal segment exteriorized, and after the lesion creation it was washed with saline and repositioned in the abdominal cavity. In both groups was performed suturing a single point in the center of the lesion using 2-0 nylon, in order to endorse the lesion created. The sutures of the muscle layers and subcutaneous tissue were performed with continuous suture with Polyglactin 910 n0 2 and n0 00 respectively, and the skin was sutured continuously using nylon n0 0. The postoperative evaluation consisted of daily physical assessment and collection of material for laboratory analysis (blood, serum and peritoneal fluid) weekly for four weeks. After 30 days, the animals underwent another laparotomy to verify the presence of intra-abdominal adhesions, macroscopic aspect of the lesions and implant, and intestinal biopsy. The collection of material for the completion of laboratory tests was performed at moments M0- pre-operative, M7- 7 days post-operatively, M14- 14 days post-operatively, M21- 21 days post-operatively, M28- 28 days post-operatively. The laparotomy was performed in ventral position following the technique of grid that´s promoted less surgical trauma, but this approach offered to surgeon less surgical field, causing some difficulty for exteriorization of the intestinal segment. The clinical outcome was satisfactory showing advantage when compared literature. During the surgical reassessment verified the presence of fibrous scar tissue between the muscle layers that determined some trouble to access the abdominal cavity. There were no intra-abdominal adhesions in both groups. The macroscopic aspect of lesions in the experimental group was characterized by extensive area of redness covered serous tissue, and the control group there was little scar tissue overlying the suture material. The intestinal biopsies and suture were performed easily. The histological evaluation showed in both groups intense eosinophilic infiltration, presence of collagen in the maturation stage in addition to evidence of intense neovascularization. The laboratory results showed statistically significant differences in blood levels of bilirubin, hematocrit, monocyte, urea, and into peritoneal fluid, these differences were manifested in protein density and leukocytes. Concluded that the focal serosal abrasion of the large colon as the single factor promoting intra-abdominal fibrous adhesions was insufficient. Furthermore, the use of equine pericardium as protecting areas of intestinal injury, even without prove their effectiveness in minimizing intra-abdominal adhesions, it appears feasible and possible, because there were no statistically difference (hematological, biochemical or peritoneal fluid) between the groups, or changes that would determine deleterious effects to the animals.

DNA double strand break (DSB) repair is critical to maintain genomic integrity and cell viability. DSBs can occur during the course of cell cycle during replication or transcription, or by exogenous agents such as chemicals or ionizing radiation. For my thesis, I studied homologous recombination (HR), which has two sub-pathways: Homology Directed Repair (HDR) and Single Strand Annealing (SSA). HDR involves strand invasion of a homologous template to prime DNA synthesis; SSA involves annealing of homologous segments flanking a DSB. Background data showed that depletion of XAB2 protein by RNA interference reduced both HDR and SSA events. XAB2 protein contains 15 tetratricopeptide repeat (TPR) motifs, which likely enable protein-protein interactions. While XAB2 is speculated to have a role in transcription coupled repair and pre-mRNA splicing, its role in HR pathway is uncertain. The overall hypothesis for my thesis is that XAB2 mediates a specific step of HR (5’-3’ end resection), and the TPR motifs present in XAB2 enable the protein to function in a complex during HR. By using an end resection assay and cell biology analysis, I found that XAB2 is essential for 5’ – 3’ end resection, an intermediate step common to both HDR and SSA pathways. With a functional complementation assay I developed, I have shown that specific TPR regions are critical for XAB2 functions in HR. Overall, my research demonstrates that XAB2 protein has a key role in the 5’-3’ end resection step of HR, and its function in HR requires specific sets of its TPR regions.

Homologous DNA recombination and rearrangement has been modeled with a class of four-regular rigid vertex graphs called assembly graphs which can also be represented by double occurrence words. Various invariants have been suggested for these graphs, some based on the structure of the graphs, and some biologically motivated. In this thesis we use a novel method of data analysis based on a technique known as partial-clustering analysis and an algorithm known as Mapper to examine the relationships between these invariants. We introduce some of the basic machinery of topological data analysis, including the construction of simplicial complexes on a data set, clustering analysis, and the workings of the Mapper algorithm. We define assembly graphs and three specific invariants of these graphs: assembly number, nesting index, and genus range. We apply Mapper to the set of all assembly graphs up to 6 vertices and compare relationships between these three properties. We make several observations based upon the results of the analysis we obtained. We conclude with some suggestions for further research based upon our findings.

Thesis (Ph. D.)--Case Western Reserve University, 2004. / [School of Medicine] Department of Pathology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

P[Kappa]a is not responsible for the improved activity. Hence, stabilization of an enediolate intermediate may be important for catalysis. In the second part of this work, the Chloroflexus aurantiacus J-10-fl heterohexameric 4-OT tautomerase was employed in random and rational directed evolution studies to introduce a CaaD activity. Genetic selection and a high throughput screening assay were used to identify mutants. Genetic selection was unsuccessful due to plasmid instability in the host strain. A small mutant library in the screening assay precluded the identification of any mutants with CaaD activity. Finally, rational design using structure-function relationships was investigated and a single mutant was discovered for hh4-OT that incorporated CaaD activity into the enzyme, the [alpha]L9R hh4-OT, this mutant has been characterized kinetically and the evolutionary implications for the tautomerase superfamily are described. / text

Repair of DNA double-strand breaks is critical to the maintenance of genomic stability, and failure to repair these DNA lesions can cause loss of chromosome telomeric regions, complex translocations, or cell death. In humans this can lead to severe developmental abnormalities and cancer. A central pathway for double-strand break repair is homologous recombination (HR), a mechanism that operates during the S and G2 phases of the cell cycle and primarily utilizes the replicated sister chromatid as a template for repair. Most knowledge of HR is derived from work carried out in prokaryotic and eukaryotic model organisms. To probe the HR pathway in human cells, we performed a genome-wide siRNA-based screen; and through this screen, we uncovered cellular functions required for HR and identified proteins that localize to sites of DNA damage. Among positive regulators of HR, we identified networks of pre-mRNA-processing factors and canonical DNA damage response effectors. Within the former, we found RBMX, a heterogeneous nuclear ribonucleoprotein (hnRNP) that associates with the spliceosome, binds RNA, and influences alternative splicing. We found that RBMX is required for cellular resistance to genotoxic stress, accumulates at sites of DNA damage in a poly(ADP-ribose) polymerase 1-dependent manner and through multiple domains, and promotes HR by facilitating proper BRCA2 expression. Screen data also revealed that the mammalian recombinase RAD51 is commonly off-targeted by siRNAs, presenting a cautionary note to those studying HR with RNAi and highlighting the vulnerability of RNAi screens to off-target effects in general. Candidate validation through secondary screening with independent reagents successfully circumvented the effects of off-targeting and set a new standard for reagent redundancy in RNAi screens.

Homologous recombination (HR) is a faithful mechanism for the repair of double-stranded DNA breaks (DSBs) and plays a critical role in maintaining the integrity of genomic DNA. The product of the Breast Cancer Susceptibility 2 (BRCA2) gene functions as a recombination mediator in HR-directed repair of DSBs. BRCA2 interacts directly with RAD51, the central recombinase of HR, through highly conserved repetitive motifs of 30-40 amino acids, named BRC repeats, and regulates the formation of the RAD51-ssDNA nucleoprotein filament. There is significant variability in the number of BRC repeats among taxa. However, all mammalian BRCA2 orthologs have eight BRC repeats, which display different characteristics in in vitro studies of RAD51-ssDNA nucleoprotein filament. To test the importance of the number of BRC repeats and to evaluate the role of individual BRC repeats in HR, BRCA2 variants bearing different combinations of BRC repeats were generated using BAC-recombineering, expressed in murine hybridoma cells, and assayed for the ability to stimulate HR using a gene targeting assay. The BRCA2 variant bearing BRC repeats 1 to 4 decreased the efficiency of HR and increased the level of Rad51 protein, whereas the BRCA2 variant bearing BRC repeats 5 to 8 significantly stimulated HR, but had no effect on the level of Rad51. These results supported the hypothesis that BRC repeats are not functionally equivalent, but rather have different, perhaps reinforcing functions in HR. / Canadian Institutes of Health Research

Oxidative stress and histone deacetylase (HDAC) inhibition has been implicated as potential mechanisms in valproic acid (VPA) teratogenicity. Reactive oxygen species (ROS) can target DNA to cause oxidative DNA damage and DNA double strand breaks (DSBs) which can be repaired through homologous recombination (HR). HR is not an error free process and can result in detrimental genetic changes. In this present study we evaluated the role of HDAC inhibition in VPA-initiated HR. HDAC inhibition may indirectly alter repair activity as a result of increased expression of genes involved in HR or indirectly by causing DNA damage which initiates repair. The first objective was to investigate the ability of VPA to cause HDAC inhibition in the Chinese hamster ovary (CHO) 33 cell line. Using immunblotting, an increase in acetylated histone H3 and H4 protein levels was observed throughout 24 hr exposure to 5 mM VPA. Secondly, to investigate whether VPA affects the activity of DNA DSB repair, CHO 33 cells were transfected with either the endonuclease I-SceI plasmid to induce a site specific DSB or the empty plasmid, pGem. However, no increase in the difference in HR between VPA and media exposed I-Sce1 transfected cells compared to cells transfected with pGem was observed, which suggests that VPA does not affect DNA repair activity. Thirdly, to determine if VPA-induced HDAC inhibition increases susceptibility to DNA damage, immunocytochemistry revealed an increase in the number of γ-H2AX foci throughout 24 hr exposure to 5 mM VPA. To determine if oxidative stress may play a role in mediating VPA-induced DNA DSBs, another recombination study was carried out in which cells were pretreated with 400 U/ml of PEG-catalase prior to VPA treatment. The observed protective effect of PEG-catalase against VPA-induced HR and the generation of intracellular ROS by VPA suggest ROS may also play a role in VPA-initiated HR. However, in our DNA oxidation study, no increase in the oxidized nucleosides, 8-hydroxy-2'-deoxyguanosine and 5-hydroxycytosine was observed after VPA treatment. These studies suggest that HDAC inhibition and ROS signalling may play other roles in DNA maintenance and cell cycle arrest in initiating DNA DSBs and HR repair. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2009-08-12 14:27:16.327