Eukaryotic cells repair DNA double-strand breaks (DSBs) through both non-homologous and homologous recombination pathways. The initiation of homologous recombination requires the generation of 3' overhangs, which are essential for the formation of Rad51 protein-DNA filaments that catalyze subsequent steps of strand invasion. Experiments in budding yeast show that resection of the 5' strand at a DSB is delayed in strains lacking any components of the Mre11/Rad50/Xrs2 (MRX) complex¹ . In meiosis, a specific class of hypomorphic mutants of mre11 and rad50 (Rad50S) are completely deficient in 5' resection and leave Spo11 covalently attached to the 5' strands of DNA breaks². Similar to mre11S and rad50S mutants, sae2 deletion strains fail to resect 5' strands at meiotic DSBs and accumulate covalent Spo11 adducts³;⁴. In addition, Sae2 and MRX were also found to function cooperatively to process hairpin-capped DNA ends in vivo in yeast. sae2 and mrx null strains show a severe defect in processing these structures and accumulate hairpin-capped DNA ends⁵;⁶. The Longhese laboratory has also shown that Sae2 deletion strains show a delay in 5' strand resection, similar to rad50S strains⁷. Recently, Bettina Lengsfeld in our laboratory demonstrated that Sae2 itself possesses nuclease activity and that MRX and Sae2 act cooperatively to cleave single-stranded DNA adjacent to DNA hairpin structures⁸. In vitro characterization of Sae2 showed that the central and N-terminal domains are required for MRX-independent nuclease activity and that the C-terminus is required for cooperative activities with MRX. Sae2 also acts independently of MRX as a 5' flap endonuclease on branched structures in vitro. Our studies investigate whether MRX, Sae2, and Exo1 function cooperatively in DNA resection using recombinant, purified proteins in vitro. We developed assays utilizing strand-specific Southern blot analysis to visualize DNA end processing of model DNA substrates using recombinant proteins in vitro. Our results demonstrate that MRX and Sae2 cooperatively resect the 5' end of a DNA duplex together with the Exo1 enzyme, supporting a role for these factors in the early stages of homologous recombination and repair. / text

Dieses Manuskript entstand aus Vorlesungen über Geodätische Berechnungen an der Hochschule für Technik und Wirtschaft Dresden. Da diese Lehrveranstaltung im ersten oder zweiten Semester stattfindet, werden noch keine Methoden der höheren Mathematik benutzt. Das Themenspektrum beschränkt sich deshalb weitgehend auf elementare Berechnungen in der Ebene. Nur im Kapitel 7 kommen einige Methoden der Vektorrechnung zum Einsatz.

No description available.

Background: The goal of the study was to evaluate the masticatory efficacy in patients who had been provided with resection prostheses after tumor removal in the maxillary/ mandibular region. These patients complained of impairment of masticatory function. Patients and Methods: 3 groups of patients were compared under clinical-experimental conditions. A uniform chewing material was masticated by the participants under standardized conditions. A sieving procedure was used to evaluate the masticatory efficacy. Analysis of the particle sizes and particle masses obtained was performed with the aid of computers. Results: The results showed that the masticatory efficacy of the patients with resection prostheses was the lowest of the 3 groups compared. The number of existing supporting zones and the location of the defect were found to be important influencing factors. Recording of the dietary habits of all patients was performed using a standardized dietary questionnaire. These data were analyzed using the corresponding software of the German Nutrition Society. With regard to the patients with resection prostheses, it was revealed that they often switched to food that did not require mastication. Conclusions: A nutritional guideline for patients with resection prostheses was developed, which is available for downloading free of charge on the Internet. / Hintergrund: Ziel der Untersuchung war die Prüfung der Kaueffektivität bei Patienten, die mit Resektionsprothesen nach Tumorentfernung im Kieferbereich versorgt worden waren. Diese Patienten klagten über eine Einschränkung der mastikatorischen Funktion. Patienten und Methoden: Unter klinisch xperimentellen Bedingungen erfolgte der Vergleich von 3 Patientengruppen. Unter standardisierten Bedingungen zerkleinerten die Patienten einheitliches Kaugut. Zur Bewertung der Kaueffektivität wurde ein Siebverfahren eingesetzt. Die Auswertung der ermittelten Partikelgrößen und Partikelmassen erfolgte computergestützt. Ergebnisse: Die Ergebnisse zeigten, dass im Vergleich der 3 Gruppen die Kaueffektivität der Patienten mit Resektionsprothesen am geringsten war. Die Zahl der vorhandenen Stützzonen des Restgebisses und die Defektlokalisation wurden als bedeutsame Einflussfaktoren ermittelt. Die Erfassung der Ernährungsgewohnheiten aller Patienten erfolgte mittels eines standardisierten Ernährungsfragebogens. Diese Daten wurden mit der zugehörigen Software der Deutschen Gesellschaft für Ernährung ausgewertet. Bei den Patienten mit Resektionsprothesen zeigte sich, dass diese auf Nahrungsmittel ausweichen, die kein Kauen erfordern. Schlussfolgerungen: Es wurde eine Ernährungsrichtlinie für Patienten mit Resektionsprothesen abgeleitet, die zum kostenfreien Herunterladen im Internet zur Verfügung steht. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

This is a prospective, non-randomized pilot study comparing the cost-utility of hepatic resection, radiofrequency ablation (RFA), systemic chemotherapy, and symptom control only for the treatment of colorectal liver metastases. Seven patients underwent hepatic resection, 7 underwent RFA, 20 received chemotherapy, and 6 received symptom control alone. Liver resection provided an average of 2.51 QALY’s compared to 1.99 QALY’s for RFA, and 1.18 QALY’s for chemotherapy, and 0.82 QALY’s for symptom control alone. The costs were $20,122, $ 15,845, $15,069, and $3,899, respectively. The cost-utilities of liver resection and RFA were similar at $8,027 and $7,965 per QALY, respectively, although patients receiving RFA generally had more advanced disease. The cost-utility of chemotherapy was $12,751/QALY and the cost-utility of symptom control alone was $4,788/QALY. RFA is still a relatively new. However, if long-term survival proves promising, it may prove to be a viable alternative to liver resection.

Background: The complexity of neurosurgical interventions demands innovative training solutions and standardized evaluation methods that in recent times have been the object of increased research interest. The objective is to establish an education curriculum on a phantom‑based training system incorporating theoretical and practical components for important aspects of brain tumor surgery. Methods: Training covers surgical planning of the optimal access path based on real patient data, setup of the navigation system including phantom registration and navigated craniotomy with real instruments. Nine residents from different education levels carried out three simulations on different data sets with varying tumor locations. Trainings were evaluated by a specialist using a uniform score system assessing tumor identification, registration accuracy, injured structures, planning and execution accuracy, tumor accessibility and required time. Results: Average scores improved from 16.9 to 20.4 between first and third training. Average time to craniotomy improved from 28.97 to 21.07 min, average time to suture improved from 37.83 to 27.47 min. Significant correlations were found between time to craniotomy and number of training (P < 0.05), between time to suture and number of training (P < 0.05) as well as between score and number of training (P < 0.01). Conclusion: The training system is evaluated to be a suitable training tool for residents to become familiar with the complex procedures of autonomous neurosurgical planning and conducting of craniotomies in tumor surgeries. Becoming more confident is supposed to result in less error‑prone and faster operation procedures and thus is a benefit for both physicians and patients.

This is a prospective, non-randomized pilot study comparing the cost-utility of hepatic resection, radiofrequency ablation (RFA), systemic chemotherapy, and symptom control only for the treatment of colorectal liver metastases. Seven patients underwent hepatic resection, 7 underwent RFA, 20 received chemotherapy, and 6 received symptom control alone. Liver resection provided an average of 2.51 QALY’s compared to 1.99 QALY’s for RFA, and 1.18 QALY’s for chemotherapy, and 0.82 QALY’s for symptom control alone. The costs were $20,122, $ 15,845, $15,069, and $3,899, respectively. The cost-utilities of liver resection and RFA were similar at $8,027 and $7,965 per QALY, respectively, although patients receiving RFA generally had more advanced disease. The cost-utility of chemotherapy was $12,751/QALY and the cost-utility of symptom control alone was $4,788/QALY. RFA is still a relatively new. However, if long-term survival proves promising, it may prove to be a viable alternative to liver resection.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 112-123).

<p>Every living cell faces a multitude of DNA threats in its lifetime because damage to DNA is intrinsic to life itself. A double-strand break (DSB) is the most cytotoxic type of DNA damage and is a potent inducer of chromosomal aberrations. Defects in DSB repair are a major driver of tumorigenesis and are associated with numerous developmental, neurological and immunological disorders. To counteract the deleterious effects of DSBs, organisms have evolved a homologous repair (HR) mechanism that is highly precise. The key to its error-free nature lies in its use of a homologous template in restoring the DSB and its preferential occurrence during late S and G2 phase of the cell cycle when identical sister chromatids are available as templates for repair. However, HR can also engage homologous chromosomes and ectopic substrates that share homology, resulting in mitotic loss-of-heterozygosity (LOH) and unwanted chromosomal aberrations. In this case, understanding of the underlying mechanisms and molecular factors that influence accurate sequence transfer and exchange between two homologous substrates becomes crucial. </p><p>The focus of this dissertation is examination of the genetic factors and molecular processes occurring at early intermediate steps (DNA end resection and DNA synthesis) of mitotic recombination in Saccharomyces cerevisiae. To model DSB repair, we established a unique plasmid-based assay with a small 8-base pair (bp) gap in the middle of an 800-bp plasmid substrate. To delineate the molecular structures of strand exchange intermediates during HR, we used a 2% diverged plasmid substrate relative to a chromosomal repair template to generate mismatch-containing heteroduplex DNA (hetDNA) intermediates. The assay was performed in a mismatch repair (MMR)-defective background allowing hetDNA to persist and to segregate into daughter cells at the next round of replication. Unexpectedly, even when MMR was inactivated, sequence analysis of the recombinants revealed patches of gene conversion and restoration reflecting mismatch correction within hetDNA tracts. We showed that, in this system, MMR and nucleotide excision repair (NER) correct mismatches via two different mechanisms. While mispairing of nucleotides triggers MMR, NER is recruited by the subtle 6-methyladenine mark on the plasmid substrate, leading to coincident correction of mismatches. The methylation marks on the plasmid were acquired from the bacterial host’s native restriction-modification system during plasmid propagation. </p><p>Formation of hetDNA occurs when a plasmid substrate engages the chromosomal template for repair, forming a D-loop intermediate. D-loop extension requires DNA synthesis by DNA polymerase/s. Translesion synthesis (TLS) polymerases have been implicated in HR in both chicken DT40 cells and fruit fly, but not in yeast. This class of polymerases is known for its low fidelity due to a lack of exonuclease domain and is commonly used for lesion bypass and in extending ends with mismatches. We reported for the first time a requirement of Polζ-Rev1 and Polη (TLS polymerases in S. cerevisiae) for completing gap repair. Moreover, gap-repair efficiency suggested that these two polymerases function independently. We concluded that TLS polymerases are involved in either extending the invading 3’ end and/or in the gap-filling process that completes recombination. </p><p>DNA resection of a DSB serves as a primary step to generate a 3’ single-stranded DNA (ssDNA) for subsequent homologous template invasion, but this process has mostly been studied in the absence of a repair template or when downstream HR steps are disabled. To analyze the individual contributions of identified nucleases to DSB resection in the context of repair, we established a chromosomal assay; the substrate size was increased to 4 kilobases (kb) and 85 SNPs were present at ~50 bp intervals. In this chromosomal assay, resection and DNA synthesis influence the length of hetDNA tracts in the final recombinants, allowing these two steps to be analyzed. We specifically focused on synthesis-dependent strand annealing (SDSA) events, where hetDNA reflects DNA synthesis and extent of resection. Our main conclusions are as follows. DNA end resection on the annealing end of NCO products generated by SDSA is not as extensive as one might expect from resection measured in single-strand annealing (SSA) assays. In addition, although the two long-range resection pathways (Sgs1-Dna2 and Exo1) can support recombination in a redundant manner, hetDNA was significantly reduced upon loss of either. End processing of DSBs is predominantly 5’ to 3’, but we also observed loss of sequences (greater than 8 nt but less than 40 nt) at the 3’ termini. We have tested and ruled out the involvement of Mre11 and Polε proofreading activity. Lastly, Pol32 functions as a subunit of Polδ to promote extensive repair synthesis during SDSA. hetDNA tract lengths were significantly shorter in the absence of the Pol32 subunit of Polδ, providing direct evidence that Polδ extends the invading end during HR. Together, this work advances our understanding of how resection nucleases and DNA polymerase/s function to regulate mitotic recombination outcome and influence the molecular patterns of NCOs.</p> / Dissertation

Racional: O carcinoma hepatocelular é uma neoplasia maligna agressiva com elevada morbidade e mortalidade. Objetivo: Revisão da literatura sobre o diagnóstico e o manejo do carcinoma hepatocelular nos vários estágios da doença. Método: Revisão da literatura utilizando a base Medline/PubMed e literatura adicional. Resultados: O carcinoma hepatocelular é geralmente complicação da cirrose hepática. As hepatites virais crônicas B e C também são fatores de risco para o surgimento do carcinoma hepatocelular. Quando associado à cirrose hepática, o carcinoma hepatocelular geralmente surge a partir da evolução de um nódulo regenerativo hepatocitário que sofre degeneração maligna. O diagnóstico é efetuado através de tomografia computadorizada de abdome com contraste endovenoso (efeito wash in e wash out), e a ressonância magnética pode auxiliar nos casos que não possam ser definidos pela tomografia computadorizada. O único tratamento potencialmente curativo para o carcinoma hepatocelular é a ressecção do tumor, seja ela realizada através de hepatectomia parcial ou de transplante. Infelizmente, apenas cerca de 15% dos carcinomas hepatocelulares são passíveis de tratamento cirúrgico. Pacientes portadores de cirrose hepática estágio Child B e C não devem ser submetidos à ressecção hepática parcial. Para esses pacientes, as opções terapêuticas curativas restringem-se ao transplante de fígado, desde que selecionáveis para esse procedimento, o que na maioria dos países dá-se através dos Critérios de Milão (lesão única com até 5 cm de diâmetro ou até três lesões de até 3 cm de diâmetro). A sobrevida em 5 anos para pacientes transplantados para o carcinoma hepatocelular pode alcançar 70% Conclusão: Quando diagnosticado em seus estágios iniciais, o carcinoma hepatocelular é potencialmente curável. O conhecimento das estratégias de 17 diagnóstico e tratamento do carcinoma hepatocelular a fim propiciam sua identificação precoce e a indicação de tratamento apropriado. / Introduction: Hepatocellular carcinoma is an aggressive malignant tumor with high lethality. Aim: A literature review on diagnosis and management of hepatocellular carcinoma was performed. Methods: Literature review utilizing databases Medline/PubMed. Results: Hepatocellular carcinoma is a common complication of hepatic cirrhosis. Chronic viral hepatitis B and C also constitute as risk factors for development of hepatocellular carcinoma. In patients with cirrhosis, hepatocelular carcinoma usually develops from a malignant transformation of a dysplastic regenerative nodule. Diagnosis is confirmed through computed tomography scan with intravenous contrast (wash in and wash out effect), and magnetic resonance may be helpful in some instances. Curative treatment for hepatocellular carcinoma may be performed through partial liver resection or liver transplantation. Only 15% of all hepatocellular carcinomas are localized and amenable to operative treatment. Patients with Child C liver cirrhosis are not amenable to partial liver resections. The only curative treatment for hepatocellular carcinomas in patients with Child B or C cirrhosis is liver transplantation. In most countries, only patients with hepatocellular carcinoma under Milan Criteria (single tumor with up to 5 cm diameter or up to three nodules with a maximum diameter of 3 cm) are considered candidates for liver transplant. Five-year survival following liver transplantation for hepatocellular carcinoma may reach 70%. Conclusion: Hepatocellular carcinoma is a potentially curable neoplasm if discovered in its initial stages. Clinicians and surgeons should be familiar with strategies for early diagnosis and treatment of hepatocellular carcinoma as a way to decrease mortality associated with this malignant neoplasm.