Thesis (Ph.D)--Boston University / Characteristics of deoxyribonuclease II from calf thymus and spleen were described and verified by the following experimental procedures. Nuclei from calf thymus were prepared free of cytoplasm both by washing homogenized tissue in sucrose solutions, and by density gradient centrifugation in organic solvents. The action of the enzyme from nuclei and from a thymus homogenate was found to be similar when measured by light scattering and hyperchromic shift. Deoxyribonuclease II from calf spleen was purified by ammonium sulfate precipitation. Further purification of the enzyme on column chromatography resulted in fractions similar to those reported in the literature. The effect of sodium sulfate on the activity of splenic DNase II, purified by ammonium sulfate precipitation, was measured by the production of acid-soluble nucleotides from deoxyribonucleic acid during hydrolysis. The effect of the sulfate ion was also measured by viscometry, as was the effect of magnesium chloride, manganese chloride, sodium citrate and iodoacetic acid. The substances were all found to be inhibitory, which is in agreement with the results reported in the literature. Deoxyribonucleases active at an acid pH were extracted from representatives of four invertebrate phyla (Echinodermata, Mollusca, Arthropoda and Annelida) and compared with vertebrate deoxyribonuclease II with respect to ionic requirements and pH optima. [TRUNCATED]

Unrepaired, or misrepaired, DNA damage can be carcinogenic or mutagenic; thus functional DNA damage repair pathways are essential for the safeguarding of the genome. SNM1B is a 5' to 3' exonuclease implicated in the repair of damaged DNA, particularly the repair of interstrand crosslinks. Genetic studies have identified SNPs in the SNM1B gene as related to cancer risk. One of these (rs11552449) encodes a single amino acid change, H61Y. This study shows that WT and H61Y SNM1B have comparable in vitro biochemical and biophysical characteristics. The structures of both WT and H61Y C-terminally truncated SNM1B (Δ-SNM1B) were solved to 2.8 and 3.1 Å, respectively, and reveal similar structural properties. The structure of WT Δ-SNM1B was also reported (to 1.8 Å) with two 2'-deoxy-5'- adenosine monophosphate molecules in the active site. The structure of SNM1B shows an accessible extended active site, which may facilitate the binding of a variety of non-canonical DNA substrates. Accordingly, in vitro, WT and H61Y SNM1B are able to exonucleolytically process a wide range of structurally diverse DNA substrates. By utilising SNM1B depleted cell lines, this study also shows that SNM1B is required for DNA repair in response to treatment with DNA-crosslinking genotoxic agents (including cisplatin and SJG-136). This study also identifies the novel double strand break repair factor, EXD2, as having intrinsic 3' to 5' exonuclease activity. EXD2 was shown to have enzymatic activity on a variety of substrates in vitro, including replication fork intermediates, 'nicked' or 'gapped' DNA duplexes, and RNA based substrates. Together with the cellular data this suggests a role for EXD2 in nucleolytically processing RNA or DNA-based intermediates in damage repair pathways.

Retrotransposons constitute around 40% of the mammalian genome and their aberrant activation can have wide ranging detrimental consequences, both throughout development and into somatic lineages. DNA methylation is one of the major epigenetic mechanisms in mammals, and is essential in repressing retrotransposons throughout mammalian development. Yet during normal mouse embryonic development some cell lineages become extensively DNA hypomethylated and it is not clear how these cells maintain retrotransposon silencing in a globally hypomethylated genomic context. In this thesis I determine that hypomethylation in multiple contexts results in the consistent activation of only one gene in the mouse genome - Tex19.1. Thus if a generic compensatory mechanism for loss of DNA methylation exists in mice, it must function through this gene. Tex19.1-/- mice de-repress retrotransposons in the hypomethylated component of the placenta and in the mouse germline, and have developmental defects in these tissues. In this thesis I examine the mechanism of TEX19.1 mediated genome defence and the developmental consequences upon its removal. I show that TEX19.1 functions in repressing retrotransposons, at least in part, through physically interacting with the transcriptional co-repressor, KAP1. Tex19.1-/- ES cells have reduced levels of KAP1 bound retrotransposon chromatin and reduced levels of the repressive H3K9me3 modification at these loci. Furthermore, these subsets of retrotransposon loci are de-repressed in Tex19.1-/- placentas. Thus, my data indicates that mouse cells respond to hypomethylation by activating expression of Tex19.1, which in turn augments compensatory, repressive histone modifications at retrotransposon sequences, thereby helping developmentally hypomethylated cells to maintain genome stability. I next aimed to further elucidate the role of Tex19.1 in the developing hypomethylated placenta. I determine that Tex19.1-/- placental defects precede intrauterine growth restriction of the embryo and that alterations in mRNA abundance in E12.5 Tex19.1-/- placentas is likely in part due to genic transcriptional changes. De-repression of LINE- 1 is evident in these placentas and elements of the de-repressed subfamily are associated with significantly downregulated genes. If retrotransposon de-repression is contributing to developmental defects by interfering with gene expression remains to be determined, however I identify a further possible mechanism leading to placental developmental defects. I determine that Tex19.1-/- placentas have an increased innate immune response and I propose that this is contributing to the developmental defects observed. Developmental defects and retrotransposon de-repression are also observed in spermatogenesis in Tex19.1-/- testes, the molecular basis for which is unclear. I therefore investigate the possibility that the TEX19.1 interacting partners, the E3 ubiquitin ligase proteins, may be contributing to the phenotypes observed in Tex19.1- /- testes. I show that repression of MMERVK10C in the testes is dependent on UBR2, alongside TEX19.1. Furthermore, I have identified a novel role for the TEX19.1 interacting partner, UBR5, in spermatogenesis, whose roles are distinct from those of TEX19.1. The work carried out during the course of this thesis provides mechanistic insights into TEX19.1 mediated genome defence and highlights the importance of protecting the genome from aberrant retrotransposon expression.

abstract: Deoxyribonucleic acid (DNA) has been treated as excellent building material for nanoscale construction because of its unique structural features. Its ability to self-assemble into predictable and addressable nanostructures distinguishes it from other materials. A large variety of DNA nanostructures have been constructed, providing scaffolds with nanometer precision to organize functional molecules. This dissertation focuses on developing biologically replicating DNA nanostructures to explore their biocompatibility for potential functions in cells, as well as studying the molecular behaviors of DNA origami tiles in higher-order self-assembly for constructing DNA nanostructures with large size and complexity. Presented here are a series of studies towards this goal. First, a single-stranded DNA tetrahedron was constructed and replicated in vivo with high efficiency and fidelity. This study indicated the compatibility between DNA nanostructures and biological systems, and suggested a feasible low-coast method to scale up the preparation of synthetic DNA. Next, the higher-order self-assembly of DNA origami tiles was systematically studied. It was demonstrated that the dimensional aspect ratio of origami tiles as well as the intertile connection design were essential in determining the assembled superstructures. Finally, the effects of DNA hairpin loops on the conformations of origami tiles as well as the higher-order assembled structures were demonstrated. The results would benefit the design and construction of large complex nanostructures. / Dissertation/Thesis / Ph.D. Biochemistry 2012

No description available.

In order to gain a greater understanding of the genetic makeup of the Maltese population, mitochondrial DNA HVR1 and HVR2, and Y-chromosomal and autosomal STRs were amplified in a representative sample of the Maltese population. The results showed that the Maltese have close genetic ties with Sicily and mainland Italy both from a matrilineal and a patrilineal perspective, whilst no conclusive evidence was found for a Phoenician link between the Maltese and the Lebanese population. In order to try and gain an insight into the Maltese population throughout time, a study was conducted on three Maltese archaeological burial places dating from the Neolithic to the Roman period. The study extracted and amplified ancient DNA sequences from these three sites and compared the resulting mtDNA sequences with the modern Maltese population. The results showed that aDNA survives in the Maltese archaeological record, and that some haplotypes found during the Roman period in Malta are also found in the modern day population, whilst other haplotypes present in the archaeological samples are not visible in the modern Maltese population.

ATM is a large and complex gene, identified as the recessive gene mutated in individuals with the childhood syndrome ataxia telangiectasia. While AT patients are rare and the disease has many severe symptoms, including cerebellar degeneration, immunodeficiency and a very high cancer risk, the relatively common carriers of one mutation are clinically normal. However, there is some evidence that these heterozygotes, too, have a high relative risk of cancer, especially breast cancer in women. The cellular radio sensitivity of carriers is intermediate between that of normal individuals and that of AT patients, and this may suggest the cells are predisposed to tumorigenic transformation. ATM is believed to be involved in DNA damage response, but the mechanisms by which it works are not fully understood. In this study, DNA from 412 Scottish women with breast cancers were screened for two ATM mutations, known to be relatively common in Celtic populations. In attempting to estimate the burden in Scotland of breast cancers in AT heterozygotes, it is shown here that even these two most common ATM mutations in the UK account for only a small proportion of all ATM mutations. None of these mutations were found, suggesting that these particular mutations do not confer a predisposition to breast cancer. Also presented are the results of various immunological studies. The ATM protein is shown to be localised to specific structures within the nucleus of a normal cell. It appears to be found in a number of different forms, varying in size and state of glycosylation. It sediments at a relatively low speed, suggesting that it is normally bound in a large protein complex. This work indicates that a large-scale screening study will be required to establish whether or not AT carriers are at an increased risk of cancer, and reveals that the ATM product may be present in more than one form, depending on whether the cell' stimulated to divide.

Tese (doutorado)—Universidade de Brasília, Faculdade de Medicina, 2008. / Submitted by Ruthléa Nascimento (ruthlea@bce.unb.br) on 2008-10-22T15:47:58Z No. of bitstreams: 1 2008_AliceMeloRibeiro.pdf: 765803 bytes, checksum: 64d83181d608218897c1293017932928 (MD5) / Approved for entry into archive by Georgia Fernandes(georgia@bce.unb.br) on 2008-11-17T13:35:28Z (GMT) No. of bitstreams: 1 2008_AliceMeloRibeiro.pdf: 765803 bytes, checksum: 64d83181d608218897c1293017932928 (MD5) / Made available in DSpace on 2008-11-17T13:35:28Z (GMT). No. of bitstreams: 1 2008_AliceMeloRibeiro.pdf: 765803 bytes, checksum: 64d83181d608218897c1293017932928 (MD5) / As proteínas de choque térmico (HSPs) são reconhecidas como importantes moléculas na modulação do sistema imunológico e são altamente conservadas entre os diferentes organismos. A vacina DNA-hsp65 de Mycobacterium leprae demonstrou efeitos profiláticos e imunoterapêuticos contra diversas doenças como, por exemplo, tuberculose, artrite e leishmaniose. Neste trabalho, avaliamos a eficácia e o potencial imunomodulador na imunização e no tratamento de camundongos infectados experimentalmente com o fungo patogênico Paracoccidioides brasiliensis, o agente etiológico da Paracoccidioidomicose, a mais importante micose endêmica na América Latina. A vacina DNA-hsp65 conferiu proteção aos animais contra o P. brasiliensis em ensaios de imunização ou de tratamento como indicado por: redução significativa no pulmonar fúngica carga (UFCs); diminuição da perda da função pulmonar e na presença de colágeno em granulomas (revelada por análises histológicas do tecido pulmonar); reestabelecimento proliferativo das células de baço; perfil de resposta imunológica de padrão Th1 com níveis elevados de IL-12, IFN - g, TNF-α e IGg2a e baixos níveis de IL-4, IL-10 e IgG1. Esses dados, em conjunto, indicam que, em BALB/c, tanto a imunização como o tratamento com a vacina DNA-hsp65 conferem proteção no curso da Paracoccidioidomicose. Nossos resultados abrem novas perspectivas sobre a prevenção e tratamento para outras micoses sistêmicas. _____________________________________________________________________________________ ABSTRACT / Heat shock proteins (HSPs) are recognized as important molecules in the modulation of the immune system and are highly conserved among different organisms. The DNA-hsp65 vaccine from Mycobacterium leprae (M. leprae) has been shown to have prophylactic and immunotherapeutic effects against various diseases, for instance, tuberculosis, arthritis and leishmaniasis. In this work, we evaluated the effectiveness and immunomodulatory potential of the DNA-hsp65 immunization and treatment in model BALB/c mice infected with Paracoccidioides brasiliensis (P. brasiliensis), the etiological agent of Paracoccidioidomycosis, the most important endemic mycosis in Latin America. The DNA-hsp65 vaccine conferred protection against this pathogen for both, the prophylactic and therapeutic assays, as indicated by: a significant reduction in pulmonary fungal burden (CFUs); a decrease in pulmonary function loss and in the presence of collagen in granulomas (revealed by histological analyses of the pulmonary tissue); the reestablishment of spleen cells proliferation; the Th1 pattern immune response with higher levels of IL-12, IFN- γ, TNF- and IGg2a and lower levels of IL-4, IL-10 and IgG1. Together, these findings indicate that, in mice, the immunization and treatment with the DNA-hsp65 vaccine protect mice against Paracoccidioidomycosis. Our results open new perspectives on prevention and treatment of other systemic mycoses.

Cells of ataxia telangiectasia (AT) individuals are hypersensitive to a variety of DNA damaging agents such as ionizing radiation and bleomycin, presumed to be due to an intrinsic defect in repair of DNA damage. The nature of the DNA lesion(s) to which AT cells are abnormally sensitive, and the defect in DNA repair are presently unclear. The major part of this project aimed at investigating the sensitivity of AT cells to DNA double-strand breaks (dsb) generated by restriction endonucleases (RE), thereby verifying the hypothesis that AT cells are deficient in the processing of dsb. AT lymphoblastoid cell lines (AT-PA and AT-KM) used in this study were initially characterized and found to be approximately 3 times more sensitive to ionizing radiation in the induction of micronuclei (Mn) and chromosomal aberrations (CA) compared with a normal lymphoblastoid cell line (N-SW). Other cellular characteristics were observed in AT-PA cells following-irradiation such as normal induction and rejoining of dsb and reduced inhibition of DNA synthesis. By using SLO poration, RE were introduced into the AT and normal cell lines and the yield of CA resulting from RE-induced dsb were subsequently investigated. The frequencies of CA induced by Pvu II were 2 - 4 fold higher in AT-PA than in N-SW cells at both 5 h and 24 h sampling times. The enhanced frequency of CA in AT cells treated with Pvu II was principally a result of an increase of chromatid aberrations, rather than chromosome aberrations at 24 h. higher frequencies of chromatid exchanges appeared in AT-PA than in N-SW cells. The results suggest that AT cells are characterized by a defect in dsb processing that converts a higher number of dsb into CA than in the normal cell line. With respect to the different end-structures of RE-induced dsb, cohesive-ended dsb generated by BamH I and Pst I were found to induce lower frequencies of CA than blunt-ended dsb generated by Pvu II and EcoR V in both the AT cell lines and the normal cell line. The results support the previous observations that cohesive-ended dsb are less clastogenic than blunt-ended dsb (Bryant 1984). Although inducing lower frequencies of CA than Pvu II and EcoR V, BamH I and Pst I induced higher number of CA in both AT-PA and AT-KM cells when compared with N-SW cells, again indicating a defect in processing cohesive-ended dsb exists in AT cells. A potent DNA repair inhibitor, Ara A, was found to potentiate the production of CA by RE in AT and normal cells. The enhancement ratios (by ara A) for CA induced by Pvu II and Pst 1 were higher in N-SW cells than in AT-PA and AT-KM cells. Ara A appeared to have no effect on the frequencies of CA induced by BamH I in any of the cell lines tested. Based on these findings, a mechanism for the rejoining of RE-induced dsb in which DNA repair synthesis may be involved is proposed, and it is postulated that dsb in AT cells are subjected to greater end degradation. Inhibition of DNA synthesis was observed in normal cells after treatment with Pvu II and EcoR V, while EcoR I and BamH I had only minor effect. AT-PA cells were found to be resistant to RE-induced inhibition of DNA synthesis, as in the case of ionizing radiation. This result suggests that RE-induced blunt-ended dsb mimic radiation-induced lesions in supressing DNA synthesis in normal cells and that AT cells respond to RE-induced dsb in a similar way to damage induced by ionizing radiation. Finally, when a nuclear extract from N-SW cells was introduced into Pvu Il-treated AT-PA cells, it was able to confer a normal frequency of CA. In contrast, neither whole cell nor nuclear extracts from normal cells influenced the production of CA induced by y-rays. These findings provide evidence for the presence of factor(s) in normal nuclear extract which complements the defect in processing of RE-induced dsb in AT cells.

Glucose isomerase (E.C. 5.3.1.4) was extracted from Lactobacillus brevis N.CD.O 474 grown in xylose, containing medium with a yield of cells (dry weight) of 2.3 - 3.3gll of medium and 300-310 glucose isomerase units. Several methods for releasing the intracellular enzyme were investigated and the specific activity recovery was highest with the heat autolysis method. The crude extract preparation was further purified by nucleic acid precipitation with MnCl2, protein fractionation by ammonium sulphate and dialysis followed by chromatography on CM-cellulose, DEAE-cellulose and gel filtration on Sephadex G-200. A final purification of 24 fold was achieved with about 25% activity recovery in 4 purification steps as follows: enzyme extraction by heat autolyis, MnCl2 treatment (nucleic acid precipitation), ammonium sulphate (2-3.6M2pH 7.0) protein precipitation and CM-cellulose chromatography. A mol. wt. of approximately 120,000 was calculated for the purified enzyme by gel filtration (Sephadex G-200) which dissociated in small subunits with mol wt. of 54,000-42,600 calculated by electrophoresis on 5% polyacrylamide - 3% SDS-8M urea. The purified enzyme was immobilised with a PEI-derivative of nylon (polyethyleneimine) and the kinetic properties of both free and immobilized enzyme were investigated. Apparent Km values for the free purified enzyme were 7.4 x 10^-3M (D-xylose); 2.8M (D-glucose); 1.9M (D-fructose). The corresponding apparent V values were 0.45; 0.015 and 0.022 mumoles min-1. mug enzyme-1 respectively. Investigations were also carried out into several other possibilities of assaying glucose isomerase activity. Parameters for the coupled reaction assay system using sorbitol dehydrogenase -NADH were optimised.