Syntheses and characterizations of the in vivo replicative bypass and mutagenic properties of the minor-groove O2-alkylthymidine lesions.

Endogenous metabolism, environmental exposure, and treatment with some chemotherapeutic agents can all give rise to DNA alkylation, which can occur on the phosphate backbone as well as the ring nitrogen or exocyclic nitrogen and oxygen atoms of nucleobases. Previous studies showed that the minor-groove O(2)-alkylated thymidine (O(2)-alkyldT) lesions are poorly repaired and persist in mammalian tissues. In the present study, we synthesized oligodeoxyribonucleotides harboring seven O(2)-alkyldT lesions, with the alkyl group being a Me, Et, nPr, iPr, nBu, iBu or sBu, at a defined site and examined the impact of these lesions on DNA replication in Escherichia coli cells. Our results demonstrated that the replication bypass efficiencies of the O(2)-alkyldT lesions decreased with the chain length of the alkyl group, and these lesions directed promiscuous nucleotide misincorporation in E. coli cells. We also found that deficiency in Pol V, but not Pol II or Pol IV, led to a marked drop in bypass efficiencies for most O(2)-alkyldT lesions. We further showed that both Pol IV and Pol V were essential for the misincorporation of dCMP opposite these minor-groove DNA lesions, whereas only Pol V was indispensable for the T→A transversion introduced by these lesions. Depletion of Pol II, however, did not lead to any detectable alterations in mutation frequencies for any of the O(2)-alkyldT lesions. Thus, our study provided important new knowledge about the cytotoxic and mutagenic properties of the O(2)-alkyldT lesions and revealed the roles of the SOS-induced DNA polymerases in bypassing these lesions in E. coli cells

Design and Implementation of Digital Information Security for Physical Documents

The objective of this thesis is to improve the security for physical paper documents. Providing information security has been difficult in environments that rely on physical paper documents to implement business processes. Our work presents the design of a digital information security system for paper documents, called CryptoPaper , that uses 2-dimensional codes to represent data and its security properties on paper. A special scanner system is designed for CryptoPaper which uses image recognition techniques and cloud-based access control to display plaintext of encrypted and encoded data to authorized users

Mechanisms of regulated lung surfactant secretion

Scope and Method of Study:This project was to study the molecular mechanisms of lung surfactant secretion from three aspects. The first part was to investigate the interaction between SNARE proteins and annexin A2. GST-tagged protein pull-down assay was the major technique utilized. The physical interaction of recombinant GST-tagged SNARE proteins with annexin A2 were tested. To confirm the interaction between annexin A2 and SNAP-23, additional methods were used, including co-immunoprecipitation, mammalian two-hybrid assays. Immunocytochemistry was used to study the co-localization of annexin A2 and SNAP-23. Deletion and site-directed mutagenesis were used to identify the binding sites for annexin A2 on SNAP-23. Furthermore, an in vitro bio-membrane fusion assay was utilized to study the functional interaction between annexin A2 and SNAP-23. The second part was to identify the v-SNARE protein involved in regulated surfactant secretion. Various VAMP genes were amplified from alveolar type II cells by using RT-PCR. The expression of VAMP proteins were detected with Western blotting. Immunohistochemistry and immunocytochemistry were utilized to study the localization of VAMP in lung and type II cells. In the third part, the proteomic profile of lamellar bodies was analyzed. Lamellar bodies were isolated from rat lungs and the proteins were separated with 1-D and 2-D SDS-PAGE. The proteins were applied to MALDI-TOF MS, and identified by searching the peptide spectra against the Mass Spectrometry protein sequence Database (MSDB) using the Mascot web based search engine.Findings and Conclusions:I.1. SNAP-23 specifically binds with annexin A2 in a Ca2+-dependent manner.2. The cysteine rich domain of SNAP-23 is required for its binding with annexin A2.3. SNAP-23 is required in annexin A2 tetramer mediated membrane fusion between isolated lamellar bodies and the plasma membrane.II.1. VAMP-2 and -8 are expressed in type II cells and lamellar bodies.2. VAMP-2 is localized on lamellar bodies and VAMP-8 is partially localized on lamellar bodies.III.1. Forty-four proteins in lamellar bodies were identified.2. Proteins involved in membrane trafficking and Ca2+-binding may play roles in lamellar body biogenesis and fusion with the plasma membrane