Little is actually known about the transcriptional regulation of spider silk as most studies have focused on the material properties of silks. We isolated and mapped the TuSp1 core promoter from the black widow spider, Latrodectus hesperus. Using a genomic DNA walking strategy, we have isolated an upstream segment (581 bp) of genomic DNA containing the promoter as well as the first exon of the TuSp1 gene. This upstream regulatory element was able to initiate transcription in insect cells when placed upstream the promoterless firefly luciferase reporter gene. Initiation of transcription was orientation dependent, as insertion of this upstream regulatory module in the reverse orientation led to inefficient transcriptional initiation. Only 170 bp of upstream sequence was required for strong transcriptional initiation, showing that core promoter resides within the first 170 bp of upstream 5' -flanking DNA. We also demonstrate the bHLH factor SGSF1 can repress gene transcription of the TuSp1 core promoter, implying SGSF I might participate in the transcriptional regulation of the TuSp1 gene in vivo.

Belonging to the diverse order Araneae, the black widow spider Latrodectus 4 hesperus produces high-performance silks with a broad range ofbiological functions and mechanical properties. The cob weaver spider spins different fibers by using seven specialized glands located in its abdomen. Egg case silk originates from the tubuliforrn gland and to date, no proteins that participate in the assembly process of egg case silk proteins have been identified. The goal of this project was the expression, purification, and characterization of such protein products. De novo sequencing of peptides from in-solution tryptic digestion of black widow spider dragline silk, the most studied type of silk, identified a novel cysteine-rich nonfibroin- like peptide that we named cysteine-rich component or CRC- 1. Further analysis of a large pool of nucleic acid sequences deposited in our custom eDNA database revealed 4 additional sequences with similarities to each other at the amino acid level called CRC-2, CRC-3, CRC-4, and CRC-5, suggesting a new family of proteins. Specifically, Q-PCR analysis revealed that the CRC-5 mRNAs were predominantly expressed in the tubuliform and aggregate glands. Since the aggregate gland manufactures a more complex aqueous solution compared to the tubuliforrn gland, we focused these studies on the tubuliform gland and resultant egg case fibers. Westem blot analysis using a cross-reactive polyclonal anti-CRC-1 antiserum conoborated the presence of CRC-5 in the tubulifmm gland and egg case silk, supporting the colocalization ofTuSpl, a tubuliform gland-specific protein, and CRC-5. Thus, we have demonstrated that these two proteins are present within tubuliform silks. In vitro studies suggested that recombinant CRC-5 displayed enzymatic activity similar to a sulfhydryl oxidase. Collectively, our findings provide new insights into novel proteins that have a potential role in the silk assembly and extrusion pathway of egg case silk fibers.

Despite the variety of successful reports of the preservation, recovery, and identification of archaeological proteins in general, there are few positive reports regarding mass spectrometry-based identification of ceramic-bound proteins. In large part, this shortage is due to the lack of consideration for the unique taphonomic histories of such residues and, in general, methods development. Further, because negative results are rarely published, there is no baseline to which results can be compared. This paper attempts to address these challenges via a multi-pronged approach that uses mass spectrometry and complementary approaches to evaluate ceramic-bound protein preservation in both controlled, actualistic experiments, and in archaeological artifacts. By comparing the results obtained from protein-spiked, experimentally-aged ceramic to those obtained from both faunal and ceramic archaeological materials, an enhanced perspective on protein preservation and subsequent recovery and identification is revealed. This perspective, focusing on taphonomy, reveals why negative results may be the norm for ceramic artifacts when non-targeted methods are employed, and provides insight into how further method development may improve the likelihood of obtaining positive results.

The purpose of this study was to completely characterize the protein moiety in the caroteno complex in C. poinsettae, determine if the distribution and level of protein in the pigment-protein complex in membranes of the wild type and in a colorless mutant could account for the differences in the stability of the membrane, and to determine if this protein is common to other pigmented and non-pigmented organisms. Also, electron microscopy of cell membranes of C. poinsettiae which had been exposed to gold-labelled antibody against the protein moitey of the pigment-protein complex, demonstrating that the protein is randomly distributed in the membranes of both wild type and colorless mutant.

This study was conducted at the installation of the Rural Academic Unit-Tiahuanaco of the BCU, located in the community of Achaca, third municipal section of Ingavi province, department of La Paz. It is 57 km from the La Paz-Desaguadero international highway, at 68 degrees 42 minutes 28 seconds latitude South by 16 degrees 35 minutes 41 seconds longitude West, at an altitude of 3856 meters above sea level. The study went from October 2000 to September 2001. The study consisted of determining the concentrations of the biochemical components in llama seminal plasma at three ages. Components studied were glucose, inorganic phosphate, creatinine, total protein, albumin, globulins, cholesterol, calcium, potassium, sodium, and magnesium. Twelve male llamas of 3, 4, and 5 years were selected and acquired from the Choquecota area of the Carangas province, department of Oruro. Four animals were chosen at each age and were subjected to a training period of semen collection during 2 months, using the artificial hindquarters designed for this effect. The 6 that best responded to the training were used for the investigation. Eight collections were obtained from each animal over the course of the study, and they were used for laboratory analysis. The results were analyzed using a hierarchical factorial design that involved a mixed analysis (nested and crossed) of the factors of age and collections. (The averages of two collections corresponding to each week were analyzed.) Each weekly collection average per age was an experimental unit. Four experimental units were obtained for each age, and the analysis of the data was done with the SAS statistics package version 6.12. From the analyses done the following results were obtained: the concentrations of glucose (6.246 [plus or minus] 0.716 mg/dl), creatinine (3.459 [plus or minus] 1.27 mg/dl), cholesterol (67.28 [plus or minus] 18.21 mg/dl), potassium (8.249 [plus or minus] 1.78 mEq/L), and sodium (123.187 [plus or minus] 18.39 mEq/L) did not show significant differences between ages or collections (p>0.05). The concentrations of calcium (12.138 [plus or minus] 3.64 mg/dl) and magnesium (1.943 [plus or minus] 0.52 mEq/L) showed significant (p<0.05) differences in age only and not in collections. Globulins (1.574 [plus or minus] 0.51 g/dl) showed differences between collections (p<0.05) but not between ages. Total protein (3.732 [plus or minus] 0.45 g/dl), albumin (2.158 [plus or minus] 0.46 g/dl), and inorganic phosphate (9.42 [plus or minus] 2.42 mg/dl) showed differences both between ages and between collections (p<0.05).

In the first part of this thesis, two chromosome Y specific genes ( SRYand TSPY) were chosen as the molecular targets to investigate the characteristics of fetal-specific DNA fragments in maternal plasma. By employing the touch down ligation-mediated PCR coupled with cloning and sequencing, the end property and the fragment species of fetal DNA were studied. / Noninvasive prenatal detection of fetal chromosomal aneuploidies is a much sought-after goal in fetomaternal medicine. The discovery of fetal DNA in the plasma of pregnant women has offered new opportunities for this purpose. However, the fact that fetal DNA amounts to just a minor fraction of all DNA in maternal plasma makes it challenging for locus-specific DNA assays to detect the small increase in sequences derived from a trisomic chromosome. On the other hand, although the clinical applications of plasma DNA for prenatal diagnosis are expanding rapidly, the biological properties of circulating DNA in plasma remain unclear. Recently, next-generation sequencing technologies have transformed the landscape of biomedical research through the ultra-high-throughput sequence information generated in a single run. Massively parallel sequencing allows us to study plasma DNA at an unprecedented resolution and also precisely detect fetal chromosomal aneuploidies in a locus-independent way. / Our group has demonstrated the use of massively parallel sequencing to quantify maternal plasma DNA sequences for the noninvasive prenatal detection of fetal trisomy 21. In the second part of this thesis, the clinical utility of this new sequencing approach was extended to the prenatal detection of fetal trisomy 18 and 13. A region-selection method was developed to minimize the effects of GC content on the diagnostic sensitivity and precision for the prenatal diagnosis of trisomy 13. To facilitate the next-generation sequencing-based maternal plasma DNA analysis for clinical implementation, two measures, i.e., lowering the starting volume of maternal plasma and barcoding multiple maternal plasma samples, were investigated. / Taken together, the results presented in this thesis have demonstrated the clinical utility of massively parallel sequencing of maternal plasma DNA and have also provided us a better understanding of the biology of circulating DNA molecules. / The third part of this thesis focuses on the massively parallel paired-end sequencing of plasma DNA. By analyzing millions of sequenced DNA fragments, the biological properties of maternal plasma DNA were elucidated, such as the size distribution of fetal-derived and maternally-contributed DNA molecules and the potential effect of epigenetic modification on DNA fragmentation. Moreover, the plasma DNA from hematopoietic stem cell transplant patients was characterized by paired-end sequencing approach. These sequencing data not only confirmed the predominant hematopoietic origin of cell-free DNA but also revealed the size difference between hematologically-derived and other tissue-derived DNA molecules in plasma. / Zheng, Wenli. / Adviser: Lo Yu Ming Dennis. / Source: Dissertation Abstracts International, Volume: 73-03, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 261-275). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Leung Ka-Kit. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 129-131). / Abstracts in English and Chinese. / Abstract --- p.1 / Chapter CHAPTER 1. --- introduction --- p.14 / Chapter 1.1 --- Preamble --- p.14 / Chapter 1.2 --- Biological background --- p.14 / Chapter CHAPTER 2. --- previous related work --- p.18 / Chapter 2.1 --- Protein functionality analysis --- p.18 / Chapter 2.1.1 --- Analysis from primary structure --- p.18 / Chapter 2.1.2 --- Analysis from tertiary structure --- p.20 / Chapter 2.2 --- Secondary structure prediction --- p.21 / Chapter 2.3 --- Motivation - Challenges from protein complexity --- p.22 / Chapter CHAPTER 3. --- mathematical representations for protein properties and sequence alignment --- p.24 / Chapter 3.1 --- Secondary structure sequence model --- p.24 / Chapter 3.2 --- Substitution matrix --- p.26 / Chapter 3.3 --- Gap --- p.26 / Chapter 3.4 --- Similarity measurement --- p.27 / Chapter 3.5 --- Geometric Model for Protein --- p.28 / Chapter CHAPTER 4. --- overall system design --- p.30 / Chapter 4.1 --- System architecture and design --- p.30 / Chapter 4.2 --- System environment --- p.32 / Chapter 4.3 --- Experimental data --- p.32 / Chapter CHAPTER 5. --- adaptive dynamic programming (adp)- general global alignment consideration --- p.35 / Chapter 5.1 --- t-triangles cutting --- p.35 / Chapter 5.1.1 --- Theoretical time and memory requirements of ADP with z-triangles cutting --- p.43 / Chapter 5.1.1.1 --- Study of parameters affecting h in case 1 --- p.44 / Chapter 5.1.1.2 --- Study of parameters affecting h in case 2 --- p.45 / Chapter 5.1.2 --- Experimental results of ADP with z-triangles cutting --- p.46 / Chapter 5.2 --- Constructing the path matrix by expansion --- p.51 / Chapter 5.2.1 --- Time and memory requirements of EXPAND --- p.57 / Chapter 5.2.2 --- Experimental results and discussions --- p.58 / Chapter CHAPTER 6. --- adp - global alignment of sequences with consecutive repeated characters --- p.65 / Chapter 6.1 --- Estimation of similarity upper bound (Ba) --- p.65 / Chapter 6.1.1 --- Sequence composition (SC) consideration --- p.65 / Chapter 6.1.2 --- Implementation of SC --- p.67 / Chapter 6.1.3 --- Experimental results --- p.69 / Chapter 6.1.4 --- Overall trend of change of structures (OTCS) --- p.74 / Chapter 6.1.5 --- Uninformed search --- p.76 / Chapter 6.2 --- Short-cut --- p.80 / Chapter 6.2.1 --- Time and memory requirements --- p.86 / Chapter 6.2.2 --- Experimental results and discussions --- p.86 / Chapter CHAPTER 7. --- ga based topology discovery --- p.87 / Chapter 7.1 --- Chromosome encoding --- p.87 / Chapter 7.2 --- Non-sequential order penalty --- p.88 / Chapter 7.3 --- Fitness function --- p.88 / Chapter 7.4 --- Genetic operators --- p.88 / Chapter 7.4.1 --- Hop operator --- p.89 / Chapter 7.4.2 --- Inverse operator --- p.89 / Chapter 7.4.3 --- Shift operator --- p.90 / Chapter 7.4.4 --- Selection pressure --- p.90 / Chapter 7.5 --- Selection of progeny --- p.91 / Chapter 7.6 --- Implementation --- p.91 / Chapter 7.6.1 --- Size of population and generation --- p.91 / Chapter 7.6.2 --- Parallelization --- p.91 / Chapter 7.6.3 --- Crowding Handling --- p.92 / Chapter 7.6.4 --- Selection of progeny --- p.92 / Chapter 7.7 --- Results of alignment with GA exploration on topological order --- p.93 / Chapter CHAPTER 8. --- FILTERING OF FALSE POSITIVES --- p.103 / Chapter 8.1 --- Alignment Segments to Gap Ratio (ASGR) --- p.103 / Chapter 8.2 --- Tolerance --- p.104 / Chapter 8.3 --- Overall trend of change of structures (OTCS) --- p.104 / Chapter 8.4 --- Results and discussions --- p.105 / Chapter CHAPTER 9. --- SECONDARY STRUCTURE PREDICTION --- p.111 / Chapter 9.1 --- 3-STATE SECONDARY STRUCTURE PREDICTION IMPROVEMENT --- p.111 / Chapter 9.2 --- 8-state secondary structure prediction --- p.117 / Chapter 9.3 --- Iterative Subordinate Voting (IS V) --- p.117 / Chapter 9.4 --- ISV Results and discussion --- p.119 / Chapter CHAPTER 10. --- CONCLUSIONS --- p.123 / Chapter 10.1 --- Contributions --- p.123 / Chapter 10.2 --- Future Work --- p.126 / Chapter 10.2.1 --- Using database indexing --- p.126 / Chapter 10.2.2 --- 3-state secondary structure prediction improvement --- p.127 / appendix --- p.128 / Chapter ´Ø --- Interpretation on the dp一filter results --- p.128

There are two contributions of this thesis. The first contribution, described in chapters one through six, involves studing the relationship between the protein packing structure of bacteriorhodopsin (bR) and its function as a proton pump. In 2002, a novel crystallization method published by Bowie and Farham resulted in an unusual antiparallel monomeric packing structure of bicelle bacteriorhodopsin (bcbR) crystals, the spectroscopic properties of which had not been studied. In this thesis, these bicelle bR crystals are investigated to better understand how the changes in the protein tertiary structure affect the function. Specifically: Does the retinal Schiff base retain its ability to isomerize in this unusual protein packing structure of bR? How is the hydration of its binding pocket affected? Does the protein retain the ability to undergo the photocycle and pump protons? If so, how are the rates of the deprotonation/reprotonation of the Schiff base affected by the antiparallel monomer packing structure of the protein? Is Asp85 still the proton acceptor during the deprotonation process of the photocycle? The second contribution of the thesis, described in chapter seven, describes the surface attachment and growth of the biofilm formed by the pathogenic bacterium Streptococcus pneumoniae using attenuated total reflection/Fourier transform infrared spectroscopy (ATR/FTIR). This organism was chosen for its clinical significance; it is one of the organisms suspected in forming biofilms in individuals who develop otitis media, one of the most common causes of ear infections of childhood. In contrast to previous ATR/FTIR experiments examining the formation of biofilms on surfaces, this method is unique in that it combines two techniques - ATR/FTIR and Epifluorescence microscopy which when used together allow for the simultaneous monitoring of the IR spectrum of the S. pneumoniae biofilm as it develops and as provides a method for quantifying total and viable cell counts at various stages during the development.

The goal of the following thesis research was to develop a systematic approach for the refinement of low-resolution protein models, as a part of the protein structure prediction procedure. Significant progress has been made in the field of protein structure prediction and the contemporary methods are able to assemble correct topology for a large fraction of protein domains. But such approximate models are often not detailed enough for some important applications, including studies of reaction mechanisms, functional annotation, drug design or virtual ligand screening. The development of a method that could bring those structures closer to the native is then of great importance. The minimal requirements for a potential that can refine protein structures is the existence of a correlation between the energy with native similarity and the scoring of the native structure as being lowest in energy. Extensive tests of the contemporary all-atom physics-based force fields were conducted to assess their applicability for refinement. The tests revealed flatness of such potentials and enabled the identification of the key problems in the current approaches. Guided by these results, the optimization of the AMBER (ff03) force field was performed that aimed at creating a funnel shape of the potential, with the native structure at the global minimum. Such shape should facilitate the conformational search during refinement and drive it towards the native conformation. Adjusting the relative weights of particular energy components, and adding an explicit hydrogen bond potential significantly improved the average correlation coefficient of the energy with native similarity (from 0.25 for the original ff03 potential to 0.65 for the optimized force field). The fraction of proteins for which the native structure had lowest energy increased from 0.22 to 0.90. The new, optimized potential was subsequently used to refine protein models of various native-similarity. The test employed 47 proteins and 100 decoy structures per protein. When the lowest energy structure from each trajectory was compared with the starting decoy, we observed structural improvement for 70% of the models on average. Such an unprecedented result of a systematic refinement is extremely promising in the context of high-resolution structure prediction.

Orientador: Marcos Antonio Machado / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-09T03:27:58Z (GMT). No. of bitstreams: 1 Winck_FlaviaVischi_M.pdf: 4501613 bytes, checksum: 37c71381bde27b6bd814d565ab527886 (MD5) Previous issue date: 2007 / Resumo: A bactéria Xanthomonas axonopodis pv. citri (Xac) é o agente causador do cancro cítrico, doença que leva a severas perdas econômicas devido à contaminação e à erradicação de plantas de citros. Com o seqüenciamento completo do genoma de Xac, vários genes supostamente envolvidos com a patogenicidade de Xac foram identificados. Os genes ligados à resposta de hipersensibilidade (hrp) e avirulência (avr) em geral estão relacionados à patogenicidade de Xanthomonas, entretanto, poucos estudos funcionais destes genes de Xac foram feitos. Foram construídas linhagens mutantes de Xac para a perda de função dos genes hrpF e avrXacE2 e, nas análises in vivo, foi verificado que hrpF está envolvido na patogenicidade de Xac e é essencial para a manifestação dos sintomas primários da doença. A mutação de avrXacE2 não provocou alterações na capacidade de Xac em provocar os sintomas do cancro, portanto, este gene não parece ser essencial para a patogenicidade da bactéria, podendo não estar envolvido diretamente na patogenicidade de Xac. Os genes hrpF e avrXacE2 foram clonados em vetores de expressão e foram realizados testes de indução da expressão destas proteínas em sistemas heterólogos. Somente a proteína AvrXacE2 foi expressa, purificada e submetida a teste de interação com as proteínas citoplasmáticas da linhagem mutante de Xac para o gene avrXacE2. Os testes de interações não confirmaram a identificação de proteínas com afinidade específica pela proteína recombinante AvrXacE2. A proteína HrpF não foi super-expressa em sistema heterólogo. Nas análises de proteoma comparativo da linhagem de Xac selvagem versus linhagem mutante para o gene hrpF, foram detectadas alterações na expressão de proteínas citoplasmáticas e "pericelulares". Com base nas observações pode-se supor que HrpF possa influenciar processos celulares relacionados à respostas à situações de estresse e não somente atuar na translocação de moléculas efetoras via T3SS. A partir do que foi exposto neste trabalho, sugere-se que as técnicas de estudos funcionais de genes e análises proteômicas podem conjuntamente permitir que novos mecanismos relacionados a patogenicidade de Xac sejam interpretados. Com os mutantes produzidos neste estudo, espera-se criar condições para novos ensaios funcionais visando a melhor compreensão da patogenicidade de Xac e buscar novas formas de combate ao cancro cítrico / Abstract: The bacterium Xanthomonas axonopodis pv. citri (Xac) is the causative agent of the citrus canker disease, which leads to economic losses due the contamination and erradication of citrus plants. The complete sequencing of its genome identified a number of genes supposedly involved with pathogenicity. Genes that code for hipersensitivity response (hrp) and avirulence (avr), in general, are related to the pathogenicity of Xanthomonas, however, only a few functional studies of these genes in Xac have been made. Here we report findings based on genomics and proteomics methods for Xac. Mutant strains of Xac for genes hrpF and avrXacE2 and in vivo assays demonstrated that hrpF is strongly involved in the pathogenicity of Xac and is essential for the manifestation of the primary symptoms of the citrus canker. On the other hand, the lack of avrXacE2 expression did not result in modifications in the capacity of Xac to elicite the symptoms of canker, therefore, this gene does not seem to be essential for the pathogenicity of the bacterium. The genes hrpF and avrXacE2 were cloned in expression vectors and tests of induction of the expression of these proteins in heterologous systems were carried out. The protein AvrXacE2 was expressed, purified and tested on interaction assays with cytoplasmic proteins of the mutant of Xac for the gene avrXacE2. The tests of interactions had not confirmed the identification of proteins with specific affinity for the recombinant protein AvrXacE2. The protein HrpF was not overexpressed in heterologous system. In the comparative proteome of the wild versus mutant strains for hrpF, modifications in the cytoplasmic protein expression and "pericellular" expression levels were detected. We postulate that, besides acting as a translocator of molecules through T3SS, HrpF may influence stress-related cellular responses. Thus, it is an opportune time to highlight the new and different ways in which HrpF serves Xac function. Moreover, we can assume that the techniques of functional genomics and proteomics analyses will clarify the mechanisms of pathogenicity used by Xac to cause citrus canker and, thus, enable the search for additional information to control the disease / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular