No description available.

The tremendous increase in biological sequence data presents us with an opportunity to understand the molecular and cellular basis for cellular life. Comparative studies of these sequences have the potential, when applied with sufficient rigor, to decipher the structure, function, and evolution of cellular components. The accuracy and detail of these studies are directly proportional to the quality of these sequences alignments. Given the large number of sequences per family of interest, and the increasing number of families to study, improving the speed, accuracy and scalability of MSA is becoming an increasingly important task. In the past, much of interest has been on Global MSA. In recent years, the focus for MSA has shifted from global MSA to local MSA. Local MSA is being needed to align variable sequences from different families/species. In this dissertation, we developed two new algorithms for fast and scalable local MSA, a three-way-consistency-based MSA and a biclustering -based MSA. The first MSA algorithm is a three-way-Consistency-Based MSA (CBMSA). CBMSA applies alignment consistency heuristics in the form of a new three-way alignment to MSA. While three-way consistency approach is able to maintain the same time complexity as the traditional pairwise consistency approach, it provides more reliable consistency information and better alignment quality. We quantify the benefit of using three-way consistency as compared to pairwise consistency. We have also compared CBMSA to a suite of leading MSA programs and CBMSA consistently performs favorably. We also developed another new MSA algorithm, a biclustering-based MSA. Biclustering is a clustering method that simultaneously clusters both the domain and range of a relation. A challenge in MSA is that the alignment of sequences is often intended to reveal groups of conserved functional subsequences. Simultaneously, the grouping of the sequences can impact the alignment; precisely the kind of dual situation biclustering algorithms are intended to address. We define a representation of the MSA problem enabling the application of biclustering algorithms. We develop a computer program for local MSA, BlockMSA, that combines biclustering with divide-and-conquer. BlockMSA simultaneously finds groups of similar sequences and locally aligns subsequences within them. Further alignment is accomplished by dividing both the set of sequences and their contents. The net result is both a multiple sequence alignment and a hierarchical clustering of the sequences. BlockMSA was compared with a suite of leading MSA programs. With respect to quantitative measures of MSA, BlockMSA scores comparable to or better than the other leading MSA programs. With respect to biological validation of MSA, the other leading MSA programs lag BlockMSA in their ability to identify the most highly conserved regions.

published_or_final_version / Pathology / Master / Master of Philosophy

published_or_final_version / Microbiology / Master / Master of Philosophy

This study was conducted to set up a systematic approach utilizing advantages of both wet lab and bioinformatic methodologies to study biodegradation abilities and microbial bacterial-functional relationship within bioremediation communities. Firstly, 11pharmaceuticals and personal care products (PPCPs)were selected as target chemicals for establishing an effective determination process in analyzing trace-level concentrations in the environment, and understanding the removal routes during pollutants removal process in wastewater treatment process using activated sludge. Ultra performance liquid chromatography-tandem mass spectrometry was utilized to develop a rapid, sensitive and reliable method without solid phase extraction pre-concentration for trace analysis of 11 PPCPs in influent and effluent from municipal wastewater treatment plants. Shorten the detection time and significant reduction of detection cost were achieved due to the omitting usage of solid phase extraction (SPE)process and avoiding the consumption of hydrophiliclipophilic balancced (HLB)cartridge. Research on removal routes of ten selected PPCPs in activated sludge found activated sludge hardly removed carbamazepine. Biodegradation was the sole route to remove acyclovir, metronidazole, benzylparaben, ethylparaben, methylparaben and propylparaben. Both adsorption and biodegradation were involved in the removal of ranitidine and benzophenone-3, while fluoxetine could be totally removed by adsorption in activated sludge. Secondly, as the target microbial community, activated sludge community was used to set up the global bioinformatic analysis process. Both metagenomic and metatranscriptomic approaches were processed to characterize microbial structure and gene expression of activated sludge community. The taxonomic profile showed thatactivated sludge was dominated by Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Verrucomicrobiaphyla. Gene expression annotation of nitrogen removal revealed that denitrification-related genes sequences dominated in both DNA and cDNA datasets while nitrifying genes were also expressed in relative high levels. Specially, ammonia monooxygenase and hydroxylamine oxidase demonstrated the high cDNA/DNA ratios, indicating strong nitrification activity. Ammonia-oxidizing bacteria present mainly belonged to Nitrosomonas and Nitrosospira species. A fast method to construct local sub-databases has been established for the quick similarity search and annotation of huge metagenomic datasets. The conducted tests showed sub-database annotation pipeline achieved a speedup of ~150-385 times, and got exactly the same annotation results with those of the direct NCBI-nr database BLAST-MEGAN method. This approach provides a new time-efficient and convenient annotation similarity search strategy for laboratories without access to high performance computing facilities. Thirdly, bisphenol A(BPA), which has a partially known biodegradation pathway and relevant bioremediating genes, was chosen as a model to establish a pipeline for systematical understanding the pathways and gene/bacteria relationships in an enriched microbial community. 11 new metabolites were detected during BPA degradation. Thereby, a novel pathway of degrading BPA metabolite was proposed. Sphingomonas strains were dominant taxa in initial degradation of BPA, while the other taxa were competing BPA metabolites during degradation. Metagenomic binning results showed a cytochrome P450 monooxygenase system, which was previously reported BPA mediator, was sharing by two Sphingomonas strains, showing the undergoing mechanism of competition of the two strains. The observations suggested bacterial specialization may occur in that community that each taxon was selected to degrade certain metabolite in a community economical way. / published_or_final_version / Civil Engineering / Doctoral / Doctor of Philosophy

The nucleotide sequence of the PRD1 terminal protein gene has been determined. The coding region for PRD1 terminal protein is 777 base pairs long and encodes 259 amino acid residues (29,326 daltons). The deduced amino acid sequence of PRD1 terminal protein reveals no overall homology with other known terminal proteins or related proteins. A closer examination revealed a highly conserved amino acid sequence, YSRLRT, exist among all identified DNA terminal proteins including PRD1, PZA, Nf, φ29 and adenovirus. This is the first conserved amino acid sequence that has been found in all identified DNA terminal proteins. Not only is the YSRLRT sequence conserved, but its spatial location is similar as well. Therefore, the significance of the YSRLRT conserved sequence is suggested by both its conservative spatial location and high degree of homology across species. To study the structure-function relationship of the YSRLRT sequence of PRD1 terminal protein, in vitro site-directed mutagenesis was performed to determine the role of each amino acid in this conserved region. The PRD1 terminal protein and DNA polymerase genes were cloned into phagemid pEMBLex3, and the recombinant plasmid used for constructing mutants. Eleven PRD1 terminal protein mutant clones were examined for their priming complex formation activities. Our results have strongly demonstrated that the positive charge residue of arginine-174 plays an important role for PRD1 terminal protein function. There are 13 tyrosine residues in the predicted PRD1 terminal protein. It was of interest to known which tyrosine is actually linked to terminal nucleotide of the PRD1 DNA. We used a new approach involving replacing the tyrosine residues with phenylalanine residues in the carboxyl terminal portion of the protein. From analyses, the tyrosine-190 has been determined to be the most likely linkage site between terminal protein and PRD1 DNA.

We address the problem of the DNA sequences developing a "dynamical" method based on the assumption that the statistical properties of DNA paths are determined by the joint action of two processes, one deterministic, with long-range correlations, and the other random and delta correlated. The generator of the deterministic evolution is a nonlinear map, belonging to a class of maps recently tailored to mimic the processes of weak chaos responsible for the birth of anomalous diffusion. It is assumed that the deterministic process corresponds to unknown biological rules which determine the DNA path, whereas the noise mimics the influence of an infinite-dimensional environment on the biological process under study. We prove that the resulting diffusion process, if the effect of the random process is neglected, is an a-stable Levy process with 1 < a < 2. We also show that, if the diffusion process is determined by the joint action of the deterministic and the random process, the correlation effects of the "deterministic dynamics" are cancelled on the short-range scale, but show up in the long-range one. We denote our prescription to generate statistical sequences as the Copying Mistake Map (CMM). We carry out our analysis of several DNA sequences, and of their CMM realizations, with a variety of techniques, and we especially focus on a method of regression to equilibrium, which we call the Onsager Analysis. With these techniques we establish the statistical equivalence of the real DNA sequences with their CMM realizations. We show that long-range correlations are present in exons as well as in introns, but are difficult to detect, since the exon "dynamics" is shown to be determined by theentaglement of three distinct and independent CMM's. Finally we study the validity of the stationary assumption in DNA sequences and we discuss a biological model for the short-range random process based on a folding mechanism of the nucleic acid in the cell nucleus.

The spinal cord is a complex structure responsible for processing sensory inputs and motor outputs. As such, the developmental and spatial organization of cells is highly organized. Diseases affecting the spinal cord, such as Amyotrophic Lateral Sclerosis (ALS), result in the disruption of normal cellular function and intercellular interactions, culminating in neurodegeneration. Deciphering disease mechanisms requires a fundamental understanding of both the normal development of cells within the spinal cord as well as the homeostatic environment that allows for proper function. Biological processes such as cellular differentiation, maturation, and disease progression proceed in an asynchronous and cell type-specific manner. Until recently, bulk measurements of a mixed population of cells have been key in understanding these events. However, bulk measurements can obscure the molecular mechanisms involved in branched or coinciding processes, such as differential transcriptional responses occurring between subpopulations of cells. Measurements in individual cells have largely been restricted to 4 color immunofluorescence assays, which provide a solid but limited view of molecular-level changes. Recently, developments in single cell RNA-sequencing (scRNA-Seq) have provided an avenue of accurately profiling the RNA expression levels of thousands of genes concomitantly in an individual cell. With this increased experimental precision comes the ability to explore pathways that are differentially activated in subpopulations of cells, and to determine the transcriptional programs that underlie complex biological processes. In this dissertation, I will first review the key features of scRNA-Seq and downstream analysis. I will then discuss two applications of scRNA-seq: 1) the in vitro differentiation of mouse embryonic stem cells into motor neurons, and 2) the effect of the ALS-associated gene SOD1G93A expression on cultured motor neurons in a cellular model of ALS.

by Pong Tsang Wai Hai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 98-105). / Acknowledgements --- p.i / Abstract --- p.ii / Abbreviations --- p.v / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Biological functions and structure of GH --- p.1 / Chapter 1.2 --- Application of recombinant GH --- p.2 / Chapter 1.3 --- Expression of eukaryotic gene in E.coli --- p.4 / Chapter 1.4 --- Methods for increasing expression of a cloned gene --- p.6 / Chapter 1.4.1 --- Changing the 5' end codons of the cDNA to E.coli preferred codons --- p.6 / Chapter 1.4.2 --- Optimization of distance between SD sequence and the initiation codons --- p.6 / Chapter 1.4.3 --- "Construction of a short ""dummy"" cistron at the 5' end of the cloned gene to improve attachment of ribosome" --- p.7 / Chapter 1.4.4 --- Increasing the copy number of recombinant expression plasmid --- p.8 / Chapter 1.4.5 --- Optimizing high density cell expression --- p.9 / Chapter 1.5 --- Quantitating the expression of cloned gene --- p.10 / Chapter 1.6 --- Inclusion bodies formation --- p.11 / Chapter 1.7 --- The purification of eukaryotic polypeptides synthesized as inclusion bodies --- p.12 / Chapter 1.7.1 --- Solubilization of the inclusion bodies --- p.13 / Chapter 1.7.2 --- Refolding the polypetides and disulfide bond formation --- p.13 / Chapter 1.8 --- Expression of secreted recombinant protein --- p.14 / Chapter 1.9 --- Purpose of present study --- p.15 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- General techniques --- p.16 / Chapter 2.1.1 --- Chemical Synthesis of DNA linkers and primers --- p.16 / Chapter 2.1.2 --- Manipulation of DNA --- p.16 / Chapter 2.1.3 --- Electro-elution of DNA from Agarose Gel --- p.17 / Chapter 2.1.4 --- Preparation of Competent Cells and Transformation --- p.18 / Chapter 2.1.5 --- Screening of the Expressed Clones --- p.19 / Chapter 2.1.6 --- Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.21 / Chapter 2.1.7 --- Western blot analysis --- p.21 / Chapter 2.2 --- Purification procedures --- p.23 / Chapter 2.2.1 --- Growing up the cells in large scale --- p.23 / Chapter 2.2.2 --- Harvesting of cells from large scale culture --- p.23 / Chapter 2.2.3 --- Sonication of the cells --- p.24 / Chapter 2.2.4 --- Washing of the inclusion body --- p.24 / Chapter 2.2.5 --- Solubilization of the inclusion bodies --- p.25 / Chapter 2.2.6 --- Renaturation of r-gcGH --- p.26 / Chapter 2.2.6.1 --- Step down dilution mehtod --- p.26 / Chapter 2.2.6.2 --- Rapid dilution method --- p.26 / Chapter 2.2.7 --- Separation by reverse phase chromatography --- p.27 / Chapter 2.2.7.1 --- Octadodecylsilica (ODS) column separation --- p.27 / Chapter 2.2.7.2 --- Fast performance Liquid Chromatography(FPLC) --- p.28 / Chapter 2.3 --- Characterization methods --- p.29 / Chapter 2.3.1 --- Radioimmunoassay --- p.29 / Chapter 2.3.1.1 --- Iodination of r-gcGH --- p.29 / Chapter 2.3.1.2 --- Binding assay --- p.31 / Chapter 2.3.2 --- Preparation of anti-r-gcGH serum --- p.32 / Chapter 2.3.3 --- Determination of amino acid composition and N-terminal sequence of r-gcGH --- p.32 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Recombinant plasmids construction --- p.34 / Chapter 3.1.1 --- Basic construction of plasmid producing gcGH in E.coli --- p.34 / Chapter 3.1.2 --- N-terminal modification of gcGH cDNA --- p.38 / Chapter 3.1.3 --- Constuction of a short 'dummy' cistron at the 5'end of gcGH cDNA --- p.40 / Chapter 3.1.4 --- Optimization of distance between ribosomal binding site and initiation codon --- p.42 / Chapter 3.1.5 --- Increasing expression level by increasing plasmid copy number --- p.44 / Chapter 3.1.6 --- Optimizing the high density expression by changing the promoter --- p.48 / Chapter 3.1.7 --- Construction of excretion plasmid for gcGH production from E. coli --- p.48 / Chapter 3.2 --- Quantitation and qualitation of the expressed protein --- p.51 / Chapter 3.3 --- Effect of IPTG on induction of r-gcGH in pp5 --- p.57 / Chapter 3.4 --- Stability of overproducing strain pp5 during continuous culture --- p.59 / Chapter 3.5 --- Stability of overproducing strain ppADH4 during continuous culture --- p.61 / Chapter 3.6 --- "Optimization of culture condition for high level expression strains,pp5 and ppADH4" --- p.64 / Chapter 3.7 --- Purification of r-gcGH --- p.67 / Chapter 3.7.1 --- Distribution of r-gcGH as Soluble and insoluble protein in E. coli --- p.67 / Chapter 3.7.2 --- Isolation and cleaning of the inclusion bodies --- p.69 / Chapter 3.7.3 --- Solubilization and renaturation of r-gcGH --- p.71 / Chapter 3.7.4 --- Purification of r-gcGH by chromatography --- p.73 / Chapter 3.8 --- Characterization of r-gcGH --- p.78 / Chapter 3.8.1 --- Amino acid composition and N-terminal sequence determination --- p.78 / Chapter 3.8.2 --- Immunological property of r-gcGH --- p.81 / Chapter 3.8.3 --- Physical Property of r-gcGH --- p.84 / Chapter 3.8.4 --- Stability of r-gcGH --- p.84 / Chapter 3.9 --- Expression and purification of r-gcGH in excretion vector ppSP14 --- p.86 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Evaluation of expression strains --- p.88 / Chapter 4.1.1 --- Strain pKgcGH2 --- p.88 / Chapter 4.1.2 --- Strain pKgcGH2-17 --- p.88 / Chapter 4.1.3 --- Strain pSD78 --- p.89 / Chapter 4.1.4 --- "Strains pLl,pL2 and pL4" --- p.90 / Chapter 4.1.5 --- "Strains pp5,pplA,pp2I and pp4Q" --- p.90 / Chapter 4.1.6 --- Strain ppADH4 --- p.91 / Chapter 4.1.7 --- Strain ppSP14 --- p.91 / Chapter 4.2 --- Disulfide bond formation during refolding process --- p.92 / Chapter 4.2.1 --- Renaturaion in the presence of L-arginine and thiol reagent in oxidized form --- p.93 / Chapter 4.2.2 --- Renaturation in the presence of thiol reagent and 3M guanidine hydrochloride --- p.94 / Chapter 4.3 --- Stability of the r-gcGH --- p.94 / Chapter 4.4 --- Further studies --- p.96 / References --- p.98

Jiang Qing Lan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (p. 171-188). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.