Sequence and Structure Based Protein Folding Studies With Implications

As the expression of the genetic blueprint, proteins are at the heart of all biological systems. The ever increasing set of available protein structures has taught us that diversity is the hallmark of their architecture, a fundamental characteristic that enables them to perform the vast array of functionality upon which all of life depends. This diversity, however, is central to one of the most challenging problems in molecular biology: how does a folding polypeptide chain navigate its way through all of the myriad of possible conformations to find its own particular biologically active form? With few overarching structural principles to draw upon that can be applied to all protein architecture, the search for a solution to the protein folding problem has yet to produce an algorithm that can explain and duplicate this fundamental biological process. In this thesis, we take a two-pronged approach for investigating the protein folding process. Our initial statistical studies of the distributions of hydrophobic and hydrophilic residues within α-helices and β-sheets suggest (i) that hydrophobicity plays a critical role in helix and sheet formation; and (ii) that the nucleation of these motifs may result in largely unidirectional growth. Most tellingly, from an examination of the amino acids found in the smallest β-sheets, we do not find any evidence of a β-nucleating code in the primary protein sequence. Complementing these statistical analyses, we have analyzed the structural environments of several ever-widening aspects of protein topology. Our examination of the gaps between strands in the smallest β-sheets reveals a common organizational principle underlying β-formation involving strands separated by large sequential gaps: with very few exceptions, these large gaps fold into single, compact structural modules, bringing the β-strands that are otherwise far apart in the sequence close together in space. We conclude, therefore, that β-nucleation in the smallest sheets results from the co-location of two strands that are either local in sequence, or local in space following prior folding events. A second study of larger β-sheets both corroborates and extends these findings: virtually all large sequential gaps between pairs of β-strands organize themselves into an hierarchical arrangement, creating a bread-crumb model of go-and-come-back structural organization that ultimately juxtaposes two strands of a parental β-structure that are far apart in the sequence in close spatial proximity. In a final study, we have formalized this go-and-come-back notion into the concept of anti-parallel double-strandedness (DS), and measure this property across protein architecture in general. With over 90% of all residues in a large, non-redundant set of protein structures classified as DS, we conclude that DS is a unifying structural principle that underpins all globular proteins. We postulate, moreover, that this one simple principle, anti-parallel double-strandedness, unites protein structure, protein folding and protein evolution.

A FUNCTIONAL, COMPARATIVE AND CLINICAL ANALYSIS OF SPERM-BORNE OOCYTE ACTIVATING FACTOR, PAWP

Successful fertilization depends upon the activation of metaphase II arrested oocytes by sperm-borne oocyte activating factor (SOAF). Failure of oocyte activation is considered as the cause of treatment failure in a proportion of infertile couples. SOAF induces the release of intracellular calcium in oocyte which leads to meiotic resumption and pronuclear formation. Calcium release is either in the form of single calcium transient in echinoderm and amphibian oocytes or several calcium oscillations in ascidian and mammalian oocytes. Although the SOAF attributes are established, it is not clear which sperm protein(s) play such role. Sperm postacrosomal WW binding protein (PAWP) satisfies a developmental criteria set for a candidate SOAF. This study shows that recombinant human PAWP protein or its transcript acts upstream of calcium release and fully activates the amphibian and mammalian oocytes. Interference trials provided evidence for the first time that PAWP mediates sperm-induced intracellular calcium release through a PPXY/WWI domain module in Xenopus, mouse and human oocytes. Clinical applications of PAWP were further investigated by prospective study on the sperm samples from patients undergoing intracytoplasmic sperm injection (ICSI). PAWP expression level, analyzed by flow cytometry, was correlated to ICSI success rate and embryonic development. This study also explored the developmental expression of the other SOAF candidate, PLCζ in male reproductive system and its function during fertilization. Our findings showed for the first time that PLCζ most likely binds to the sperm head surface during epididymal passage and is expressed in epididymis. We demonstrated that PLCζ is also compartmentalized early in spermiogenesis and thus could play an important role during spermiogenesis. Detailed analysis of in vitro fertilization revealed that PLCζ disappears from sperm head during acrosome reaction and is not detectable during sperm incorporation into the oocyte cytoplasm. In conclusion, this dissertation provides evidence for the essential non-redundant role of sperm PAWP in amphibian and mammalian fertilization; recommends PAWP as a biomarker for prediction of ICSI outcomes in infertile couples; and proposes that sperm PLCζ may have functions other than inducing oocyte activation during fertilization.

IMPROVING THE PERFORMANCE OF DATABASE-CENTRIC APPLICATIONS THROUGH PROGRAM ANALYSIS

Modern software applications are becoming more dependent on database management systems (DBMSs). DBMSs are usually used as black boxes by software developers. For example, Object-Relational Mapping (ORM) is one of the most popular database abstraction approaches that developers use nowadays. Using ORM, objects in Object-Oriented languages are mapped to records in the database, and object manipulations are automatically translated to SQL queries. As a result of such conceptual abstraction, developers do not need deep knowledge of databases; however, all too often this abstraction leads to inefficient and incorrect database access code. Thus, this thesis proposes a series of approaches to improve the performance of database-centric software applications that are implemented using ORM. Our approaches focus on troubleshooting and detecting inefficient (i.e., performance problems) database accesses in the source code, and we rank the detected problems based on their severity. We first conduct an empirical study on the maintenance of ORM code in both open source and industrial applications. We find that ORM performance-related configurations are rarely tuned in practice, and there is a need for tools that can help improve/tune the performance of ORM-based applications. Thus, we propose approaches along two dimensions to help developers improve the performance of ORM-based applications: 1) helping developers write more performant ORM code; and 2) helping developers configure ORM configurations. To provide tooling support to developers, we first propose static analysis approaches to detect performance anti-patterns in the source code. We automatically rank the detected anti-pattern instances according to their performance impacts. Our study finds that by resolving the detected anti-patterns, the application performance can be improved by 34% on average. We then discuss our experience and lessons learned when integrating our anti-pattern detection tool into industrial practice. We hope our experience can help improve the industrial adoption of future research tools. However, as static analysis approaches are prone to false positives and lack runtime information, we also propose dynamic analysis approaches to further help developers improve the performance of their database access code. We propose automated approaches to detect redundant data access anti-patterns in the database access code, and our study finds that resolving such redundant data access anti-patterns can improve application performance by an average of 17%. Finally, we propose an automated approach to tune performance-related ORM configurations using both static and dynamic analysis. Our study shows that our approach can help improve application throughput by 27--138%. Through our case studies on real-world applications, we show that all of our proposed approaches can provide valuable support to developers and help improve application performance significantly.