Studies have highlighted a growing national problem regarding the number of untested Sexual Assault Kits (SAKs). A 2011 National Institute of Justice report revealed Los Angeles alone had 10,000 untested SAKs. This backlog has fueled the need for specific and efficient testing of SAK evidence. In traditional workflows, serology tests are used to indicate the presence of a targeted bodily fluid and prioritize samples for genetic analysis. However, given the lack of sensitivity and specificity of modern serological assays, current SAK workflows often skip serological identification altogether for a “direct to DNA” approach. While these Y-Screen workflows achieve rapid screening of samples for the presence of a detectible male contributor, they do not provide any serological information. As a result, samples lack what can be critical investigative context. Improved serological capabilities with enhanced sensitivity and specificity would provide greater confidence in results for the confirmatory identification of seminal fluid. At a minimum, forensic biologists should understand the limitations associated with traditional serological approaches to seminal fluid identification when processing SAK samples. Current serological techniques based on antigen-antibody binding have exhibited both sensitivity and specificity limitations. False positive results for semen can be obtained by non-target biological fluids such as breast milk, urine, and vaginal fluid, or by non-specific binding events. This study evaluates a promising emerging technique that combines high specificity protein biomarker detection with targeted mass spectrometry. This research targeted human-specific peptide markers for seminal fluid proteins and peptide standards to perform quantification of seminal fluid peptide targets using an Agilent 6495 mass spectrometer coupled to a 1290 series liquid chromatograph. This approach has shown to be both more specific and sensitive in identifying a bodily fluid compared to current immunological based approaches. Thus, this proteomic workflow was used to evaluate authentic false positive rates of current immunochromatographic techniques for seminal fluid identification. Self-collected vaginal swabs collected from participants not engaging in barrier-free vaginal intercourse with male partners were tested using various immunochromatographic assays designed to detect both semenogelin (Sg) (RSID™-Semen) and prostate specific antigen (PSA) (ABAcard® p30 Test and SERATEC® PSA Semiquant). Similarly, three seminal fluid biomarkers (semenogelin 1, semenogelin 2, and prostate specific antigen) were used for seminal fluid identification via mass spectrometry. Any samples producing positive results on any immunochromatographic assay were evaluated to determine whether the target protein was actually present at levels above the reported sensitivity limits of the lateral flow tests. Additionally, Sperm HY-LITER™ Express was used to microscopically confirm the absence of spermatozoa in all samples producing positive immunochromatographic results. In addition to using the quantitative proteomic assay to estimate the rate of authentic false positive results associated with lateral flow assays, this research sought to establish the correlation (or lack thereof) between absolute quantitation of seminal fluid markers and the ability to successfully generate DNA profiles. Self-collected post-coital swabs from donors engaging in barrier free vaginal intercourse with male partners over varied periods of time between 1-8 days after intercourse were collected. All samples were analyzed using the quantitative seminal fluid protein mass spectrometry assay, once again targeting SgI, SgII, and PSA. Both autosomal STR profiles (GlobalFiler™) and Y-STR profiles (Yfiler™ Plus) were subsequently generated. With regard to immunochromatographic assay false positive rates, a total of 17 false positives for semen were observed (n=150), 14 of which were consistent with PSA and 3 with Sg, for a corresponding total false positive rate of 9.3% and 2%, respectively (11.3% overall). These samples were all confirmed to be sperm negative with mass spectrometry and microscopic analysis. This data supports the use of current immunochromatographic assays for the presumptive detection of seminal fluid while also providing further support for the improved specificity of alternative serological approaches using mass spectrometry identification of biological targets. With regard to the relationship between quantitative levels of target seminal fluid peptides and the ability to generate STR profiles from vaginal swabs collected at various post coital intervals, a total of 61 post-coital samples were tested. Of these, 48 samples had a seminal fluid target greater than the limit of quantitation for the mass spectrometry assay and 26 produced an STR (n=9) and/or Y-STR (n=10) profile. A correlation between peptide quantitation and ability to generate a genetic profile was unable to be determined from this initial sample set. Overall, however, it has been demonstrated that the use of proteomic mass spectrometry for the identification of seminal fluid targets (with its enhanced sensitivity and specificity) would enable forensic practitioners to make better use of serological information during the analysis of challenging sexual assault samples.

This research is concerned with analyzing a set of viral genomes to elucidate the underlying characteristics and determine the information-theoretic aspects of the genomic signatures. The goal of this study thereof, is tailored to address the following: (i) Reviewing various methods available to deduce the features and characteristics of genomic sequences of organisms in general, and particularly focusing on the genomes pertinent to viruses; (ii) applying the concepts of information-theoretics (entropy principles) to analyze genomic sequences; (iii) envisaging various aspects of biothermodynamic energetics so as to determine the framework and architecture that decide the stability and patterns of the subsequences in a genome; (iv) evaluating the genomic details using spectral-domain techniques; (v) studying fuzzy considerations to ascertain the overlapping details in genomic sequences; (vi) determining the common subsequences among various strains of a virus by logistically regressing the data obtained via entropic, energetics and spectral-domain exercises; (vii) differentiating informational profiles of coding and non-coding regions in a DNA sequence to locate aberrant (cryptic) attributes evolved as a result of mutational changes and (viii) finding the signatures of CDS of genomes of viral strains toward rationally conceiving plausible designs of vaccines. Commensurate with the topics indicated above, necessary simulations are proposed and computational exercises are performed (with MatLabTM R2009b and other software as needed). Extensive data gathered from open-literature are used thereof and, simulation results are verified. Lastly, results are discussed, inferences are made and open-questions are identified for future research. / by Sharmistha P. Chatterjee. / Thesis (Ph.D.)--Florida Atlantic University, 2013. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Class I phosphoinositide 3-kinases (PI3K) and mammalian target of rapamycin complexes 1/2 (mTORC1/2) are enzymes that play important roles in elementary biology and disease. As a consequence, numerous small-molecule inhibitors of their catalytic activity have been developed and these have shown clinical utility in certain cancers. Unfortunately, acquired resistance to these therapies is a common phenomenon and often occurs relatively quickly following treatment. Our understanding of how resistance develops is hampered by the difficulty of measuring the circuitry and plasticity of the signalling networks that these and other kinases signal within. Advances in mass spectrometric technologies have rendered the routine quantitative interrogation of the phosphoproteome (the set of phosphorylated proteins expressed in a particular biological system at a specific time) more tractable than ever before. The aim of this project therefore, was to improve upon existing mass spectrometry (MS)-based phosphoproteomics methods, and to utilise these to contribute to our understanding of kinase signalling networks and examine their plasticity in models of acquired resistance to PI3K and mTORC1/2-targeted therapies. Novel approaches for the enrichment of phosphopeptides from complex biological matrices (and their analysis by MS) were designed, tested and optimised. These methods were then used to systematically characterise a kinase signalling network comprising the Akt/PI3K/mTOR and MEK/ERK signalling axes in MCF7 breast cancer cells. The biological relevance of this network was confirmed through the assessment of its dynamics upon EGF and IGF-1 stimulation. Finally, the plasticity of this network following chronic treatment with targeted PI3K and mTORC1/2 inhibitors (GDC-0941 and KU-0063794) was examined in cell-line models of acquired resistance to these two compounds. This revealed that these cells each remodelled this network in a different manner, thus indicating that the initial conditions of the system were not the sole determinant of how resistance was acquired.

While the central role of spermatozoa in sexual reproduction and fertilization is well understood, many functional attributes of sperm have yet to be elucidated at the molecular level. One key to ultimately understand the molecular basis of sperm function is to comprehensively characterize its biochemical composition. This crucial information has been lacking, as molecular characterization of the sperm cell cannot be assessed by classic gene expression assays since mature spermatozoa are transcriptionaly inert. Whole-cell shotgun proteomic approaches have revolutionized the molecular analysis of sperm form and function. We have utilized improved methodologies to re-analyze the D. melanogaster sperm proteome and characterize five additional Drosophila species sperm proteomes. This methodology, which included a 1D SDS-PAGE prefractionation step, resulted in good reproducibility between biological replicates and high quality sperm proteomes. An interspecific analysis of the sperm proteomes revealed that despite variation in protein composition, Drosophila sperm proteomes have a consistent functional profile and 519 proteins were identified a being conserved across the melanogaster subgroup within a phylogenetic framework. Evolution of the sperm proteome was explored in Mus musculus through the utilization of targeted proteomic datasets completed, which provided subcellular localizations for sperm components. This study resulted in several novel findings, including evidence for accelerated evolution as well as an enrichment of positive selection on genes found in the cell membrane and acrosome. This may be a result of the selective pressures encountered by these membrane proteins during sperm development, maturation and transit through the female reproductive tract where the sperm cell membrane, and eventually the acrosome, are exposed to the extracellular milieu and are available for direct cell-cell interactions. These findings not only reveal the varying evolutionary pressures acting on a single cell type but also highlights the utility of the proteomics technique in clarifying protein interaction and evolutionary history.

In the last decade omics technologies enabled detailed and system-wide analysis of complex biological samples. Genomics, transcriptomics and metabolomics all benefited tremendously from technological advances in their respective fields. Proteomics was revolutionised by mass spectrometry, which allowed simultaneous identification of thousands of proteins in cells, tissues and organisms. And this mainly qualitative revolution, quickly turned quantitative. This work had two main objectives. Firstly, to apply the state of the art instrumentation, data analysis and bioinformatics methods to better our understanding of basic cell biology in a model organism Saccharomyces cerevisiae. Specifically, to quantitatively describe the effects of perturbations, such as adverse environmental conditions or chaperone gene deletions, on protein abundances in the cell. Additionally, it was aimed to demonstrate and evaluate the ability of a new timeof-flight mass spectrometer to perform large-scale absolute quantification. First, it was found that yeast cells are remarkably robust to deletions of major chaperone hub proteins (Ssa1p or Ssb1p deletions). This ability was attributed to network structure and redistribution of folding workload among other related chaperones rather than simple functional redundancy. Second, to build on the first set of results, a detailed time resolved description of yeast proteome dynamics in response to heat stress was provided for the wild type and Ssb1p chaperone mutant strains. In this study, for the first time in the literature, temporal expression patterns of many hallmark heat shock proteins were elucidated. Globally, a slow and sustained proteome remodelling or 'buffering' was revealed in both strains. However, it was also shown that the cells knocked out for the Ssb1p chaperone respond to heat in a distinctly different manner to the wild type strain. Finally, consistent and reproducible absolute quantification of multiple yeast proteomes was demonstrated using a new commercial time-of-flight mass spectrometer with ion mobility separation capabilities. The data obtained revealed global differences in cellular protein content between various chaperone prefoldin mutants as well as differential expression of a set of proteins promising to be interesting targets for further investigations.

WRKY transcription factors (TFs) belong to a large family of regulatory proteins in plants that modulate many plant processes. Extensive studies have been conducted on WRKY-mediated defense response in Arabidopsis thaliana and many crop species. This study aims to investigate the potential roles and contributions of WRKY TFs regulation in improving defense response in the resynthesized Arabidopsis allotetraploids (Arabidopsis suecica) from two related autotetraploid progenitors, Arabidopsis thaliana (At4) and Arabidopsis arenosa (Aa). Upon infection by Pseudomonas syringae (Pst), the allotetraploids has showed enhanced resistance against the pathogen when compared to the parents. Rapid induction of WRKY18, WRKY40, WRKY38, WRKY53, WRKY6; MAP kinase pathway related genes, WRKY33, PAD3; SA-pathway related genes, ICS1, EDS1, PBS3, MYB31; was evident in response to Pst and salicylic acid treatment in the allotetraploids. Cleaved amplified polymorphic sequences analysis further revealed that the AtWRKY18, AaWRKY40, AtWRKY33, and AtWRKY60 alleles expressed at higher levels when compared to their respective homoeologs in the allotetraploids, suggesting potential altered protein-protein interaction networks in the hybrids. Therefore, a split-luciferase complementation assay was used to characterize and quantify protein-protein interaction among these homoeologous WRKYs in the allotetraploids. Results showed that preferential protein-protein interactions exist for the cis-interacting AtWRKY18/AtWRKY18 homodimer or trans-interacting AtWRKY18/AaWRKY40 heterodimer when compared to the respective interacting complexes. In addition, differential affinities of WRKY18 and WRKY40 homo- and hetero- dimers toward the W-boxes at the WRKY60 promoter were observed. In the allotetraploids, PR1 expression was repressed under basal state when compared to the progenitors. Although PR1 is expressed at a higher level in A. thaliana, its expression fold change was higher and faster in the all otetraploids upon salicylic acid treatment. Transient expression of WRKY18 or WRKY40 homodimer in various combinations induced differential expression of PR1 gene in their respective wrky18 and wrky40 Arabidopsis thaliana mutants. In contrast, similar PR1 induction by homodimer in various combinations was observed when they were transiently expressed in the allotetraploids. In addition, transgenic AtWRKY18 overexpression plant displayed enhanced disease resistance against Pst when compared to AaWRKY18 overexpression lines. Such enhanced disease resistance was found to associate with the higher expression of PR1 and PR2 in AtWRKY18 transgenic lines. Moreover, differential Pst-induced expression of the direct targets (ICS1, EDS1 and PBS3) of WRKY18 in the Arabidopsis AtWRKY18 and AaWRKY18 overexpressors supported a biological difference between the At and Aa homodimers in mediating the targets regulation, thus contributing to the difference in disease responses. Overall, our findings suggested that the rapid differential alleles expression and altered protein-protein or protein-DNA interactions of WRKY transcription factors could contribute to the improved defense in the allotetraploids, providing a molecular basis of for heterotic phenotype development in hybrids.

Every living organism possesses a genome that contains within it a unique set of genes, a substantial number of which encode proteins. Over the last 20 years, it has become apparent that organismal complexity arises not from the specific complement of genes per se, but rather from interactions between the gene products - in particular, interactions between proteins. As an inevitable consequence of the crowded cellular interior, most protein-protein interactions are fleeting. However, many are significantly more long-lived and result in stable protein complexes, in which the constituent subunits are obligately dependent on their binding partners. Despite the abundance of protein complexes and their critical importance to the cell, we currently have an incomplete understanding of the mechanisms by which the cell ensures their correct assembly. In the chapters that follow, I have attempted to improve our understanding of the regulatory systems underlying assembly of protein complexes, and the way in which assembly as a whole affects the behaviour of the cell. The thesis opens with an extended literature review covering the currently available methods for characterising protein complexes. After this introduction, chapters 2-4 are concerned with regulatory mechanisms and biological implications common to the assembly of all protein complexes. Chapter 5 diverges from this work, and describes a family of evolutionarily related proteins that regulate the behaviour of condensins and cohesins. Bacterial and archaeal genomes contain far less non-coding DNA than eukaryotes, and coding genes are often packaged into discrete units known as operons. The proteins encoded within operons are usually functionally related, either through participation in metabolic pathways or as subunits of heteromeric protein complexes. Since protein complexes assemble via ordered pathways, we reasoned that there might be a signature of assembly order present in operons, the genes of which are translated in sequential order. By comparing computationally predicted assembly pathways with gene order in operons, we demonstrated this to be the case for the large majority of operon-encoded complexes. Within operons, gene order follows assembly order, and adjacent genes are substantially more likely to share a physical interface than those further apart. This work demonstrates that efficient assembly of complexes is of sufficient importance as to have placed major constraints on the evolution of operon gene order. Following this study of bacterial operons, I present results from research investigating how patterns of protein degradation in eukaryotes are influenced by the formation of protein complexes. This showed that, whilst most proteins display exponential degradation kinetics, a sizeable minority deviate considerably from this pattern, instead being more consistent with a two-step degradation process. These proteins are predominantly members of heteromeric complexes, and their two-step decay profiles can be explained using a model under which bound and unbound subunits are degraded at different rates. Within individual complexes, we find that non-exponentially decaying proteins tend to form larger interfaces, assemble earlier, and show a higher degree of coexpression, consistent with the idea that bound subunits are degraded at a slower rate than unbound or peripheral subunits. This model also explains the behaviour of proteins in aneuploid cells where one or more chromosomes have been duplicated. In general, protein abundance scales with gene copy number, so that the immediate effect of duplicating a chromosome is to double the abundance of the proteins encoded on it. However, previous analyses of mass spectrometry data, as well as my own, have shown that the abundance of many proteins on duplicated chromosomes is significantly attenuated compared to what one would expect. These proteins, like those with non-exponential degradation patterns, are very often members of larger complexes. Since the overall concentration of a protein complex is constrained by that of its least abundant members, duplicating a single subunit will predominantly increase the unbound, unstable fraction of that subunit. The results from this work strongly suggest that the apparent attenuation of many proteins observed in aneuploid cells is indeed a consequence of the failure of these proteins to assemble into complexes. Finally, I present a study concerning an important, universally conserved family of protein complexes, namely the SMC-kleisins. Two members of this family, condensin and cohesin, are responsible for two hallmarks of eukaryotic chromatin organisation: the formation of condensed, linear chromosomes, and sister chromatid cohesion during cell division. Unlike other SMC-kleisins, condensin and cohesin possess a number of regulators containing HEAT repeats. By developing a computational pipeline for searching and clustering paralogous repeat proteins, I was able to demonstrate that these regulators form a distinct sub-family within the larger class of HEAT repeat proteins. Furthermore, these regulators arose very early in eukaryotic history, hinting at a possible role in the origin of modern condensins and cohesins.

氧自由基曾被認為是有氧代謝过程的無用副產物。然而，在正常條件下，氧自由基也可以修飾蛋白質的結構與功能，同時能在多種細胞代謝過程中作為重要的信號分子。半胱氨酸的巰基極易被氧自由基、氮自由基以及其它的親電子分子所氧化，且其可逆的氧化反應對於與氧化還原相關的調控與信號傳遞是極其重要的。雖然僅有特定的一小部分半胱氨酸可以被氧化修飾，我們至今對於影響半胱氨酸對氧化還原敏感性的决定因素所知甚少。對於蛋白质半胱氨酸氧化修饰的比較分析及預測，不仅可以提高我們對半胱氨酸氧化還原敏感性的决定因素的認識，同時也能對後續关于重要的氧化還原敏感蛋白的實驗驗證起指導作用。 / 本研究中，本人主要做了如下三部分的工作：第一部分， RedoxDB數據庫的構建。这也是首個針對已被实验验证的蛋白氧化還原修飾的數據庫。第二部分，氧化還原敏感的半胱氨酸位點的特征分析及預測。本人分析了基於序列的各種可能與半胱氨酸氧化還原敏感性相關的特征，並發現其中三個特征可用於預測氧化還原敏感的半胱氨酸位點。基於這三個特征，我開發了RSCysPred 一个基於支持向量機且只依赖蛋白序列的預測氧化還原敏感的半胱氨酸位點的新方法。第三部分，可逆二硫鍵的分析。可逆二硫鍵的形成是蛋白質可逆氧化還原修飾的最主要的類型之一。我从RedoxDB中获取相关数据，然后針對功能位點標簽以及靜電特征等方面的特征，對可逆二硫鍵與結構二硫鍵進行了詳盡的比較分析。結果表明相比結構二硫鍵，可逆二硫鍵具有一些显著不同的特征。进一步的分析显示這些特征可用於可逆二硫鍵的預測。 / Reactive oxygen species (ROS) has been regarded as unwanted by-product of aerobic metabolism. However, under normal conditions, ROS can modify the structure and function of proteins and may also act as important signaling molecules in various cellular processes. Cysteine thiol groups of proteins are particularly susceptible to oxidation by ROS/RNS and other electrophilic molecules, and their reversible oxidation is of critical roles for redox regulation and signaling. Despite the fact that only a small fraction of cysteine residues undergone oxidative modification, the determinants of cysteine redox-sensitivity is poorly understood to date. Comparative analyses and computational prediction of protein cysteine oxidative modification will not only enhance our understanding about the determinants of cysteine redox-sensitivity, it may also facilitate further experimental investigation of important redox proteins. / This thesis includes the following three parts of work. Part I: Construction of RedoxDB - a curated database of protein oxidative modification. It is the first database for experimentally verified protein oxidative modification events. Part II: Feature analysis and computational prediction of redox-sensitive cysteines. I analyzed various sequence-based features potentially related to cysteine redox-sensitivity, and identified three features for efficient computational prediction of redox-sensitive cysteine. Based on these features, I developed RSCysPred, a SVM-based tool for predicting redox-sensitive cysteine using primary protein sequence only. Part III: Study on reversible disulfide formation, which is the major type of protein oxidative modification. By using data retrieved from RedoxDB, I performed extensive comparison between reversible disulfide-bonded cysteines and structural disulfide-bonded cysteines with regard to the functional site signatures and electrostatic properties. The results support that reversible disulfides show several remarkable differences compared with structural disulfides. Further analyses indicated that these features could be used for efficient prediction of reversible disulfide. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Sun, Mingan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 77-89). / Abstract also in Chinese. / Thesis/Assessment Committee --- p.I / Statement: --- p.I / Abstract of thesis entitled --- p.II / 摘要 --- p.IV / Acknowledgements --- p.V / Table of contents --- p.VI / List of Tables --- p.IX / List of Figures --- p.X / List of Abbreviations --- p.XI / CHAPTER 1 --- p.1 / General Introduction --- p.1 / Chapter 1.1 --- Generation of ROS and oxidative stress --- p.2 / Chapter 1.2 --- Protein cysteine oxidative modification --- p.3 / Chapter 1.2.1 --- Protein oxidative modification --- p.3 / Chapter 1.2.2 --- Special properties of cysteine --- p.3 / Chapter 1.2.3 --- Reversible cysteine oxidation and its function --- p.4 / Chapter 1.2.4 --- Specificity of cysteine oxidative modification --- p.5 / Chapter 1.3 --- Experimental approaches for detecting protein oxidative modification --- p.6 / Chapter 1.4 --- Computational prediction of protein oxidative modification --- p.7 / Chapter 1.5 --- Project Objectives --- p.8 / CHAPTER 2 --- p.11 / RedoxDB: a Curated Database of Experimentally Verified Protein Oxidative Modification --- p.11 / Chapter 2.1 --- Introduction --- p.12 / Chapter 2.2 --- Database concept --- p.13 / Chapter 2.3 --- Database interface and tools --- p.15 / Chapter 2.4 --- Discussion --- p.16 / CHAPTER 3 --- p.23 / Computational Prediction of Redox-Sensitive Cysteines Using Sequential Distance and Other Sequence-based Features --- p.23 / Chapter 3.1 --- Introduction --- p.24 / Chapter 3.2 --- Methods and Materials --- p.26 / Chapter 3.2.1 --- Datasets --- p.26 / Chapter 3.2.2 --- Feature extraction --- p.28 / Chapter 3.2.3 --- Support vector machines (SVMs) implementation and parameter optimization --- p.29 / Chapter 3.2.4 --- Performance assessment --- p.30 / Chapter 3.2.5 --- Statistical analyses --- p.30 / Chapter 3.3.6 --- Webserver implementation --- p.30 / Chapter 3.3 --- Results --- p.31 / Chapter 3.3.1 --- Performance evaluation by 10-fold cross-validation --- p.31 / Chapter 3.3.2 --- Evaluation of the most efficient features --- p.33 / Chapter 3.3.3 --- Comparison with current structure-based method --- p.34 / Chapter 3.3.4 --- Performance evaluation using OSCTdb --- p.36 / Chapter 3.4 --- Discussion --- p.37 / CHAPTER 4 --- p.49 / Comparative Analysis of Reversible and Structural Disulfides to Reveal Their Distinct Characteristics --- p.49 / Chapter 4.1 --- Introduction --- p.50 / Chapter 4.2 --- Materials and methods --- p.52 / Chapter 4.2.1 --- Datasets --- p.52 / Chapter 4.2.2 --- Functional site signatures --- p.53 / Chapter 4.2.3 --- Electrostatic properties --- p.55 / Chapter 4.2.4 --- Support vector machines (SVMs) implementation and parameter optimization --- p.56 / Chapter 4.2.5 --- Performance assessment --- p.56 / Chapter 4.2.6 --- Statistical analyses --- p.56 / Chapter 4.3 --- Results --- p.56 / Chapter 4.3.1 --- General features of the functional site signatures for reversible-SS Cys and structural-SS Cys --- p.56 / Chapter 4.3.2 --- Differences in amino acid composition are related to several physical-chemical properties --- p.58 / Chapter 4.3.3 --- Electrostatic characteristics of disulfide-bonded Cys --- p.60 / Chapter 4.3.4 --- Comparison of S-S distance --- p.61 / Chapter 4.3.5 --- Predictive power of newly identified features --- p.62 / Chapter 4.4 --- Discussion --- p.63 / Chapter 4.4.1 --- Reversible disulfides show distinct characteristics compared with structural disulfides --- p.63 / Chapter 4.4.2 --- Chances and challenges for computational prediction of reversible disulfides --- p.64 / CHAPTER 5 --- p.79 / Conclusions and Perspectives --- p.79 / Chapter 5.1 --- Contributions and conclusions from this thesis research --- p.80 / Chapter 5.2 --- Future perspectives --- p.81 / Reference --- p.82 / List of Publications --- p.95

Circadian clocks are endogenous pacemakers found in many organisms including plants, generating approximately 24h rhythms. Knowledge about the plant circadian clock plays a role for crop improvement. The plant circadian clock and its downstream outputs have been studied in detail by transcriptomics, however post-transcriptional and post-translational aspects are still to be researched. In addition, it has recently been shown that a protein modification remains rhythmic when rhythmic transcription is absent. This gives evidence for the existence of two oscillators: a transcription-translation feedback loop and a non-transcriptional oscillator. The aim of this PhD is to gain knowledge about circadian changes in abundance and phosphorylation of proteins as well as protein-protein interaction using the model plant Arabidopsis thaliana. I used high-throughput proteomics and phosphoproteomics methods to identify hundreds of phosposites that change in abundance in WT plants as well as dozens of proteins that exhibit circadian changes in their abundance. I also found significant temporal changes in protein phosphorylation in the transcriptionally arrhythmic mutant CCA1-Ox, albeit with dynamics different from the WT, demonstrating that without transcriptional rhythms, protein modification can still undergo rhythmic changes to some extent. In addition, I found reproducibly that the majority of changing phosphopeptides are most abundant at dawn and this is independent of the presence of a functional transcriptional oscillator. Roles of different kinases and affected phosphoproteins are discussed. I chose one of the rhythmically phosphorylated proteins, the bifunctional enzyme F2KP, for further functional experiments. In vitro experiments demonstrate that the rhythmic phosphosite is important for the activity of the enzyme. This is discussed in the light of circadian regulation of carbon metabolism. In addition to these studies on circadian protein abundance and modification, I investigated time-of-day dependent protein-protein interaction of the clock protein GIGANTEA (GI). Using an interaction proteomics timecourse, I identified about 100 potential new interactors of GI, some of which are candidates for links between diel timing and carbon metabolism. These results will help to generate hypotheses for explaining the surprising pleiotrophy of gi mutants.

Parkinson’s Disease (PD) is the second most common neurodegenerative disorder after Alzheimer’s and old age is the strongest risk factor for developing PD. PD has traditionally been seen as a motor disorder, but its non-motor symptoms such as dysautonomia, sensory dysfunction, sleeping problems and neuropsychiatric features equally add to the disease burden. There is no cure for PD and this is probably a reflection of our poor understanding of the disease pathogenesis. One way of tackling this is to focus on the small, but significant number of PD patients with a family history compatible with Mendelian autosomal inheritance (10-15%). Hereditary and sporadic PD share important clinical and neuropathological features, and there is reasonable hope that dissecting molecular pathways of PD gene products will have more general implications for the pathophysiology of PD associated neurodegeneration and help device new treatment strategies. Mutations in the FBXO7 gene have recently been shown to cause an autosomal recessive early onset Parkinsonian-pyramidal syndrome and FBXO7 has been designated as PARK 15 (Di Fonzo et al., 2009). FBXO7 is a member of the F-box protein family, which functions as the variable subunit of Skp1-Cullin1-F-box protein (SCF) E3 ubiquitin ligase complexes and as such dictate substrate specificity. The canonical outcome of ubiquitylation is proteasomal degradation and my working hypothesis is that FBXO7 may be involved in protein quality control in the brain. A perturbation thereof may be a first step towards FBXO7 dependent disease. At the time of starting with my PhD project, little was known about the molecular function of FBXO7 and how mutations in FBXO7 result in neurodegeneration. In order to learn more and dissect the signalling pathway of FBXO7 I have used tagged stable overexpression cell lines of the FBXO7 wildtype as well as human disease mutant proteins for tag-pulldowns followed by mass-spectrometry to identify interacting partners and possible substrates. With this approach I have been able to confirm the interaction between FBXO7 and its core SCF E3 ligase partners as well as some of the previously reported interacting partners. I have been able to show that not only the FBXO7 wildytpe protein, but also all of the so far reported human disease mutants are able to assemble into an SCF complex. Hence, my fist conclusion is that the human disease mutants do not exert their pathogenicity by SCF complex disruption. Next, a knock-in (KI) mouse model of one of the pathogenic FBXO7 mutations (R378G) was generated and evaluated by molecular and biochemical approaches as well as motor and behaviour phenotyping. In particular, I have used the Fbxo7 mouse model for extensive proteomic screens to identify wildtype (wt) and KI Fbxo7 interactors: endogenous Fbxo7 immunoprecipitations from mouse brain lysates and subsequent fingerprint mass-spectrometry; differential whole proteome: ex vivo differential dimethyl labelling of wt and KI brain samples, and Fbxo7-dependent ubiquitinome analysis: quantitative di-GLY capture proteomics combining in vivo SILAC labelling with antibody-based affinity enrichment of “di-GLY remnant motifs”- containing peptides prior to proteomic profiling of the wild-type in comparison to the homozygous R379G Fbxo7 KI ubiquitinome in MEF lysates. The di-GLY remnant motif is the signature peptide of ubiquitinylated protein sites at peptide level after tryptic digestions. Some of my findings are: • For the first time I show that endogenous Fbxo7 actually assembles into an Skp1-Cullin1-Fbxo7 complex and that the pathogenic R378G does not disrupt SCFFbxo7-KI complex formation in vivo. This is true for the Fbxo7 KI mouse model, but also for patient derived immortalized cell lines carrying the R378G FBXO7 mutation.• Endogenous Fbxo7 interacts with the Sumo E3 ligase complex RanBP2/ RanGAP1*Sumo1/Ubc9 complex. • In the differential enrichment of ubiquitylated protein species in SILAC labelled wild-type and homozygous R379G Fbxo7 KI MEFs, I have clearly identifies 2 highly conserved lysine residues, which are conserved amongst VDAC 1, 2, and 3 in mouse as well as human homologous, to be preferentially ubiquitinylated in a Fbxo7 wild-type background (in collaboration with Dr. Patrick Pedrioli, MRC Programme leader).• There is a significant difference in motor performance between wildtype and homozygous R379G KI Fbxo7 mice at 10 months of age (in collaboration with Dr. Steve Martin, Neuroscience Division, Dundee). • Furthermore, I have successfully set up an in vitro FBXO7 dependent ubiquitinylation assays.