Malaria is a life-threatening disease caused by Plasmodium parasites that are spread through the bites of infected mosquito vectors. It is a worldwide pandemic that threatens 3.4 billion people annually. Currently, there are only a few validated Plasmodium drug targets, while drug resistance continues to rise. This marks the urgency for the development of novel parasite-specific therapeutics. Plasmodium falciparum diverges from the paradigm of the eukaryotic cell cycle by undergoing multiple rounds of DNA replication and nuclear division without cytokinesis. A better understanding of the molecular switches that coordinate the progression of the parasite through the intraerythrocytic developmental stages will be of fundamental importance for the design of rational intervention strategies. To achieve this goal, we performed an isobaric tag-based approach for a system-wide quantitative analysis of protein expression and site-specific phosphorylation events of the Plasmodium asexual developmental cycle in the red blood cells. This study identified 2,767 proteins, 1,337 phosphoproteins, and 6,293 phosphorylation sites. Approximately 34% of identified proteins and 75% of phosphorylation sites exhibit changes in abundance as the intraerythrocytic cycle progresses. Because the links between Plasmodium protein kinases as key cell cycle regulators to cellular events are largely unknown, it is of importance to define their cognate physiological substrates. To test the hypothesis that genetic screening would be a useful approach for discovery of candidate substrates of a protein kinase, we used the orphan kinase PfPK7 as a model. Our comparison of the phosphoproteome profiles between the wild-type 3D7 and PfPK7- parasites identified 146 proteins with 239 phosphorylation sites exhibiting decreased phosphorylation in the absence of PfPK7 at the developmental stages where nuclear division and merozoite formation occur. Further analysis of the decreased phosphorylated events revealed three motifs that are enriched among phosphorylated sites in proteins that are down regulated. In vitro kinase assays were done to validate the potential substrates of PfPK7 and to elucidate the signaling events that are regulated by PfPK7. In parallel to our experimental analysis, we used a computational approach for substrate prediction from our phosphoproteome dataset. This analysis identified 43 distinct phosphorylation motifs and a range of proline-directed potential MAPK/CDK substrates. To identify substrates/ interactors of Plasmodium CDK-like kinases, we also used HA-tagged CDK-like kinases, PfPK6 and Pfmrk lines. Co-immunoprecipitation of the HA-tagged PfPK6 and Pfmrk baits, followed by mass spectrometric analyses, identified the components of the protein interaction complexes of these kinases. Our analyses of HA-PfPK6 and HA-Pfmrk immunoprecipitates identified 15 and 21 proteins in the interaction complex, respectively. The ability of recombinant PfPK6 and Pfmrk to interact and/or utilize any of the proteins identified in the interaction complex as substrates was verified through in vitro kinase assays and pull-down analysis. This study is the most comprehensive definition of the constitutive and regulated expression of the Plasmodium proteome during the intraerythrocytic developmental cycle, and offered an insight into the dynamics of phosphorylation during the asexual cycle progression [1]. In summary, this study has 1) defined the constitutive and regulated expression of the Plasmodium proteome during its asexual life cycle, 2) demonstrated that fluctuation and reversible phosphorylation is important for the regulation of P. falciparum*s unique cell cycle, 3) provided the foundation for quantitative phosphoproteomic analysis of kinase negative mutants to understand their function, 4) provided a major step towards defining kinase-substrate pairs operative within parasite*s signaling networks, and 5) generated a preliminary interactome for PfPK6.

Biofouling is the accumulation of sessile marine organisms, such as algae, tube worms and barnacles on man-made substrata and has negative economic and ecological implications. Ship hulls are readily fouled, which significantly increases drag while decreasing ship fuel efficiency when moving through water. Fouled hulls have also become important vectors of invasive species. These problems are minimized when hulls are painted with a toxic anti-fouling or non-toxic foul-release coating. Due to recent restrictions of anti-fouling paint use, research and development of non-toxic alternatives has increased. Novel hull coating efficiency is often quantified by the critical removal stress value of barnacles from the coating. Barnacle adhesive cement protein content is thought to be responsible for barnacles’ incredible ability to adhere underwater. The expression level and type of adhesive proteins has eluded scientists due to their extreme insolubility within cured barnacle cement. Identification of these proteins may provide insight to the adhesion of fouling species and aid coating development. Barnacles are a cosmopolitan organism, able to withstand a wide range of environmental conditions, yet foul-release coating research had not previously incorporated environmental factors as variables in determining coating performance. Temperature is known to affect protein structure and function and is also a formative factor of barnacle larvae survival and development. Even so, the interaction between temperature and barnacle adhesion to has not previously been explored. We examined the effect of temperature on barnacle adhesion to foul-release coatings. After observing differences in critical removal stress due to temperature, we attempted to attribute these differences to specific proteins within the adhesive using 2D SDS PAGE. Gel image analysis determined that there were significant differences in cement protein expression between barnacles raised within different temperatures. Preliminary protein identification with Mass Spectronomy (MALDI TOF/TOF) was performed, however further research and a larger barnacle genomic database is needed to elucidate barnacle cement protein sequences.

Ciliates are important model organisms that have been used to study many aspects of cellular biology, including telomeres, histone modifications, and ribozymes. These unicellular eukaryotes house both a germline genome and a somatic genome in distinct nuclear structures within a single cell. One of their most unique features is their ability to undergo complex programmed genome rearrangements, during which their germline genome is fragmented and rearranged to form a new somatic genome. This rearrangement process results in a highly specialized somatic genome with many polyploid short chromosomes that are rich with genes. While all ciliates can undergo this developmental process, Oxytricha trifallax experiences particularly complex rearrangements that result in a more radically unconventional structure in its somatic genome. Much of the previous work studying Oxytricha has been focused on the complex rearrangements that it undergoes during sexual development and the mechanisms that allow it to perform these genome rearrangements events at the level of accuracy required for proper somatic function afterwards. Due to this particular focus on Oxytricha sexual development, the rest of Oxytricha’s unique biology has not been studied to the same degree. For my thesis I examined two aspects of Oxytricha biology that have not been well understood. In Chapter 1 I report the results of a proteomic survey of both types of nuclei found within the vegetative cell, the somatic macronucleus and the germline micronucleus. We performed mass spectrometry on enriched samples of both nuclear types and analyzed the enrichment of proteins between the two. Despite some mitochondrial contamination, we found that many categories of functional proteins were enriched in one of the two nuclei. We validated the appropriate nuclear localization of specific proteins from each subcategory through imaging Our results confirmed many previously predicted aspects of the two nuclei and provide a valuable resource for further studies on nuclear proteins in Oxytricha. In Chapter 2 I describe various features of a comparative analysis between the somatic genomes of multiple strains of Oxytricha trifallax. Previous work from the lab has focused primarily on the reference strains JRB310 and JRB510, which are most commonly used due to their ability to mate. We generated four new draft assemblies of the somatic genomes of strains JRB27, JRB39, SLC89, and SLC92. Many metrics demonstrate that these new assemblies are largely complete. Our analyses of these new strains revealed that there are numerous strain-specific chromosomes in Oxytricha that can encode genes. While they do not seem to encode core genes that would be missing otherwise, they are prime candidates for further examination to identify mating type-related genes.

Oxidative stress is commonly associated with diet-induced metabolic syndrome (MetS) and left ventricular cardiac remodeling, but much remains unknown about the role of redox signaling, sensors, and switches in mediating the effects of high fat and sugar intake. In this work, I describe and apply an optimized method for quantifying changes in reversible protein-cysteine oxidation in the heart. This method uses isobaric tagging of cysteine thiols and mass spectrometry in a modified biotin switch on whole tissue lysate. Analyzing the resulting data with systems biology approaches helped delineate redox pathways playing a role in disease development, while cysteine-specificity provided exact targets for mutation-based mechanistic studies. Initial findings in a mouse model for MetS, wherein C57Bl6J mice were fed a high fat/high sucrose diet, identified energy pathways as the primary target of changing reversible cysteine oxidation. In follow-up studies, our collaborators helped validate the pathophysiological role of two particular cysteines in complex II; their early reversible oxidation and later irreversible oxidation contributed to decreased ATP output from cardiac mitochondria. A subsequent, more robust study revealed a weakness in our original method. While investigating the role of hydrogen peroxide-induced oxidative post-translational modifications (OPTMs) in the development of MetS sequelae, analysis of four mouse groups, each with an n=5, revealed that measurements of reversibly oxidized cysteine thiols were highly variable compared to those of all available thiols. Thus, I developed a strategy to address the source of variability and, in the process, improved many additional steps in the switch protocol. Finally, in an effort to clarify the role of the most stable reversible OPTM, glutathionylation (RSSG), we characterized the HFHS diet response in mice engineered to have more or less RSSG via genetic manipulation of glutaredoxin-1 expression. Those with more RSSG suffered worsened cardiac function, making them an ideal model for future studies with the methods optimized in this work. Studying the progression from poor diet to cardiac involvement in these and other mouse models using the methods described herein will aid in the design of diagnostics and targeted therapies against the growing burden of metabolic CVD.

The ovarian hormone 17β-estradiol (E2) is one of the central regulators of the female reproductive system. E2 is also a pleiotropic regulator since it can exert its non-reproductive role on other organ systems. E2 is neuroprotective, it maintains body's energy homeostasis, participates in various repair mechanism and is required for neural development. However, there is a substantial evidence suggesting that there might be a molecular reprogramming of E2's action when it is supplied exogenously after E2 deprivation. Though the length of E2 deprivation and age has been linked to this phenomenon, the molecular components and how they activate this reprogramming is still elusive. Our main goal was to perform global proteomics and metabolomics study to identify the molecular components and their interaction networks that are being altered in the brain and serum after a short-term E2 treatment following ovariectomy (OVX) in Sprague Dawley rats. One of the strength of our global study is that it gave us extensive information on the brain proteome itself by identification of a wide number of proteins in different brain sections. By analyzing the differentially expressed proteins, our proteomics study revealed 49 different networks to be altered in 7 sections of the brain. Most of the perturbed networks were involved in cell metabolism, neural development, protein synthesis, cellular trafficking and degradation, and several stress response signaling pathways. We assessed the neuroenergetic status of the brain based on E2's response to various energy generating pathways, including glycolysis, TCA cycle, and oxidative phosphorylation, and several signaling pathways. All energetics pathways were shown to be downregulated in E2 treatment, which suggests that E2 exerts its neuroprotective role by restoring energy homeostasis in OVX rat model by regulating complex signaling and metabolic networks. Our second focus was to determine the metabolite response (amino acids and lipids) after E2 treatment in the brain and serum by employing targeted metabolomics study. We have found that in rat brain cortex there was significant upregulation of a wide number of amino acids suggesting alternate route of metabolism. Another alternate explanation is that E2 replacement replenished the amino acid pool in the tissue. Pathway enrichment analysis revealed upregulation of several pathways, including amino sugar metabolism, purine metabolism, and glutathione metabolism. By combining proteomics and metabolomics in two different biological matrices we were able to gather a vast array of information on how E2 replacement after E2 deprivation can confer neuroprotection. Our findings will help to create a foundation of basic science to be used for developing potentially effective hormone therapies.

The recognition of small organic compounds and metabolites is essential for living systems, enabling the cell to sense environmental stimuli and respond appropriately. Developing quantitative models of living systems which can incorporate these environmental stimuli would accordingly benefit from comprehensive mapping of interactions between proteins and small molecules of interest. While high-throughput experimental methods provide a wealth of interaction data, the scale of chemical space currently precludes comprehensive enumeration of protein-compound interaction space. Computational methods can help to bridge this gap by inferring proteome-scale protein-compound interactomes, elucidating structural features within protein families which mediate specificity of binding to specific small molecules, and inferring the affinity of binding for specific protein-compound interactions. In this thesis, we attempt to use, and in some cases develop, methods to study protein-compound interactions at these three scales. First, we describe recent work in extending our structure-based algorithm for predicting protein-compound interactions throughout the proteome to include a wider array of small molecules. We demonstrate that this method performs comparably to existing methods and describe an online database storing the results of this analysis. We also report several case studies illustrating how this database can be used along with cautionary vignettes indicating areas where the method fails and directions for future improvement. We subsequently analyze druggable pockets occurring within protein-protein interfaces (PPIs) to assess whether they are less structurally conserved than analogous pockets of conventional drug sites. We find that PPI interfacial pockets are associated with fewer expected off-targets than conventional drug sites, however that this finding is specific to individual protein families, rather than a general feature of interfacial PPI pockets. Finally, we use Free Energy Perturbation to predict the binding affinity of an array of small volatile odorants with an olfactory receptor from the jumping bristletail, Machilis hrabei, as well as attempt to further optimize the system in order to study the effects of mutating receptor binding site residues on binding affinity to its active ligands.

This study aimed to understand the mechanism of maize inbreds resistance to A. flavus by exploring the proteins that are differentially regulated in presence of pathogen. Silk has been hypothesized as one of the entry routes of fungal growth and so the proteome of silks was investigated by 1) performing a comparative proteomic study to identify silk proteins that are abundant in resistant maize inbreds and down-regulated or absent in susceptible inbreds, 2) identifying the up-regulated proteins in maize resistant and susceptible inbreds when challenged by A. flavus 3) by mapping the proteome of silk proteins in a A. flavus resistant inbred and 4) performing an antiungal assay to test antiungal activity of silk proteins extracted from resistant and susceptible maize inbreds. Using comparative proteomics, proteins that are contributing to the resistance phenotype and could be used for marker-assisted selection in breeding programs were identified from silks collected from resistant (Mp313E, Mp420) and susceptible (SC212m, Mp339) maize inbred 21 and 25 days after silk emergence (DAS) and also, from the silks of ears inoculated at 15 DAS and collected 6 days after inoculation (DAI). Silk proteins were extracted and analyzed by 2-dimensional gel electrophoresis (2-DE). Gel images were analyzed by PD Quest software (Bio-Rad) and proteins that were consistently different were identified using MALDI-TOF-TOF. Two candidate genes that were up-regulated in 21 and 25 DAS in resistant tissues were investigated for polymorphisms and their RNA expression was also studied. Nine proteins from all the differentially regulated proteins were mapped to chromosomes 1, 2, 4 and 6 which are known to have aflatoxin resistance QTLs. Proteome map of Mp313E silks was developed using 2-DE and multi dimensional identification technology (MudPIT) and approximately 971 identified proteins were functionally annotated from the sequences available at AgBase website. The reference map of Mp313E silks could also be used to link proteomics with trancriptomics, metabolic mechanisms and genomics. Antifungal assays using GFP-tagged A.flavus and chitinase assay on silk proteins from resistant and susceptible corn inbreds showed significant activity in the resistant line compared to the susceptible line (p<0.01). A model describing the role of silk proteins in fungal resistance is proposed.

Listeria monocytogenes is a deadly, Gram-positive foodborne pathogen that is ubiquitous in the environment. The bacterium expresses a number of virulence and stress adaptation proteins that support its pathogenic capabilities. Two-dimensional gel electrophoresis (2-DE) was used to map L. monocytogenes surface proteins, which play a central role in virulence, and to examine protein expression by L. monocytogenes grown on ready-to-eat meat, an important source of Listeria infections. A novel method for solubilization of surface proteins from L. monocytogenes for 2-DE was developed. Additionally, the unique proteome expressed by L. monocytogenes grown on a meat matrix was uncovered. The developed solubilization method will facilitate efforts to identify and routinely compare surface proteins of Listeria by 2-DE. Furthermore, the 2-DE database of proteins expressed by L. monocytogenes grown on a meat matrix will allow further understanding of the interactions of Listeria with its food environment that influence its ability to cause disease.

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