Drosophila cadherin Cad99C is a critical regulator of follicle cell microvilli during Drosophila oogenesis. I extended the functional analysis of Cad99C by identifying Drosophila Myosin VIIA, and Cad74A and Cad87A, which are homologues of human Myosin VIIA and Cadherin 23, respectively, as components of the Cad99C protein complex and by studying their interactions in follicle cell microvilli. My co-immunoprecipitation data show that the cytoplasmic tail of Cad99C interacts with Myosin VIIA, and the extracellular cadherin repeats of Cad99C interact with Cad87A and Cad74A, independent of the cytoplasmic tail of Cad99C. Genetic studies indicate that 1) Cad99C and Cad74A/Cad87A localize independently to microvilli, although their amount is affected by changes in the ratio of their concentrations. 2) All cadherins affect microvillus morphology when overexpressed although the phenotypes are different. The overexpression effect of Cad74A depends on Cad99C but not vice versa. These data point to complex interactions between the microvillus cadherins.

Reversible protein phosphorylation is an essential regulatory mechanism used by eukaryotes to coordinate the biochemical processes of the cell. The PP2A phosphatase functions to dephosphorylate specific proteins originally targeted by stimulus-activated protein kinases. The identification of a sub-network surrounding the human PP2A-Striatin holoenzyme, termed STRIPAK, provides insight into novel mechanisms for PP2A function and regulation. I reveal that STRIPAK participates in at least two mutually-exclusive sub-complexes, one of which contains the putative cortactin-binding protein, CTTNBP2NL. I show that CTTNBP2NL is enriched at the actin cytoskeleton, likely in a STRIPAK-independent manner. This study also reveals that STRIPAK interacts with a subunit of the dynein motor, at least partially, through CTTNBP2NL. This work will serve as a platform for the structural and functional characterization of STRIPAK and will ultimately assist in defining novel mechanisms of regulation and function for the human PP2A phosphatase.

The Saccharomyces cerevisiae origin recognition complex (ORC) binds to replication origins at the ARS consensus sequence (ACS), serving as a scaffold for the assembly of replication complexes needed for the initiation of DNA synthesis. I generated a genome-wide map of nucleosome positions surrounding replication origins because the precise locations of nucleosomes may influence replication. My map revealed a nucleosome-free region surrounding the ACS that is bordered by two well-positioned nucleosomes. I was able to explain differences in origin properties by clustering nucleosome profiles. I found an association between the replication time and nucleosome profile for a given origin cluster. An ORC depletion mutant nucleosome map indicated a shift in nucleosomes towards the ACS. I present the first genome-wide view of origin nucleosome architecture, indicate a relationship between chromatin structure and replication timing, and suggest a model whereby the interplay between DNA sequence and ORC binding defines the nucleosome occupancy pattern.

The erythroblastoma (ErbB) receptor family consists of four members that are implicated in many human cancers. To gain insight into their biological function, we investigated their interacting partners to derive a comprehensive protein interaction network. Using the membrane yeast two-hybrid (MYTH) system we probed the ErbB2, ErbB3, and ErbB4 interaction space and validated a subset of interacting partners using the luminescence-based mammalian interactome mapping (LUMIER) system. The integrated use of these two complementary protein interaction technologies generated high confidence data and identified many novel ErbB binding partners, a subset of which was supported by co-immunoprecipitation in the breast adenocarcinoma cell line Sk-Br-3 including the GPCR GPRC5B, the cysteine protease CAPN1, and WIF1, a secreted protein containing 5 EGF domains that may represent a novel ErbB ligand. Our systematic approach offers an unbiased systems level view that may identify novel drug targets and contribute to therapeutic research.

Neuroblastoma (NB) is the most common extra-cranial solid tumor in children. Recently discovered neuroblastoma tumor-initiating cells (NB-TICs) have many properties of cancer stem cells and form tumors with as few as 10 cells. To elucidate the signaling pathways driving NB- TIC survival and proliferation, we surveyed the phospho-tyrosine containing subset of the NB- TIC proteome. Over 300 phosphorylated proteins were identified, including 21 tyrosine kinases of which several belong to the Src kinase family. Using bioinformatics tools, several hematopoietic signaling pathways were identified, including the B cell receptor (BCR) pathway. Further proteomic approaches substantiated molecular hematopoietic features in NB-TICs. Inhibitors of BCR proximal kinases SYK and SFKs were cytotoxic to NB-TICs. Clinically utilized inhibitors of SFKs induce apoptosis in NB-TICs. Targeting hematopoietic survival pathways in NB-TICs from the bone marrow, which have thus far not been predicted to play a role in this neural malignancy, may provide new drug therapies for NB.

The pediatric sarcoma alveolar rhabdomyosarcoma (ARMS) is highly aggressive with a poor prognosis for diagnosed patients. Here, we demonstrate that targeting the unique oncogene integrin-linked kinase (ILK) in ARMS cells in conjunction with the common chemotherapy agent vincristine, a synergistic effect is found in the reduction of cell viability in vitro. This result was achieved by both RNAi-mediated depletion of ILK and using a small molecule kinase inhibitor specific for ILK. Both techniques were found to disrupt important protein interactions at the site of the centrosome. Combination ILK disruption and vincristine treatment of cells induced the expression of apoptotic markers and arrested cells in the G2/M stage of the cell cycle. Interestingly, protein levels of JNK and its target c-Jun were regulated with combined treatment. Altogether, these findings indicate that the use of molecular targets like ILK may further improve the clinical treatment of ARMS.

The aims of this thesis were to characterize the molecular ecology of the hyporheic zone – between dissolved organic matter (DOM) and microbes – and to test whether seasonal and spatial patterns existed in correlation with seasonal ecosystem processes. The hyporheic zone is an area of vertical integration between groundwater and surface water, and lateral integration between terrestrial and stream ecosystems. Colonization corers were used to collect in situ DOM and bacterial communities from the hyporheic sediments of two streams that varied in hydroperiod (i.e., permanent vs. intermittent). DOM was collected using passive samplers and analyzed using 1H NMR and fluorescence spectroscopy; bacteria were characterized using terminal-restriction fragment length polymorphism. At the permanent site, bacteria correlated significantly with seasonal environmental factors including: fall communities with DOM concentration; spring and winter communities with nitrate concentrations; and summer communities with temperature. Bacterial communities at the intermittent site were significantly correlated with flooding as a function of hydrologic connectivity. Sediment communities were discriminated between hyporheic sediments and interstitial porewaters, and shared several operational taxonomic units (OTUs). Sediment communities were more distinct when hydrologic connectivity was low, and porewater communities changed dramatically upon flooding. Fifteen out of 259 OTUs were shared across aquatic sediments, interstitial porewater and watershed soil samples. DOM was spatially and seasonally dynamic in both sites. Five key DOM groups described using 1H NMR spectroscopy revealed spatial differences between the permanent and intermittent sites. EEM-PARAFAC models confirmed that despite significantly different molecular components, the relative sources of DOM at both sites were similar, including humic-like terrestrial sources and tyrosine (microbial) sources. This study provides new knowledge on both organic matter dynamics and bacterial communities in a dynamic aquatic ecotone, and also confirmed the hypothesis that bacterial communities correlated significantly with ecosystem processes within a watershed.

To date, most genome-scale approaches designed to explore kinase pathways have been targeted towards substrate identification for individual kinases but provide little functional information and only a limited view of the interplay of kinases and their targets in key biological processes. I attempted to tackle the complexity of kinase networks using an unbiased integrated global analysis in budding yeast. I used functional genomics screens to study the yeast kinome using combinatorial genetic perturbations. I first assessed the effects of gene overexpression on the fitness of non-essential kinase deletion mutants to generate a comprehensive view of Synthetic Dosage Lethal (SDL) interactions involving yeast kinases. These data were complemented by assessing genome-wide Synthetic Lethal (SL) interactions for kinases that gave SDL interactions. By measuring >600,000 potential interactions between kinase-gene pairs, I produced a meta-network of ~1300 dosage lethal interactions and 7500 negative and positive genetic interactions. I reasoned that by combining two complementary genetic datasets for kinases, I could: 1) characterize the unexpected phenotypic outcomes that result from the interplay of gain-of-function and loss-of-function phenotypes; 2) better comprehend the complexity of kinase signaling networks and; 3) predict the function of novel genes that arise from the combined genetic network and a gold standard list of known kinase-substrate pairs in the literature. The SDL network alone was enriched for pathways known to be regulated by cognate kinases including phosphoproteins, and kinase targets and kinases that yielded informative SDL interactions were largely those with cell polarity roles. Condition-specific screens and analysis of kinase double mutants suggested that the apparent resistance of many kinases to genetic perturbation cannot be solely attributed to kinase redundancy but most likely reflects the requirement for many kinases in certain activating conditions. Next, I created the first systematic gold standard for kinase-substrate pairs and generated the first kinase interaction database, specifically curated for experiments pertaining to kinase-substrate relationships, in order to analyze the SDL network for identification of kinase targets. Also, using a novel approach that combines the gold standard and the integrated SDL-genetic interaction meta-network, I found that the integrated network is more functionally informative than either SGA or SDL networks alone. Additional integration of known kinase-substrate relationships extracted from the biochemical literature into this network was key to the identification of recurring motifs that enable accurate prediction of single mutant phenotypes. I have also identified ~2000 triplet regulatory network motifs, unraveling novel pathways regulated by kinases. I have tested several motifs using phenotypic and biochemical assays and have identified a novel gene involved in the regulation of cell wall integrity pathway, and found a new regulatory mechanism involving the mitotic exit network machinery. My study provides a general framework for predicting phenotypic outcomes from different combinations of genetic mutations, but also delineates the complexity of signaling pathways involving phosphorylation. The identified network motifs belie the simplistic notion of linear kinase cascades, and support a complex network model where multi-dimensional signaling waves dictate a phenotypic outcome. My data suggest that unraveling the complexity of network biology demands an unbiased global analysis using an integrated set of functional genomics approaches with a high quality gold standard.

The Varkud satellite (VS) plasmid is found in the mitochondria of some strains of Neurospora, and exhibits properties that may allow it to be developed as a genetic transformation vector to study mitochondrial molecular biology. An ideal transformation vector would not confer a phenotype. The overall goal of my thesis was to examine interactions of VS with its Neurospora host to identify possible phenotypes, using biochemical and proteomic approaches. Biochemical experiments provided evidence consistent with the plasmid transcript, VS RNA, being present as a ribonucleoprotein particle that can be separated from ribosomal subunits by sucrose density gradient centrifugation; however, no VS-specific proteins were identified under the purification conditions examined. During the analyses of proteomic data I obtained new insights into the consequences of the statistical methods commonly used to normalize quantitative 2D gel data. However, irrespective of the method used, the fraction of the proteome amenable to 2D gel-based proteomics revealed, at most, subtle effects of VS on the abundance of a few proteins. I also observed no differences in growth rate between strains differing by the presence or absence of VS when grown in the presence of inhibitors and stressors affecting a wide range of mitochondrial and other cellular functions. Overall, despite VS RNA being as abundant as the large and small mitochondrial ribosomal RNAs, my genetic, biochemical and proteomic investigations of the effect of VS on its host strain provides evidence that VS is a phenotypically neutral element.

Bioremediation of chlorinated solvents to a non-toxic end product can be achieved with Dehalococcoides sp., through reductive dehalogenation of the chlorinated organics. Dehalococcoides sp. are typically maintained in enrichment cultures containing multiple microorganisms, which often exhibit better growth and dechlorination rates than Dehalococcoides isolates. This thesis examines the nature of the relationships between the Dehalococcoides and the non-dechlorinating organisms in enrichment cultures. Comparative metagenomics revealed differences and similarities in taxonomy and functional gene complements between three Dehalococcoides-containing enrichment cultures. This allowed identification of pivotal supporting organisms involved in maintaining dechlorination activity through provision of nutrients and other factors to the Dehalococcoides. A Dehalococcoides pan-genus microarray was designed using available sequenced genomes as well as a draft genome generated from an in-house metagenome sequence. The array leverages homolog clustering during probe design to improve detection of the Dehalococcoides genus, including strains not utilized in the array design. A phylogenetic examination of the reductive dehalogenase gene family showed that organism and gene phylogenies are not linked, indicating vertical inheritance of reductive dehalogenases is not a primary mechanism of acquisition. Design of a universal PCR primer suite targeting a curated database of reductive dehalogenase homologous genes was used to characterize the reductive dehalogenase complement of four environmental sites and two enrichment cultures. Using an enrichment culture containing three phylogenetically distinct dechlorinating organisms, the interactions of niche-specific organisms were examined through single-cell genome sequencing. From the partial genome sequences, novel reductive dehalogenase genes were identified, as well as evidence of lateral gene transfer between the three dechlorinating organisms. This research primarily utilizes genomic and metagenomic datasets, which serve as metabolic blueprints for prediction of organisms’ functions. The results presented in this thesis advocate in favour of phylogenetic diversity within enrichment cultures to maintain functional redundancy, leading to more robust cultures for bioremediation efforts.