The currently known network of human protein-protein interactions (PPIs) is providing new insights into diseases and helping to identify potential therapies. However, according to several estimates, the known interaction network may represent only 10% of the entire interactome - indicating that more comprehensive knowledge of the interactome could have a major impact on understanding and treating diseases. The primary aim of this thesis was to develop computational methods to provide increased coverage of the interactome. A secondary aim was to gain a better understanding of the link between networks and phenotype, by analyzing essential mouse genes. Two algorithms were developed to predict PPIs and provide increased coverage of the interactome: FpClass and mixed co-expression. FpClass differs from previous PPI prediction methods in two key ways: it integrates both positive and negative evidence for protein interactions, and it identifies synergies between predictive features. Through these approaches FpClass provides interaction networks with significantly improved reliability and interactome coverage. Compared to previous predicted human PPI networks, FpClass provides a network with over 10 times more interactions, about 2 times more proteins and a lower false discovery rate. This network includes 595 disease related proteins from OMIM and Cancer Gene Census which have no previously known interactions. The second method, mixed co-expression, aims to predict transient PPIs, which have proven difficult to detect by computational and experimental methods. Mixed co-expression makes predictions using gene co-expression and performs significantly better (p < 0.05) than the previous method for predicting PPIs from co-expression. It is especially effective for identifying interactions of transferases and signal transduction proteins. For the second aim of the thesis, we investigated the relationship between gene essentiality and diverse gene/protein features based on gene expression, PPI and gene co-expression networks, gene/protein sequence, Gene Ontology, and orthology. We identified non-redundant features closely associated with essentiality, including centrality in PPI and gene co-expression networks. We found that no single predictive feature was effective for all essential genes; most features, including centrality, were less effective for genes associated with postnatal lethality and infertility. These results suggest that understanding phenotype will require integrating measures of network topology with information about the biology of the network’s nodes and edges.

Regional and global trends in aquaculture production, value and price are assessed for the last 30 years relative to trends in wild caught species. Based on data from the Fisheries and Aquaculture Department of the Food and Agriculture Organization of the United Nations for aquaculture production, data is extracted for the first time to address regional (Europe, North America, South America, Africa, Oceania and Asia) trends in production focused on the top five aquaculture produced species. Previous uses of the database have largely focused on global production. Of the top five species (whiteleg shrimp Penaeus vannamei, Atlantic salmon Salmo salar, silver carp Hypophthalmichthys molitrix, common carp Cyprinus carpio, and giant tiger prawn Penaeus monodon), Asia accounts for most of the global production (with the exception of Atlantic salmon Salmo salar). The central issue considered in this thesis concerns the likelihood and capacity of aquaculture production of fish and shellfish protein for human consumption relative to that of exploited wild stocks. Over the last 30 years or so, aquaculture production has risen exponentially and captures of wild caught fish have now plateaued. The relative status, rearing practices, production and basic economic perspectives of the principle aquaculture produced species globally are compared with wild caught production. The principle finding is that total global aquaculture production will exceed that of commercial wild caught species by about 2015. The significance of this is discussed in terms of current views of environmental (e.g. pollution, disease and habitat degradation) and economic (e.g. production level, farm price, marketing economics, fixed costs (facility and equipment depreciation, loan interest, land lease, fixed wages), variable costs (cost of seed stock, feed, energy)) impacts of aquaculture. Similarly, these issues are considered for the fishing industry (e.g. fishing down the food web, likelihood of expansion of bottom fisheries into deeper waters, reduction of biodiversity, declining global catches). It is concluded that aquaculture is a necessity and that if current trends continue aquaculture production can more than supplement human fish protein needs even in the given context of the rapid growing population, but that in the long term aquaculture production will itself be substantially supplemented by “rebounding” wild fishery production.

Bovine adenovirus type 3 (BAdV-3) is a non-enveloped icosahedral particle which contains a double stranded DNA genome. The genome of BAdV-3 is organized into early, intermediate and late regions. The late region is organized into seven regions L1-L7 (Reddy et.al., 1998). The L6 region of late transcription unit of BAdV-3 encodes one of the non structural protein named 33K protein. The objective of the present study was to characterize the 33K protein and to identify the viral/cellular proteins involved in the interaction with 33K protein.<p> The RT-PCR analysis revealed the presence of spliced and unsliced mRNAs encoding 33K and 22K proteins respectively in BAdV-3 infected cells. The 33K and 22K proteins share a N-terminus region of 138 amino acids. To determine the specificity of these two proteins, rabbit polyclonal antiserum was raised against peptides representing unique C- terminal regions of the proteins. Anti-33Kp serum detected two major proteins of 42 kDa and 22 kDa and five minor proteins of 39kDa, 35kDa, 29kDa, 25kDa and 19kDa in BAdV-3 infected cells or 33K transfected cells. Similarly, anti-22Kp serum detected three proteins of 41kDa, 39kDa and 37kDa in BAdV-3 infected cells. However, a protein of 39kDa and 37kDa was detected in 22K (having splice sites removed) transfected cells. The 33K protein is predominantly localized to the nucleus of BAdV-3 infected cells and is involved in stimulating the transcription from major late promoter. Analysis of mutant 33K proteins demonstrated that amino acids 201-240 and amino acid 204-231 are required for nuclear localization and MLP transactivation.<p> The adenovirus 33K protein appears to be a multifunctional protein performing different role in viral infection. Earlier study has shown that the 33K protein plays a role in viral capsid assembly and efficient capsid DNA interaction in BAdV-3 (Kulshreshtha et.al., 2004). The involvement of 33K protein in different steps of adenovirus replication may require protein protein interaction. Using 33K protein as bait in yeast two hybrid system, open reading frames (ORFs) of BAdV-3 were screened for the potential interactions with 33K protein. The 33K protein showed specific interactions with two late viral proteins- 100K and protein V (pV). The yeast two hybrid findings were validated by in vitro binding using <i>in vitro</i> synthesized transcription-translation products. It was demonstrated that the interaction of 33K with 100K and pV takes place during BAdV-3 infection. The stretch of amino acids 81-120 and 161-200 in 33K protein were involved in the interaction with pV and 100K protein.<p> For screening the cellular interactions, the 33K protein was used as a bait to screen bovine retina cDNA library. The yeast two hybrid screening revealed that the 33K protein appears to interact with bovine presenilin-1-associated protein / mitochondrial carrier homolog 1 (BoPSAP / BoMtch1) and bovine microtubule associated protein (BoMAP). However, subsequent analysis by various <i>in vitro</i> and <i>in vivo</i> assays could only confirm the interaction between 33K protein and BoPSAP/BoMtch1. In addition, the 33K protein was also shown to be colocalized with BoPSAP in mitochondria. Based on these observations, it may be possible that 33K protein may play an anti-apoptotic by interacting with BoPSAP since the human homolog of PSAP has been known to induce apoptosis.

Protein-protein interactions regulate and drive biological processes and understanding the assembly of these interactions is important. The LysR-Type Transcriptional Regulators (LTTRs) are a large family of transcriptional regulators found in prokaryotes. I have used the LTTRs as a model for protein specificity. In order to understand a residue’s contribution to oligomerization, alanine-scanning mutagenesis was used to probe the contribution of residues identified from in silico analysis of two proteins: OxyR and CynR. The contribution of the residues to oligomerization was characterized using lcI repressor fusions. In OxyR, seven residues were identified as hot spots. Moreover, these hot spots are not especially conserved. The interaction surface of OxyR was mapped onto a multiple sequence alignment of the LTTR family. This mapping identified putative contacts in the CynR regulatory domain dimer interface. Combined with the in vivo testing, three residues were identified as hot spots. The residues identified in OxyR and CynR do not overlap. To investigate the assembly of the LTTRs I used a negative-dominance assay with lcI repressor fusions. Taken together, I show that the LTTRs in E. coli K-12 are mostly specific in their interactions.

Proteins need fold to perform their biological function. Thus, understanding how proteins fold could be the key to understanding life. In the first study, the stability and structure of several !-hairpin peptide variants derived from the C-terminus of the B1 domain of protein G (PGB1) were investigated by a number of experimental and computational techniques. Our analysis shows that the structure and stability of this hairpin can be greatly affected by one or a few simple mutations. For example, removing an unfavorable charge near the N-terminus of the peptide (Glu42 to Gln or Thr) or optimization of the N-terminal charge-charge interactions (Gly41 to Lys) both stabilize the peptide, even in water. Furthermore, a simple replacement of a charged residue in the turn (Asp47 to Ala) changes the !-turn conformation. Our results indicate that the structure and stability of this !?hairpin peptide can be modulated in numerous ways and thus contributes towards a more complete understanding of this important model !-hairpin as well as to the folding and stability of larger peptides and proteins. The second study revealed that PGB1 and its variants can form amyloid fibrils in vitro under certain conditions and these fibrils resemble those from other proteins that have been implicated in diseases. To gain a further understanding of molecular mechanism of PGB1 amyloid formation, we designed a set of variants with mutations that change the local secondary structure propensity in PGB1, but have similar global conformational stability. The kinetics of amyloid formation of all these variants have been studied and compared. Our results show that different locations of even a single mutation can have a dramatic effect on PGB1 amyloid formation, which is in sharp contrast with a previous report. Our results also suggest that the "-helix in PGB1 plays an important role in the amyloid formation process of PGB1. In the final study, we investigate the forces that contribute to protein stability in a very general manner. Based on what we have learned about the major forces that contribute to the stability of globular proteins, protein stability should increase as the size of the protein increases. This is not observed: the conformational stability of globular proteins is independent of protein size. In an effort to understand why large proteins are not more stable than small proteins, twenty single-domain globular proteins ranging in size from 35 to 470 residues have been analyzed. Our study shows that nature buries more charged groups and more non-hydrogen-bonded polar groups to destabilize large proteins.

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During co-translational integration, the transmembrane (TM) sequence of a nascent membrane protein moves laterally into the ER lipid bilayer upon reaching the translocon. Our lab has previously shown that this movement is a multistep process, but it was not clear whether the observed photocrosslinking of the TM segment to translocon proteins resulted from specific interactions or simply from TM-translocon proximity. If the latter, the TM &#945;-helix will be oriented randomly with respect to translocon proteins, whereas, if the former, a specific TM helix surface would face TRAM and/or Sec61&#945;. Integration intermediates were prepared by in vitro translation of truncated mRNAs in the presence of a Lys-tRNA analog with a photoreactive moiety attached to the lysine side-chain. When photoadduct formation was monitored as a function of probe location within the TM &#945;-helix, we found that the extent of photocrosslinking to TRAM and Sec61&#945; was non-random. Thus, the TM sequence occupies a distinct location within the translocon, a result that can only be achieved through protein-protein interactions that mediate the lateral movement, positioning, and integration of the TM sequence. In the case of multi-spanning membrane proteins, it was unknown how multiple hydrophobic regions integrated into the ER membrane. By placing photoprobes within each of several TM domains of a multi-spanning membrane protein, we were able to determine at what stage of integration each TM segment was no longer adjacent to translocon proteins. Using this approach we were able to establish a mechanism of integration for multi-spanning membrane proteins co-translationally inserted into the ER membrane.

Thesis (Ph.D.)--Ohio University, August, 2009. / Release of full electronic text on OhioLINK has been delayed until September 1, 2012. Includes bibliographical references (leaves 89-107)

Thesis (Ph.D.)--Ohio University, August, 2009. / Title from PDF t.p. Release of full electronic text on OhioLINK has been delayed until September 1, 2012. Includes bibliographical references (leaves 89-107)

Zugl.: Göttingen, Univ., Diss., 2009