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Multiple versions of a parent gene can function within molecular systems as gene duplicates (paralogs) and alternatively spliced isoforms. Proteins related in this manner often serve redundant roles, though they can be selectively or randomly prescribed unique functions. The present collection of three manuscripts details the evolution of members of the deubiquitinating enzyme superfamily. The first manuscript delineates the chronology of USP4, USP15 and USP11 emergence and concludes that the presumed ancestor, USP11, is in fact a recent duplicate and that, at minimum, one copy of USP4 or USP15 is required for organismal viability. The second determines that the long and short isoforms of mammalian USP4 are maintained by natural selection to occupy discrete spatial roles. The final manuscript broadens the scope and objectively draws the genealogy of all deubiquitinating enzymes, with emphasis on significant points of functional divergence of paralogs within innate immunity and DNA repair pathways.

Squalene synthase (EC 2.5.1.2.1; SQS) is located at a branch point in the isoprenoid pathway and catalyzes the condensation of two molecules of farnesyl diphosphate to form squalene. SQS activity contributes to the formation of triterpenes and sterols, including phytosterols, brassinosteroids, cholesterol, and in potato plants, steroidal glycoalkaloids (SGAs). These compounds have diverse functions in the plant. SGAs are defense compounds that deter feeding by potato pests. The wild potato Solanum chacoense accumulates higher amounts of SGAs than cultivated potato and some of its accessions produce leptines, a rare class of SGAs that is toxic to Colorado potato beetle. Unlike most eukaryotes, higher plants have more than one gene coding for SQS. Three sqs gene homologs were isolated from S. chacoense, sqs1Sc, sqs2Sc, and sqs4Sc, that have 74 to 83% identity at the amino acid level. Some of the amino acid differences between sqs isoforms are likely to affect enzyme activity. Each of the three genes contained an intron in the 3'UTR. This feature may have a role in the nonsense-mediated decay of incomplete sqs mRNAs. A partial SQS polypeptide retaining catalytic activity but lacking the membrane anchoring domain could adversely affect a cell with the randomly distributed accumulation of squalene. The mRNA of sqs1Sc and sqs2Sc was detected in all tissues whereas sqs4Sc transcript was limited to bud tissue. The sqs2Sc transcript was less uniformly distributed in the plant than sqs1Sc and accumulated most abundantly in floral tissue. The results demonstrate that the three sqs genes have different patterns of gene expression and encode proteins with different primary structures indicating distinct roles in plant squalene metabolism. / Master of Science

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Hedgehog (Hh) signalling from the notochord induces the slow muscle cell fate in the adaxial cells of the developing zebrafish embryo. Slow muscle formation is disrupted in zebrafish ??you-type?? mutants resulting in U-shaped somites. In many you-type mutants, genes encoding components of the Hh signalling pathway are mutated. scube2, a gene not previously known to be involved in Hh signalling, is disrupted in the you-type mutant ??you??. you mutants are deficient in several Hh dependent cell types and show decreased expression of Hh target genes. The Scube (signal peptide-CUB domain-EGF-related) family of proteins act as secreted glycoproteins or cell-surface proteins and are thought to be involved in protein-protein interactions and ligand binding. At the protein level, the Scube family resembles the endocytic receptor Cubilin. Cubilin is known to interact with another endocytic receptor Megalin, which can function as an endocytic receptor for Sonic Hedgehog (SHH) in vitro. Megalin endocytosis of Shh may be an important part of the Hh signal transduction pathway. An anti-Scube2 antibody was developed during this work to investigate the intracellular localization pattern of Scube2 and facilitate the identification of potential Scube2 binding partner(s). In addition, this work identified and characterized two homologs of scube2 in zebrafish, scube 1 and scube 3. The high level of similarity amongst the Scube family of proteins and the weak phenotype of the you mutant suggested scube1 and scube3 might also be involved in slow muscle development. Loss of function experiments performed by antisense morpholino knockdown of scube1 and scube3 in the you mutant decreases the expression of Hh target genes to levels seen in embryos lacking Hh signalling and dramatically enhances the loss of slow muscle fibres compared to you mutants alone. Thus, injecting both scube1 and scube3 morpholinos into you blocks Hh signalling and these embryos fail to develop slow muscle. Inhibition of the three partially redundant scube genes inhibits Hh signalling in zebrafish embryos, thereby demonstrating the essential requirement for scube gene function in the Hh signalling pathway.

Hedgehog (Hh) signalling from the notochord induces the slow muscle cell fate in the adaxial cells of the developing zebrafish embryo. Slow muscle formation is disrupted in zebrafish ??you-type?? mutants resulting in U-shaped somites. In many you-type mutants, genes encoding components of the Hh signalling pathway are mutated. scube2, a gene not previously known to be involved in Hh signalling, is disrupted in the you-type mutant ??you??. you mutants are deficient in several Hh dependent cell types and show decreased expression of Hh target genes. The Scube (signal peptide-CUB domain-EGF-related) family of proteins act as secreted glycoproteins or cell-surface proteins and are thought to be involved in protein-protein interactions and ligand binding. At the protein level, the Scube family resembles the endocytic receptor Cubilin. Cubilin is known to interact with another endocytic receptor Megalin, which can function as an endocytic receptor for Sonic Hedgehog (SHH) in vitro. Megalin endocytosis of Shh may be an important part of the Hh signal transduction pathway. An anti-Scube2 antibody was developed during this work to investigate the intracellular localization pattern of Scube2 and facilitate the identification of potential Scube2 binding partner(s). In addition, this work identified and characterized two homologs of scube2 in zebrafish, scube 1 and scube 3. The high level of similarity amongst the Scube family of proteins and the weak phenotype of the you mutant suggested scube1 and scube3 might also be involved in slow muscle development. Loss of function experiments performed by antisense morpholino knockdown of scube1 and scube3 in the you mutant decreases the expression of Hh target genes to levels seen in embryos lacking Hh signalling and dramatically enhances the loss of slow muscle fibres compared to you mutants alone. Thus, injecting both scube1 and scube3 morpholinos into you blocks Hh signalling and these embryos fail to develop slow muscle. Inhibition of the three partially redundant scube genes inhibits Hh signalling in zebrafish embryos, thereby demonstrating the essential requirement for scube gene function in the Hh signalling pathway.

A família gênica Forkhead P {FOXP) tem sido alvo de muitos estudos envolvendo evolução do cérebro e comportamento animal. Destacam-se particularmente as investigações com o gene FOXP2, que indicam que mudanças neste gene estariam associadas com a evolução da vocalização em algumas espécies de mamíferos, incluindo o Homo sapiens. Recentemente, estudos de desordem intrínseca de proteínas (IDPs) tem ganhado ênfase no contexto evolut ivo, visto que uma correlação posit iva entre regiões de desordem e altas taxas evolutivas tem sido observada. Através de um conjunto de abordagens que inclui predizer o conteúdo de desordem e os motivos lineares de interação, bem como as taxas evolutivas, buscamos desvendar a historia evolutiva dos genes da subfamília FOXP. Concentramos nossas análises sobre regiões desordenadas das proteínas FOXPl, FOXP2, FOXP3 e FOXP4 encontradas em 77 espécies de tetrápodes. Tais regiões proteicas são normalmente negligenciadas em estudos dessa natureza, pois se localizam fora de seus tra dicionais domínios conservados, normalmente associados à função principal da proteína. Sít ios apontados estando sob seleção positiva e relaxamento da restrição seletiva mostraram-se hotspots importantes para mudanças que podem impactar na capacidade de interação das proteínas. Encontramos que os maiores valores de w são mais prevalentes em regiões desordenadas que em ordenadas. Ainda, alto e similar valor de desordem (70%) foi encontrado nas 77 proteínas ortólogas de FOXPl , FOXP2, e FOXP4, indicando a manutenção de um "padrão geral" sobre um longo tempo evolutivo. Portanto, a variabilidade tanto de aminoácidos quanto de motivos lineares dentro das regiões de desordem foi marcante. A proteína FOXP3 apresentou menor nível de desordem (30%), mas signif icante sinal de seleção positiva em alguns sítios. Composição idênt ica de resíduo de aminoácido e/ou motivos lineares em espécies filogeneticamente distantes, indica clara convergência molecular, provavelmente associada a pressões seletivas similares. Sucessivamente, nossos achados mostraram uma clara diferença na composição de motivos lineares entre mamíferos e não mamíferos, dando suporte para a importância dos estudos de evolução da interatividade proteica para as compreensões de características taxa-específicas. / Forkhead Family P (FOXP) has been target of many studies about brain and behavior evo lution among species. FOXP2 receives special attention in academic society, due associations with vocalízation evolution in mammals, including Homo sapiens. Recently, intrinsically disorder proteins studies have gained emphasis in the evolutionary context, as positive correlation between disorder regions and higher evolutionary rate has been observed. Through a set of approaches, including disorder and linear motif predictions, as well as estimate evolutionary rates, we aimed to unveil the evolutionary history of FOXP subfamily genes. We focused our ana lysis over disordered regions of FOXPl, FOXP2, FOXP3 and FOXP4 proteins retrieved in 77 tetrapods. Such protein regions are usually neglected in studies of this nature, for being localized out of the traditional conserved domains, usua lly associated with the main function of the protein. Sites indicated as under relaxation of selective constrains or positive selection have shown to be important hotspots for changes that can impact in protein interaction capability. Higher w va lues are prevalent in disordered regions than in ordered ones. Still, high and similar disorder proportion (~70%) was found among 77 orthologues proteins of FOXPl, FOXP2 and FOXP4, indicating general pattern of disorder maintenance, along tetrapod's evolutionary tree. However, amino acid and linear motifs variability within disordered regions was observed. FOXP3 protein presented lower disorder leveis (~30%), when compared with other paralogues, but signal of positive selection was observed in some sites. ldentical composition of amino acid residues and/or linear motifs is, probably, associated with similar selective pressure. Successively, ou r results showed clear differences in linear motif composition between mammals and non-mammals, supporting the importance of evolutionary studies on protein interaction for the understanding of taxa-specifics characteristics.

ABSTRACT The success of land plants can be attributed to the evolution of beneficial associations between plant roots and soil microbes. Root-microbe mutualisms extend the range of plant nutrient acquisition delivered through the hyphal network of mycorrhiza, an ancient and widespread plant symbiosis, or by the more recent adaptive innovation of nitrogen-fixing nodule symbioses. A plant’s genetic toolkit governs its selection of beneficial symbionts and the developmental extent of these intimate interactions. However, the evolutionary origins and function for only a few symbiotic signaling components have been explored. The central aim of this dissertation is to resolve the evolutionary events that contributed two, novel genetic components for establishing root symbioses, NPF1B and NPF1C. The Medicago truncatula (Mt) LATD/NIP/NPF1.7C transporter functions in root and nodule meristems and is a member of the large NPF1 gene subfamily. Here, I propose that LATD/NIP’s role in establishing nitrogen-fixing symbioses is derived from the ancient mycorrhizal signaling pathway. I used a comparative phylogenomic approach to investigate the evolutionary origins of the NPF1 gene across flowering plants and then asked whether diversifying or purifying selection forces influenced NPF1 gene retention. I postulated that such gene retention correlates with the adaptive traits of mycorrhizal or nitrogen-fixing root nodule symbiosis; to test this I measured trait correlation within my dataset. I found that the NPF1 phylogeny is comprised of five well-supported angiosperm clades, A, B, C, D1 and D2, that arose by successive duplications and have unequal gene retention. NPF1B is present as a single copy gene or lost entirely, while the other major NPF1 clades expanded to multiple genes within angiosperms. The NPF1A, B and C genes are under strong purifying selection while the NPF1D genes display positive, diversifying selection. My data revealed a statistically significant correlation of NPF1A, B, C, and D2, but not NPF1D1, gene retention with the ability of a species to form mycorrhizal associations. Additionally, the retention of the NPF1B, C, D1, D2, but not NPF1A, genes within a species is statistically correlated with its ability to form nitrogen-fixing symbiosis. Supporting this correlation, NPF1B genes are expressed in plant root tissues with and without mycorrhizal fungi yet available datasets failed to detect NPF1B expression in nodule tissues whereas the NPF1C genes are expressed in both symbiotic and non-symbiotic plant root tissues. In support of functional conservation, expression of legume LATD/NIP cDNAs from Cicer arietinum (Ca) and Lotus japonicus (Lj) restored, in part, the root and nodule defects of the Mtlatd mutant and resulted in the formation of peculiar hybrid lateral root-nodule structures while, in wild-type M. truncatula, significantly augmented root development. In L. japonicus, the disruption of LATD/NIP alters the number of lateral roots and nodules My thesis data support the hypothesis for an ancestral NPF1 gene function in establishing mycorrhizal associations in angiosperms and, consequent to the monocot-eudicot divergence, co-opted this function for accommodating nitrogen-fixing symbioses in eudicots. Successive duplications then yielded the NPF1B and NPF1C genes that, by neofunctionalization and natural selection, further refined their roles in root organogenesis and symbiosis; a prerequisite for the evolution of nodule organs.

Stress activated protein kinase (SAPK) interacting protein 1 (Sin1) is a member of a recently characterized gene family, conserved from yeast to humans. The gene copy number is strictly conserved (one Sin1 gene per genome), and the protein may be expressed ubiquitously in mammalian tissues. The Sin1 family has been implicated in several different signal transduction pathways. Originally identified as a partial cDNA and candidate Ras inhibitor, recent functional studies have revealed interactions with an interferon (IFN) receptor subunit (IFNAR2), and the SAPK JNK. Interactions have also been described between the yeast orthologues and the phosphatidylinositol kinase TOR2. Collectively, these data suggest that Sin1 has an important cellular role, and this study has investigated possible functions for this protein. As human Sin1 proteins have no paralogues within the genome, secondary structure homology was used to identify major domains within the protein. Four major domains within human Sin1 were deduced: an N-terminal domain containing a functional nuclear localization signal, a functional nuclear export signal, and a coiledcoil region; the conserved region in the middle that is likely to be a ubiquitin-like β-grasp protein binding domain; a Ras binding domain; and a pleckstrin homology-like domain that targets Sin1 to the plasma membrane and lipid rafts in vivo. Full and partial length EGFP constructs were used to examine the localization of human Sin1, and several isoforms derived from alternative splicing. All isoforms localized to the nucleus and nucleolus. Beyond this, Sin1α and Sin1ϒ had cytoplasmic staining, while Sin1 and Sin1β were also found at the plasma membrane and lipid rafts. Both the N-terminal domain and the conserved region in the middle were found to contribute to nuclear localization. Comparative genomic analysis between human, mouse, rat, dog, and chicken Sin1 genes revealed a number of conserved intronic regions, and the putative functions of these were predicted. Additionally, a putative promoter module within a CpG island and encompassing the transcription start site was predicted in all species. The human CpG island was found to have promoter activity in HEK293 cells. Using bioinformatics, genes that may be co-regulated with Sin1 were identified. These genes contained the Sin1 promoter module, and were found to co-express in large scale gene expression studies. Most of these genes were directly involved in the cellular response to pathogen infection, suggesting a conserved role for Sin1 in this pathway. Key biochemical functions of the Sin1 proteins were also identified, including the ability of Sin1 proteins to form dimers, and the ability of over-expressed Sin1 to induce apoptosis (mediated through the conserved region in the middle). Additionally, endogenous Sin1 protein levels were found to change following serum deprivation and hypoosmotic stress. Together, these studies have provided significant insight into the cellular role of Sin1, suggesting a role in inducing apoptosis as part of the IFN response to viral infection. The biological significance of the Sin1 proteins is discussed in the context of their predicted functions and the evolution of the protein family.