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During the evolution of multicellularity, the unit of selection transitions from that of the single cell to that of the integrated multicellular organism. Cellular specialization mediates this transition, as the evolution of differentiated cell types leads to fitness becoming a property of the multicellular organism. How does cellular specialization evolve? While we know that genetic changes result in the development of new cell types, it remains unclear whether the development of a new cell type can be ancestrally plastic prior to coming under developmental-genetic control via genetic assimilation. We use the volvocine green algae as a model system to address the overarching question of whether the plastic development of a new cell type preceded its fixation via genetic assimilation. Previous research on the evolution of cellular differentiation in this clade has determined that a gene necessary for somatic cell development is present in species with and without soma and that an ancestral stress response was co-opted during the evolution of somatic differentiation. This raises the possibility that somatic cells may have originated as a plastic trait prior to coming under developmental-genetic control. Here, I show that Eudorina species previously characterized as undifferentiated develop a small proportion of cells resembling soma following exposure to cold shock, an environmental stressor. We also find that the offspring of cold-shocked colonies (but not the grand-offspring) also develop somatic-like cells. We show that these cells are morphologically consistent with the somatic cells seen in closely related species that are obligately differentiated. We find that somatic-like cells in cold-shocked colonies are controlled by cell-level, temporal regulation while the production of somatic-like cells in the offspring of cold-shocked colonies are controlled by group-level, developmental regulation induced by the maternal environment. We propose that cell-level contro

Amyloid promotes a dramatic transition in protein conformation that perpetuates, giving rise to a broad variety of distinct phenotypes, ranging from pathological disorders to dynamic heritable traits. Amyloid has long been thought to be resistant to clearance by the proteostasis network, but increasing evidence is challenging this view. For example, heat shock disassembles yeast prion amyloids, revealing in vivo solubilization of these aggregates. However, the exact proteostatic niche that promotes amyloid clearance is largely unknown. We identified several environmental stresses leading to prion curing via the same mechanism as heat shock and further showed that a shared characteristic was the activation of the transcription factor heat shock factor 1 (Hsf1). Strikingly, artificial Hsf1 activation interfered with heat shock-mediated prion curing, presumably due to overexpression of a nucleotide exchange factor Sse1. Limiting Sse1, which decelerates the Hsp70 cycle, promoted chaperone loading on prion aggregates and enabled artificial Hsf1 activation to resolve prion aggregates; in contrast, it impaired resolution of stress-induced aggregates and cell growth at elevated temperature. Thus, our study demonstrates that the proteostasis network, fine-tuned for optimal dissolution of non-amyloid aggregates, can be reconfigured for solubilization of amyloid by modulating the Hsp70 cycle.

My thesis is focused on role of soft matter (membrane lipids plus water) in G-protein coupled receptor (GPCR) activation. Notably GPCRs are one of the hot topics that have attracted considerable attention in pharmacology and drug companies. About 50% of the modern drugs available in the market target GPCRs. Although GPCRS are of immense importance in pharmacology, critical information is missing about their activation mechanism. The major reason is that extensive studies have not been done in native conditions. However GPCRs are integral membrane protein and the role of membrane lipids and water is crucial in rhodopsin signaling, which has not been investigated considerably. Here we used rhodopsin, a canonical GPCR, to study the role of soft-matter (membrane lipids and water) in rhodopsin signaling. We hypothesize that rhodopsin activation involves an ensemble of states with an increase in hydrated protein volume. The opening of the protein paves the way for G-protein binding and initiation of rhodopsin signaling. Since rhodopsin is an integral membrane protein, the properties of membrane lipids could modulate rhodopsin activation. We used a novel charge-induced directed-reconstitution method to recombine rhodopsin spontaneously with artificial membranes and polymerosomes. UV-visible spectroscopy was used to characterize the light activation of the protein. We report a novel allosteric mode of rhodopsin activation, where electrostatic interaction between positively charged membrane head-groups and negatively charged Glu134 disrupts the ERY motif of the second ionic lock, and leads to constitutive activation of the protein. Our results are in agreement with the molecular dynamics simulations analysis, and are further supported by results using the ATR-FTIR spectroscopy technique. In addition we have studied the role of water in rhodopsin function using different size osmolytes. We observed a surprising size-reversal effect on the rhodopsin activation mechanism. Small

Patients with nonketotic hyperglycinemia and deficient glycine cleavage enzyme activity, but without mutations in AMT, GLDC or GCSH, the genes encoding its constituent proteins, constitute a clinical group which we call 'variant nonketotic hyperglycinemia'. We hypothesize that in some patients the aetiology involves genetic mutations that result in a deficiency of the cofactor lipoate, and sequenced genes involved in lipoate synthesis and iron-sulphur cluster biogenesis. Of 11 individuals identified with variant nonketotic hyperglycinemia, we were able to determine the genetic aetiology in eight patients and delineate the clinical and biochemical phenotypes. Mutations were identified in the genes for lipoate synthase (LIAS), BolA type 3 (BOLA3), and a novel gene glutaredoxin 5 (GLRX5). Patients with GLRX5-associated variant nonketotic hyperglycinemia had normal development with childhood-onset spastic paraplegia, spinal lesion, and optic atrophy. Clinical features of BOLA3-associated variant nonketotic hyperglycinemia include severe neurodegeneration after a period of normal development. Additional features include leukodystrophy, cardiomyopathy and optic atrophy. Patients with lipoate synthase-deficient variant nonketotic hyperglycinemia varied in severity from mild static encephalopathy to Leigh disease and cortical involvement. All patients had high serum and borderline elevated cerebrospinal fluid glycine and cerebrospinal fluid:plasma glycine ratio, and deficient glycine cleavage enzyme activity. They had low pyruvate dehydrogenase enzyme activity but most did not have lactic acidosis. Patients were deficient in lipoylation of mitochondrial proteins. There were minimal and inconsistent changes in cellular iron handling, and respiratory chain activity was unaffected. Identified mutations were phylogenetically conserved, and transfection with native genes corrected the biochemical deficiency proving pathogenicity. Treatments of cells with lipoate and with mito

Intersections of distinct biological pathways in cells allow for nodes of metabolic regulation. This work describes the discovery of the intersection of two pathways in yeast mitochondria: RNA processing and fatty acid synthesis and attachment. Analysis of the components of the pathways is presented here along with a model illustrating the connection as a potential mode of regulation of mitochondrial gene expression.A genome-wide screen of respiratory-deficient Saccharomyces cerevisiae deletion strains for defects in mitochondrial RNA processing revealed that two novel genes affect processing of mitochondrial tRNAs by RNase P. One gene encodes Htd2, an enzyme in the type II mitochondrial fatty acid synthesis pathway (FAS II). The other gene is described here as encoding Lip3, an enzyme involved in the synthesis and attachment of the co-factor lipoic acid, which is synthesized from a product of the FAS II pathway.RPM1 is the mitochondrial-encoded RNA subunit of mitochondrial RNase P. The multigenic transcription unit containing RPM1 also contains tRNApro. Maturation of RPM1 necessitates processing of the tRNA by RNase P. Thus, RNase P is required for maturation of its own RNA component, constituting a positive feedback cycle. The present work demonstrates that a product of the FAS II pathway is necessary for the assembly or activity of RNase P, as deletion of any gene encoding an FAS II enzyme results in inefficient processing of tRNApro from the transcript.Analysis of the enzymes involved in the synthesis and attachment of lipoic acid to target proteins is also described here. Disruption of any of these enzymes affects protein lipoylation and tRNA processing. Gcv3, a target of lipoylation, was found to be required for lipoylation as well as for efficient tRNA processing.A second feedback cycle controlling pyruvate dehydrogenase activity and fatty acid synthesis may be functional under certain conditions. Pyruvate dehydrogenase, which provides acetyl-CoA for the FAS

Taura syndrome (TS) is one of the most devastating diseases affecting the shrimp farming industry worldwide. The causative virus, Taura syndrome virus (TSV), has been identified. My work is centred on the development of monoclonal antibodies against TSV. I have also characterized a novel variant of the virus from Venezuela and evaluated the effect of hyperthermia on TSV infection. This work has resulted in 3 manuscripts, which constitute the core of this dissertation. The taxonomy throughout this dissertation is done according to Holthuis (1980).The first manuscript describes the production of a monoclonal antibody reacting with the Belize strain of TSV. The antibody, MAb 2C4, exhibits good sensitivity and specificity for TSV in immunohistochemistry (IHC) and dot blot immunoassay. MAb 2C4 reacted with the TSV-HI94, TSV-SI98 and TSV-BZ02 variants, but not with the TSV-VE05 and TSV-TH05 variants. This antibody adds and improves tools to those available for TSV diagnosis.Chapter three describes a relatively novel variant of TSV from Venezuela, which was characterized by our laboratory. By genetic sequencing, this new isolate exhibits a 94% similarity with TSV-HI94. IHC, dot blot immunoassay and bioassays were also performed. While processed samples reacted only weakly with the TSV monoclonal antibody MAb 1A1, the virus in its native state reacted strongly with the antibody. In bioassays, TSV-VE05 presented mortality comparable to TSV-HI94 in Penaeus vannamei. These data confirm the presence of TSV in Venezuela and that a new variant of the virus was responsible for the outbreak of TS.In chapter four, the behavior of TSV infection under hyperthermic conditions was examined. I compared the susceptibility of Kona stock P. vannamei to the infection by two variants of TSV under hyperthermic conditions (32oC). Shrimp, infected with TSV-HI94, were resistant to infection at high temperature. However, under the same hyperthermic conditions, the challenged shrimp were fully susc

Individual isolates of the fungus Nectria haematococca exist in a wide range of habitats and part of this diversity is attributed to the presence of conditionally dispensable (CD) chromosomes that carry habitat-defining genes. In the current study a new factor located on one of these CD chromosomes was found. This trait allows pea pathogenic isolates of N. haematococca to grow in homoserine, a compound present in large amounts on pea root exudates. The gene(s) for homoserine utilization (HUT) are located on the same CD chromosome that carries the cluster of genes for pea pathogenicity, the PEP cluster. The PDA1 gene, a member of the PEP cluster, is routinely used as a marker for the presence of this CD chromosome, therefore it has been called the PDA1-CD chromosome. For the purpose of identifying the HUT gene(s), a physical map of the PDA1-CD chromosome was constructed. This map, in combination with synteny analysis, and Southern hybridizations led to the identification of a region of 365Kb that is likely to contain the HUT gene. By searching the publicly available genome of N. haematococca several candidates for HUT were identified.The synteny evaluation between the PDA1-CD chromosome and a different CD chromosome that carries the MAK1 gene, for chickpea pathogenicity, revealed a region (> 463Kb) of synteny, which advocates for a common ancestor for these CD chromosomes. However a large region (~ 1 Mb) in each of the CD chromosomes was found to carry unique DNA, therefore we proposed that individual isolates of this fungus contain large regions of unique DNA located on the CD chromosomes. The localization of syntenic regions also suggests that breakage points previous identified in the MAK1-CD chromosome could potentially be "hot spots" for recombination between both CD chromosomes. Furthermore, the anchoring of the PDA1-CD map to the genome of N. haematococca allowed the identification of additional putative habitat colonization genes present on both CD chromosome