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Comparative genomic analysis of proteome reduction in the apicomplexans
(2020-05-16)
Apicomplexans are alveolate parasites which include Plasmodium falciparum, the main cause of malaria, one of the world’s biggest killers from infectious disease. Apicomplexans are characterized by a reduction in proteome size, which appears to result from metabolic and functional simplification, commensurate with their parasitic lifestyle. However, other factors may also help to explain gene loss such as population bottlenecks experienced during transmission, and the effect of reducing the overall genomic information content. The latter constitutes an ‘informational constraint’, which is proposed to exert a selective pressure to evolve and maintain genes involved in informational fidelity and error correction, proportional to the quantity of information in the genome (which approximates to proteome size).<br />
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In this dissertation, the dynamics of gene loss is examined in 41 Apicomplexan genomes using orthogroup analysis. This work shows that loss of genes involved in amino acid metabolism and steroid biosynthesis can be explained by metabolic redundancy with the host. There is a marked tendency to lose DNA repair genes as proteome size is reduced. This may be explained by a reduction in size of the informational constraint and can help to explain elevated mutation rates in pathogens with reduced genome size.<br />
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Effective population size (Ne) has a direct contribution to evolutionary changes. In these species, Ne is not well studied due to the morphological and genomic complexity. In order to measure Ne, model species P. falciparum is chosen whose mutation rate and generation time are already predicted. MSMC analysis indicates a recent bottleneck, consistent with predictions generated using allele-based population genetics approaches, implying that relaxed selection pressure due to reduced population size might have contributed to gene loss. However, the nonrandomness of pathways that are lost challenges this scenario.<br />
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Malaria is an ancient disease and yet, there is no effective cure or prevention. This study looks for new antimalarial targets to identify unique orthogroups in malaria causing Plasmodium species that infect humans, with a high proportion of membrane associated proteins. Thus, orthogroup analysis appears useful for identifying novel candidate pathogenic factors in parasites, when there is a wide sample of genomes available.<br />
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In terms of biodiversity, Apicomplexa has many unclear taxonomic structures. In this study, a statistically robust phylogeny is reconstructed by concatenating 522 genes from the core Apicomplexan genome which account for 6068 amino acid sequences. Different biases and pitfalls among alignments and phylogeny inference methods are also discussed.<br />
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Lastly, this study provides a foundation for future experimental research, specific and comparative analysis of Apicomplexan proteomes....
Transcript, structural, and histologic characterization of a novel EF-Hand protein and its isoform specific to the Ambulacraria clade
(2020-02-19)
Transcriptomic databases have become one of the main sources for protein discovery. In our studies of transcripts from normal echinoderms, we have identified several transcripts that have attracted our attention. One of these is a previously unidentified transcript (Orpin) that appeared to be upregulated during intestinal regeneration. In Chapter 2, using bioinformatics tools we: (1) identified a second Orpin sequence (2) describe their motifs and domains, and perform phylogenetic analyses that suggest that Orpins might comprise a novel subfamily of EF-hand containing proteins specific to the Ambulacraria clade. Semiquantitative RT-PCR analyses revealed that Orpin mRNAs are expressed in various tissues but no significant differential expression was found in regenerating tissues.<br />
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In Chapter 3 we expressed and purified genetically modified versions of Orpin to further characterize this EF-hand protein. We developed two protocols:<br />
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The first protocol consisted in the production of His-OBSm, which is His tagged with two additional genetic modifications: the deletion of the signal peptide encoding sequence and the addition of a 25 residues peptide from the pET200 plasmid vector. These modifications made possible the purification of a soluble recombinant version of Orpin B.<br />
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The second protocol was developed to produce a soluble recombinant Orpin that best resembled the original protein. This protocol consisted of the expression of a GST-Orpin lacking the signal peptide region. For this protocol, we tested different parameters that affect protein expression, including, additives, host cells, growth media and supplementation, and protein extraction methods. The best parameters were identified and used to obtain a soluble Orpin form. We propose that the developed strategies can be used to increase the soluble expression and purification of other EF-hand proteins that are difficult to express by standard methods.<br />
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In chapter 4 we produced antibodies against His-OBSm and used these to identify the cells expressing the protein in H. glaberrima tissues. Antibody specificity was tested through Western Blots.<br />
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Finally, we investigated the effect of the Wnt signaling pathway on Orpin mRNA expression in regenerating gut explants and found an apparent decrease expression of an Orpin B isoform following Wnt/B-catenin pathway activation....
Physiological and immunological acclimation in corals differing in life-history traits: a field experiment
(2020-05-20)
Corals can exhibit much variability in life-history strategies. A proposed categorization recognizes competitive, stress-tolerant and weedy species. 'Competitive' corals invest more energy towards growth, 'stress-tolerant' towards maintenance and 'weedy' towards reproduction. While competitive corals are declining due to climate change and anthropogenic stressors, stress-tolerant and weedy are thriving. We performed a reciprocal transplant experiment between a degraded and a more pristine reef, and measured the physiological (Symbiodinium density, chlorophyll a concentration, protein concentration) and immunological (oxidative stress) response of the stresstolerant Pseudodiploria strigosa and Orbicella annularis, and the weedy Porites astreoides. Results indicate that the studied species can rapidly and successfully acclimate to local environmental conditions. Comparisons among species suggest that the physiological and immunological response in P. astreoides, P. strigosa and O. annularis are more influenced by species-specificity than by life-history strategy....
Exploring mechanisms of behavioral flexibility and individual specialization through the comparison of members of multiple subspecies of honey bees.
(2020-04-02)
In this thesis, we explore the cognitive basis for the emergence of individual foraging strategies (IFS) across multiple honey bee subspecies using Reversal Learning assays and Free-Flying foraging problems. In Chapter 1 ...
Deciphering the Epargyreus clarus (silver spotted skipper) wing patterning genes
(2020-05-20)
Butterfly wing patterns have captivated evolutionary and developmental biologists for decades because of their vast diversity. Studies have focused on the molecular basis of pattern variation in the Nymphalidae, with little effort put toward skippers, family Hesperiidae. This study had two aims: identify the presence of Wnt genes through ISH and pharmacological drug injections, and test differentially expressed genes in three developmental stages, between wing types, and between wing compartments of E. clarus using RNA-sequencing. I hypothesized, that a simple yet modular set of genes was involved in wing pattern development of the skipper, similar to that of nymphalid butterflies and that genes such as WntA and Ubx would be differentially expressed as in nymphalids. ISH confirmed the presence of two Wnt genes and RNA-seq provided evidence of differential expression, suggesting that the genes underlining wing pattern diversity are being shared between these two distantly related families....
Growth of Proteus vulgaris in synthetic human urine for an ureolysis system
(2020-06-23)
On this thesis, a synthetic human urine formulation alike real urine and that can be ideal for P. vulgaris growth is identified. Additionally, urease activity from robust P. vulgaris is assessed electrochemically by measuring ammonia oxidation currents from synthetic human urine cultures via cyclic voltammetry. The results of this research will be fundamental for future experiments leading to the continuous growth of P. vulgaris and urease activity in real human urine....