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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....
Identification of the gut microbiota in the regenerating intestine of the sea cucumber Holothuria glaberrima and its role on intestinal regeneration
(2023-07-28)
The microbiota, the set of microorganisms associated with a particular environment or host, has acquired a prominent role in the study of many physiological and developmental processes. Here I explore a possible role of the microbiota in regeneration, focusing primarily on the regeneration of the intestine in the sea cucumber Holothuria glaberrima, an echinoderm common in local waters. In CHAPTER 1, I review the involvement of the microbiota in regeneration-related cellular events, providing examples of regenerative models that extend from the repair of tissue layers to the regeneration of complete organs or animals. In addition, I highlight the role of the microbiota in the digestive tract, in echinoderms, primarily holothuroids, which are models for regeneration studies. As a first step in these studies, I characterize the microbiota of normal and regenerating animals (CHAPTER 2). In this chapter the microbial community of sea cucumbers undergoing intestinal regeneration was studied using 16S rRNA amplicon sequencing. For this, sea cucumbers were eviscerated and left to regenerate in seawater aquaria for up to 21 days. Among the findings reported are: (i) the existence of microbial compartmentalization in normal animals which changes in regenerating animals. (ii) a strong effect of the environment, in this case the closed aquarium system, on the normal and regenerating microbiota, (iii)) an increase in species richness and evenness in regenerating animals and (iv) differential abundance of specific bacterial taxa. To determine the role of the microbiota in the regeneration process an initial approach was executed in the following chapters. CHAPTER 3, shows the experiments where the effects of various antibiotics on intestinal regeneration of H. glaberrima were evaluated. For this, eviscerated holothurians were exposed to different antibiotic cocktails and left to regenerate for 10 days. Immunohistological and histochemical analyses were performed to analyze regenerative processes, including rudiment size, extracellular matrix (ECM) remodeling, cell proliferation, and muscle dedifferentiation. Reduction in muscle dedifferentiation, ECM remodeling, and the size of their regenerating rudiments were observed in antibiotic-treated animals in comparison to untreated ones. Our results demonstrate a negative effect of antibiotics on intestinal regeneration and strongly suggest that these effects are due to alterations in the microbial community. To discard the possibility that the antibiotics directly affect holothurian metabolic activity, their effect on the metabolism of H. glaberrima tissues was assayed. A second approach to test the effect of microbiota on intestinal regeneration is shown on CHAPTER 4. Here I utilized fecal microbial transplants (FMT) to validate the effectiveness of microbial transplants in enhancing intestinal regeneration. For this, eviscerated holothuroids, exposed to antibiotics prior to evisceration, were subjected to FMT from non-eviscerated donors. Control animals received no FMT or autoclaved FMT. Bacterial samples were cultured from each group to verify if antibiotics could induce changes in the animal microbiota. Histological analysis revealed that different combinations of antibiotics, along with FMT, have specific effects on various regeneration processes. These findings support the role of the microbiota in the sea cucumber's intestinal regeneration process, reveal factors contributing to organ regeneration, and provide insight into little studied functions of the gut microbiota....
Characterizing the effects of estrogen and G15 on triple-negative Inflammatory Breast Cancer
(2023-05-15)
Inflammatory breast cancer (IBC) remains the most aggressive and lethal form of breast cancer—reporting rapid progression, poor prognosis, and a unique clinical diagnosis. IBC is frequently classified as a hormone ...
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....
Novel hydrazone compounds with broad-spectrum antiplasmodial activity and synergistic interactions with antimalarial drugs
(2023-12-12)
Malaria is a devastating disease that kills millions of people each year, and the emergence of multidrug-resistant Plasmodium parasites poses a major challenge to global malaria control efforts. This dissertation evaluated the antiplasmodial activity of seven novel hydrazone compounds (referred to as CB compounds: CB-27, CB-41, CB-50, CB-53, CB-58, CB-59, and CB-61) against multiple stages of Plasmodium parasites. All CB compounds exhibited broad-spectrum antiplasmodial activity, inhibiting the growth of drug-resistant or sensitive strains of Plasmodium falciparum blood stages with high potency. Interestingly, CB-41 showed prophylactic activity against hypnozoites and liver schizonts in the Plasmodium cynomolgi, a primate model for Plasmodium vivax. Four CB compounds (CB-27, CB-41, CB-53, and CB-61) inhibited P. falciparum oocyst formation in mosquitoes, and five CB compounds (CB-27, CB-41, CB-53, CB-58, and CB-61) hindered the in vitro development of Plasmodium berghei ookinetes. The CB compounds did not inhibit the activation of P. berghei female and male gametocytes in vitro. Six CB compounds showed no inhibition of Plasmodium glutathione S-transferase as a putative target, and no cytotoxicity was exhibited in HepG2 cells. The development and application of the Machine Learning Synergy Predictor (MLSyPred©) tool, an open-sourced and accessible tool for predicting synergistic antimalarial drug combinations, is a significant contribution to malaria research. Using predictions made by MLSyPred©, P. berghei blood stage isobologram analyses showed synergy between CB-61 and FDA-approved antimalarial drugs clindamycin and halofantrine, suggesting that CB compounds could be used in combination therapy to enhance the efficacy of existing antimalarial drugs. These findings demonstrate that CB compounds (CB-27, CB-41, CB-53, and CB-61) are promising candidates for further development as broad-spectrum antimalarial drugs that could treat multidrug-resistant malaria and prevent transmission. CB compounds and the MLSyPred© tool have the potential to make important contributions to malaria research....