Thesis advisor: Marc Muskavitch / The ubiquitin-proteasome system (UPS), composed of classes of proteins central to the process of cellular protein turnover in eukaryotes, is essential to the life cycle of the malaria parasite, Plasmodium falciparum. Although the UPS has been well characterized in other organisms, the extent of its involvement in different stages of P. falciparum growth and development has not been investigated in depth. MG132, a small-molecule proteasome inhibitor known to target the 20S proteasome core (part of the catalytic center for selective protein degradation), has been used successfully in many research studies that require proteasome inhibition. We present data supportive of the conclusion that MG132 is highly effective as a tool for P. falciparum research. In this thesis, I describe the effects of partial and complete proteasome inhibition on parasite growth and development by the use of variable concentrations of MG132. I also assess the effects of MG132 on 20S P. falciparum proteasome enzymatic activities. I have generated parasite lines that exhibit tolerance, or low-level resistance, to MG132, through intermittent compound exposure. Sequencing of the catalytic β-5 subunit of the MG132-tolerant parasites reveals non-synonymous point mutations in three tolerant parasite lines. The use of MG132 as a tool compound for study of the UPS in P. falciparum facilitates research into detailed roles of the proteasome using reversible partial and complete inhibition. MG132-tolerant lines are also valuable tools for studying the genesis of different levels of drug resistance and cross-resistance in parasite evolution. / Thesis (PhD) — Boston College, 2015. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Câncer se destaca no contexto de patologias por ser uma das doenças que mais acometem mortes por ano, sendo caracterizada como um conjunto de doenças multifatoriais que tem em comum o crescimento desordenado de células que invadem tecidos e órgãos, podendo espalhar-se para outras regiões do corpo, dando origem às metástases. Uma importante etapa da cascata metastática é a transição epitélio-mesenquimal (EMT), um processo bem orquestrado que resulta na perda do fenótipo epitelial e aquisição do fenótipo mesenquimal pelas células tumorais, que adquirem carácter invasivo e migratório, além de se tornarem mais resistentes às drogas. Durante este processo, ocorrem inúmeras alterações celulares que modificam a estabilidade proteica e/ou promovem sua translocação subcelular, o transporte de proteínas para a membrana, alterações no citoesqueleto e incluindo o envio de proteínas para degradação pelo proteassoma. A desregulação de fatores de transcrição e modificação pós traducional de proteínas são fatores que podem levar à EMT. Após a eficiente indução da EMT in vitro utilizando o inibidor de histonas deacetilase (SAHA) em células de adenocarcinoma de mama MCF-7, foram realizadas análises proteômicas envolvimento os inibidores relacionados ao sistema ubiquitina proteassoma, MG132 e P5091. A modulação por inibição de USP7 resultou em variação da expressão de diversas proteínas biomarcadoras da EMT (SNAIL, ?-Catenina, CDK1) e proteínas envolvidas no ciclo celular (P53 e CDK1). O estudo proteômico permitiu a correlação do processo da EMT por SAHA com as vias de modificações pós traducionais relacionadas ao sistema ubiquitina proteassoma, e ainda propõe USP7 como alvo de estudos detalhados para EMT com potencial proposta terapêutica / Cancer stands out in the context of pathologies because it is one of the diseases that most affect deaths per year, being characterized as a set of multifactorial diseases that has in common the disordered growth of cells that invade tissues and organs, being able to spread to other regions of the body, giving rise to metastases. An important step in the metastatic cascade is the epithelial-mesenchymal transition (EMT), a well-orchestrated process that results in the loss of the epithelial phenotype and acquisition of the mesenchymal phenotype by the tumor cells that acquire a more invasive and migratory character, and become more resistant to drugs. During this process, numerous cellular alterations occur that modify the protein stability and/or promote its subcellular translocation, the transport of proteins to the membrane, changes in the cytoskeleton and including the sending of proteins for degradation by the proteasome. Deregulation of transcription factors and posttranslational modification of proteins are factors that can lead to EMT. After an efficient induction of EMT using the histone deacetylase inhibitor (SAHA) in MCF-7 breast adenocarcinoma cells, proteomic analyzes were performed involving inhibitors related to the ubiquitin proteasome system, MG132 and P5091. Modulation by inhibition of USP7 resulted in varying expression of various EMT biomarker proteins (SNAIL, ?-Catenina, CDK1) and cell cycle (P53 e CDK1). The proteomic study allowed the correlation of the SAHA EMT process with the posttranslational modifications pathways related to the ubiquitin proteasome system and also proposes USP7 as the target of detailed studies for EMT with potential therapeutic proposal

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The intracellular sperm injection (ICSI) technique is a very effective tool for the fertilization research. In the newly established laboratory at the Faculty of Science of the Charles University it was necessary to introduce this method and define the early developmental potential of fertilized oocytes. After fertilization oocytes were incubated to the blastocyst stage with a success comparable with other laboratories (17%). The ubiquitin-proteasome system which plays a major role in a protein degradation within cells is involved in a regulatory mechanism of sperm maturation. It is also responsible for a penetration of a vitelline membrane. In these processes ubiquitin residues are localized extracellulary. High level of sperm ubiquitination correlates with their low quality. Hypotetically it can be expected that the ubiqutination of impaired sperm cells can be used as a negative marker for their recognition and degradation by 26S proteasome complex localized. Experiments in this diploma thesis were designed based on the hypothesis that the executive part of the selective mechanism is the 26S proteasome. Therefore the effect of MG132 peptide inhibition of the 20S proteasome on the pronuclei formation and subsequent early embryonic development after ICSI was studied. Inhibition of 20S proteasome...