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Enriquecimento de proteínas parceiras de KAHRP e mapeamento de proteínas organelares de Plasmodium falciparum utilizando CRISPR-Cas9, APEX2 e proteômica
Get PDFTese (doutorado)—Universidade de Brasília, Faculdade de Medicina, Programa de Pós-Graduação em Patologia Molecular, 2021.A malária é uma doença tropical, causada por espécies do gênero Plasmodium spp. O Plasmodium
falciparum é a espécie mais virulenta, pois é a única capaz de produzir o quadro clínico mais grave
da doença: a malária cerebral. De acordo com as últimas estimativas, cerca de 229 milhões de
pessoas são acometidas pela malária, e, estudos que possam viabilizar a descoberta de novos
alvos terapêuticos se tornam altamente necessários, haja vista aos crescentes casos de
resistência aos antimaláricos disponíveis. Neste sentido, a fim de poder explorar melhor os
parceiros de interação com a proteína KAHRP (do inglês, knob-associated histidine rich protein),
uma proteína relacionada à citoaderência ao endotélio do hospedeiro humano facilitando a
formação de “rosetas” em P. falciparum, foi implementado o uso de CRISPR-Cas9 juntamente com
a abordagem de marcação por proximidade mediada por ascorbato-peroxidase modificada
(APEX2), a fim de criar uma proteína quimérica da KAHRP fusionada a APEX2 por knock-in,
utilizando o sistema CRISPR-Cas9, com a finalidade de promover a biotinilação de proteínas
vizinhas parceiras. Para avaliar a funcionalidade da proteína quimérica KAHRP-Flag-APEX2 ,
ensaios de validação por Western-blot, estreptavidina-blot e imunofluorescência foram realizados.
As análises de proteômica e de bioinformática revelaram que dentre as 208 proteínas do parasita
biotiniladas vizinhas da KAHRP-Flag-APEX2, algumas delas são preditas por serem exportadas
pelo translocon PTEX presente na membrana do vacúolo parasitóforo, e, outras preditas por
serem exportadas por mecanismos não clássicos. Dentre as proteínas observadas, encontramos:
GBP130, PTP2, membros do grupo PHISTb, UIS2, Pf332, RESA, as proteínas que compõe o
complexo RhopH/CLAG (RhopH1/CLAG3.1, RhopH2 e RhopH3) e do complexo PTEX: PTEX88
e 150. Adicionalmente, seguindo o princípio da acurácia e proximidade proporcionada pelo uso da
APEX2, construções similares foram realizadas, a fim de endereçar a HA-APEX2 para algumas
organelas de P. falciparum. Pelo fato do parasita apresentar organelas especializadas, tais como
o apicoplasto e as roptrias, o conhecimento do arsenal de proteínas presentes nestas estruturas,
podem fornecer evidências para que futuras estratégias para a erradicação da doença, baseado
no desenvolvimento de quimioterápicos e/ou vacinas sejam realizadas. O uso da APEX2 tem se
mostrado altamente eficiente nesse quesito, pois permite uma descrição altamente precisa acerca
das proteínas presentes em uma organela de interesse, baseando-se na sua resolução temporal
e espacial. Assim, construções epissomais foram adicionalmente obtidas, endereçando a HA-
APEX2 para o apicoplasto (pCC1-SP(ACP)-HA-APEX2), a mitocôndria (pCC1-SP(TrxR)-HA-APEX2) e
as roptrias (pCC1-SP(RAP1)-HA-APEX2). O controle citosólico também foi obtido (pCC1-HA(cito)-
APEX2). Com a mudança da metodologia de transfecção com o uso da plataforma Amaxa II
Nucleofector II AAD-1001N (Lonza) e a determinação da ineficácia da droga anti-folato WR99210
proveniente da Sigma-Aldrich para selecionar os parasitas mutantes por [3H] hipoxantina, a obtenção
dos parasitas transfectados foi possível após 3-4 semanas de seleção. Os ensaios de validação da
expressão e da marcação por biotinilação propiciada pela APEX2 por Western-blot e
estreptavidina-blot dos respectivos compartimentos estão em andamento.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).Malaria is a tropical disease caused by Plasmodium species. P. falciparum is the most virulent
specie from this genus, because is the only one capable to produce the most life-threatening clinical
state of the illness: cerebral malaria. In agreement with the last estimative, about 229 million people
are diagnosed with malaria worldwide, and studies that might shed light into novel therapeutic
strategies turn-out very required, especially when an increasing of the numbers of resistance cases
have been reported. Therefore, in order to explore neighboring interacting proteins of KAHRP
(knob-associated histidine rich protein), a widely known protein involved with cytoadherence
properties onto host endothelium facilitating rosetting in P. falciparum, it was implemented the use
of CRISPR-Cas9 system together with proximity-tagging based on the use of ascorbate-peroxidase
modified (APEX2), in order to produce a chimeric protein of KAHRP fused to APEX2 by knock-in,
allowing biotinylation of neighboring proteins of KAHRP-Flag-APEX2. To assess the activity of this
chimeric protein, validation assays were performed, such as Western-blot, streptavidin-blot and
immunofluorescence. From our proteomic and bioinformatic analyses, a total of 208 parasite-
derived neighboring proteins of KAHRP-Flag-APEX2 were identified. Some of them are predicted
to be exported through translocon PTEX at parasitophorous vacuole membrane, and others are
predicted to be exported through non-classical mechanisms. Among these proteins, we have found
in our subproteomic dataset: GBP130, PTP2, PHISTb members, UIS2, Pf332, RESA, proteins
from RhopH/CLAG complex (RhopH1/CLAG3.1, RhopH2 and RhopH3) and PTEX complex:
PTEX88 and 150. In addition, taking advantage of the accuracy and proximity labeling allowed by
APEX2 technology, similar vector constructions were made, in order to address HA-APEX2 to
some P. falciparum compartments. As some compartments of this parasite are unique, such as
apicoplast and rhoptries, the knowledge of the proteomic arsenal from these organelles, might
provide novel insights for the disease eradication through rational drug design pipelines and/or
vaccine development. The use of APEX2 has been shown very insightful in this state-of-the-art,
because it allows an accurate description of the protein content of a given compartment, merely
based on temporal and spatial resolution of the technology. Thus, episome-based constructions
were additionally obtained addressing HA-APEX2 to apicoplast (pCC1-SP(ACP)-HA-APEX2),
mitochondrion (pCC1-SP(TrxR)-HA-APEX2) and rhoptries (pCC1-SP(RAP1)-HA-APEX2). A cytosolic
episome-based expressing APEX2 was also obtained (pCC1-HA(cito)-APEX2). After changing our
transfection platform to Amaxa II Nucleofector II AAD-1001N (Lonza) and with determination of the
WR99210 inefficacy purchased from Sigma-Aldrich to select mutant parasites through [3H]
hypoxanthine, we were able to obtain the first transfected parasites after 3-4 weeks under drug pressure.
Validation assays regarding protein expression and biotinylation proximity tagging into the
compartments provided by APEX2 through Western-blot and streptavidin-blot are under progress.Faculdade de Medicina (FM)Programa de Pós-Graduação em Patologia Molecula
FEZ proteins family (Fasciculation and Elongation protein Zeta) as bivalent transport adaptors : functional, structural and evolutionary aspects.
No full textOrientador: Jörg KobargTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: As proteínas humanas FEZ1 e FEZ2 (fasciculation and elongation protein zeta) são ortólogas da proteína UNC-76 de C. elegans e estão envolvidas no crescimento e na fasciculação dos axônios através de interações que envolvem kinesinas, mitocôndrias e vesículas sinápticas. Além disso, algumas evidências sugerem a participação de FEZ1 na etiologia da esquizofrenia, no ciclo viral, além da resistência à quimioterápicos. Sua estrutura intrinsecamente desordenada, com coiled-coil ao longo da sequência, pode contribuir para sua função. Nós exploramos a evolução molecular da família de proteínas FEZ com ênfase no ramo dos vertebrados. Através do perfil do interactoma comparado entre FEZ1 e FEZ2 de Homo sapiens e UNC-76 de C. elegans foi observado um padrão de conservação das interações proteínaproteína entre FEZ1 e UNC-76, que explicam a capacidade de FEZ1 resgatar os defeitos causados por mutações em unc-76 em nematoides, de acordo com o descrito por Bloom e colaboradores em 1997. Além disso, caracterizamos a interação entre FEZ1 e SCOCO (short coil-coiled) por SAXS (Small Angle X-ray Scattering). Essa interação já foi descrita previamente entre os seus ortólogos UNC-76 e UNC-69, que cooperam no crescimento axonal. Um estado de heterotetramérico foi observado, consistindo de duas moléculas GST-SCOCO interagindo com duas moléculas de 6xHis-FEZ1 dimerizadas. Por PAGE (Polyacrylamide Gel Electrophoresis, eletroforese em gel de poli-acrilamida), SAXS, Espectrometria de Massas e Ressonância Magnética Nuclear, constatamos que FEZ1 dimeriza envolvendo a formação de ponte dissulfeto. In vivo, este estado dimérico de forma covalente pode ser importante para o transporte mediado por kinesinas de proteínas ao longo dos microtúbulos. Assim, FEZ1 pode ser classificada como uma proteína adaptadora do transporte, dimérica e bivalente, essencial para o crescimento axonal e organização pré-sináptica normal e transporte de cargas. A agregação de novos parceiros de interação encontrada para a proteína FEZ2 poderia ser interpretada como aquisição de novas funções moleculares e pode ter ocorrido nos primeiros estágios da evolução dos cordadosAbstract: The human proteins FEZ1 and FEZ2 (fasciculation and elongation protein zeta 1) are orthologs of the protein UNC-76 from C. elegans, involved in growth and fasciculation of axons, through interactions that involve kinesins, mitochondria and synaptic vesicles. Moreover, some evidence suggests involvement of FEZ1 in the etiology of schizophrenia, in addition to the viral cycle and resistance to chemotherapy. Its structure intrinsically disordered, with coiled-coil along the sequence, can contribute to its function. We have explored the molecular evolution of the FEZ protein family with emphasis on the vertebrata branch. Analyzing the interactome profile of the FEZ1 and FEZ2 from Homo sapiens and UNC-76 from C. elegans we observed a conserved pattern of protein-protein interactions among FEZ1 and UNC-76 that explain the ability of FEZ1 to rescue the defects caused by unc-76 mutations in nematodes, according to Bloom and co-workers in 1997. Furthermore, we characterized the interaction between FEZ1 and SCOCO (short coiled-coil protein) by SAXS (Small Angle X-ray Scattering). This interaction has been previously reported between their orthologs UNC-76 and UNC-69 that cooperate in axonal outgrowth. A heterotetrameric state was observed, which consists of two GST-SCOCO molecules attached to two FEZ1 molecules. By PAGE (Polyacrylamide Gel Electrophoresis), SAXS, Mass Spectrometry and Nuclear Magnetic Resonance we defined that FEZ1 dimerizes involving formation of disulfide bond. In vivo this covalent mediated dimeric state could be important for kinesin mediated protein transport along the microtubule. Thereby, FEZ1 may be classified as a dimeric and bivalent transport adaptor, essential to axon outgrowth and normal pre-synaptic organization and transport of cargoes. The aggregation of new interaction partners found for the FEZ2 protein could be interpreted as the acquisition of new molecular functions and may have occurred in the early stages of chordate evolutionDoutoradoBioquimicaDoutor em Biologia Funcional e Molecula
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
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