1,720,981 research outputs found
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
Characterization of APRc as an immune evasion factor of Rickettsia
Get PDFSeveral pathogens, such as Rickettsia, have developed different tactics to evade immune
responses in order to subsist and proliferate within the host. Rickettsial species are resistant to the serum
bactericidal effects and can evade complement-mediated killing, suggesting that they developed
mechanisms to inhibit recognition by host serum components. Interestingly, many pathogenic bacteria
have also been shown to use non-immune immunoglobulin (Ig)-binding proteins to avoid recognition
by innate immune serum components. With this work, we provide evidence for a novel Ig-binding
protein from Rickettsia, anticipating additional immune-evasion tactics for this obligate pathogen. We
demonstrate that the rickettsial retropepsin APRc is capable of binding to immunoglobulins from
different species and classes. Also, our results suggest that the interaction of APRc with human IgG
promotes the oligomeric stabilization of APRc. Mapping the APRc region responsible for binding
revealed the segment between amino acids 150-166 as one of the interacting regions. Moreover, we
demonstrate that the interaction occurs in the Fab domain but does not entail IgG cleavage. We also
demonstrate IgG-APRc binding in serum samples. Finally, we demonstrate IgG-binding at the surface
of Rickettsia. APRc-IgG binding and its localization at Rickettsia's outer membrane anticipate APRc as
one of the proteins contributing to that activity. Altogether, we propose that APRc may act as a novel
evasin by playing a role in protecting Rickettsia from complement-mediated killing through this non immune IgG-binding activity.
Since Rickettsia are arthropod-borne pathogensthat can originate severe diseases in humans, there
is a growing concern about the increasing incidence of rickettsioses and their impact on global health.
Therefore, with the lack of reliable protective vaccines for rickettsial diseases, the development of
alternative therapeutics is critical. With this work, we provide valuable information for a deeper
understanding of the molecular mechanisms underlying rickettsial pathogenesis, contributing to the
future development of alternative therapeutics
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
A Chloroplastidial Atypical Aspartic Protease from Arabidopsis thaliana:Optimization of Heterologous Expression, Purification and Biochemical Characterization
No full textDissertação de mestrado em Bioquímica, apresentada ao Departamento Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra.As proteases aspárticas estão amplamente distribuídas por todos os reinos biológicos.
O reino das plantas apresenta um número mais elevado de proteases aspárticas do
que os restantes reinos biológicos, o que sugere que estas possam ter um papel
importante nos seus processos biológicos.
As proteases aspárticas atípicas de plantas possuem características únicas. Apresentam
uma organização muito diferente da sua estrutura primária (não possuem o domínio
PSI), possuem um elevado conteúdo em cisteínas e uma sequência de aminoácidos
única à volta da primeira tríade catalítica (hidrofóbico-hidrofóbico-DTG-serina-acídico).
Estas proteases têm localizações celulares pouco usuais e vários autores afirmam que
estas enzimas possuem uma função especializada, estando envolvidas em processos
altamente regulados.
A NP_181826 (At2g42980) é uma protease aspártica atípica cloroplastidial de
Arabidopsis thaliana. Esta protease possui um domínio de ligação a DNA, o que sugere
que o DNA poderá ter um efeito de regulação da actividade da protease. Esta protease
também apresenta uma presequência bipartida. Este tipo de presequência só foi
descrito em organismos que sofreram um processo de endossimbiose duplo, sendo
que nunca foi encontrada em plantas superiores como a Arabidopsis thaliana.
As suas características únicas e o facto de se encontrar no cloroplasto sugerem que
esta protease aspártica possa ter um papel extremamente importante neste organelo.
O objectivo deste trabalho consistiu em estabelecer e optimizar protocolos de
expressão e purificação para a produção da NP_181826, usando E.coli como sistema
heterólogo de expressão. Neste estudo, uma putativa forma madura da NP_181826
(35toDTG) foi clonada e expressa neste sistema.
Várias estratégias foram utilizadas para optimizar a sua expressão e purificação.
Quando expressa na forma solúvel, a forma recombinante 35toDTG apresentou
tendência para ser co-purificada com outras proteínas e a quantidade de proteína
obtida foi muito baixa. A expressão na forma de corpos de inclusão seguida de um processo de refolding por diálise resultou na melhor estratégia para obter quantidades
elevadas de proteína purificada.
A proteína recombinante 35toDTG apresentou actividade proteolítica contra o
substrato típico das protease aspárticas, com um pH óptimo a 3.5. Esta protease
aparenta ter uma especificidade muito restrita, sendo capaz de cortar a cadeia β
oxidada da insulina em apenas um local. A sua completa inibição na presença de
Pepstatina A e a perda total de actividade do seu mutante do local activo colocam esta
protease na família A1 das proteases aspárticas. A inibição com DTT e GSH, não
havendo qualquer inibição com GSSG, e o efeito de activação por parte do NADH e
ADT levam a concluir que o ambiente redox e a presença de nucleótidos podem
influenciar directamente a estabilidade e/ou actividade catalítica desta protease
aspártica atípica.
Os resultados experimentais sugerem que a NP_181826 poderá ter uma função
altamente específica no cloroplasto da Arabidopsis e que o ambiente redox do
cloroplasto poderá influenciar directamente a sua actividade proteolítica.Aspartic proteases are widely distributed among all the biological kingdoms. Plants
have a higher number of aspartic proteases, suggesting that in these organisms
aspartic proteases may play an important role in different biological processes.
Plant atypical aspartic proteases have very unique characteristics. They present a very
different domain organization in its primary structure (loss of the PSI domain), a higher
number of cysteine residues and a unique amino acid sequence around the first
catalytic triad (hydrophobic-hydrophobic-DTG-Serine-Acid). These proteases were
found in very unusual localizations and several authors claim that they have specialized
functions and are involved in highly regulated processes.
NP_181826 (At2g42980) is a chloroplastidial atypical aspartic protease from
Arabidopsis thaliana. This protease has a putative DNA-binding domain, suggesting
that DNA may have a regulatory influence in its catalytic activity. Also, a bipartite
presequence is present in NP_181826 primary sequence. These bipartite presequences
were only found so far in organisms that experienced a secondary endosymbiosis and
they were never described in superior plants, such as Arabidopsis. The chloroplastidial
localization and its unusual sequence features suggest that this aspartic protease may
have a critical role in the biological system.
The aim of this study was to establish and optimize an expression and purification
protocol for production of NP_181826, using E. coli as the heterologous host. A
putative mature form of NP_181826 (35toDTG) was cloned and expressed in this
system.
Several approaches were used to optimize 35toDTG expression and purification. When
expressed as soluble protein, 35toDTG was shown to co-purify with several other
proteins and the protein yields were always very low. Expression as inclusion bodies
followed by refolding by dialysis was the best strategy to obtain a higher yield of
purified protein.
Recombinant 35toDTG showed proteolytic activity towards a typical aspartic protease
substrate, at an optimum pH value of 3.5. This protease showed a very restricted
specificity, being able to cleave oxidized insulin β-chain in only one site. Its complete
6
inhibition in the presence of Pepstatin A and the total loss of proteolytic activity
observed for the active-site mutant, positions NP_181826 in the A1 family of aspartic
proteases. The inhibition effect of DTT and GSH, with no effect in the presence of
GSSG, and NADH and ATP activation effect in 35toDTG proteolytic activity suggest that
the redox environment and the presence of nucleotide species can have a direct effect
on the stability and/or catalytic activity of this atypical aspartic protease.
These experimental evidences strongly suggest that NP_181826 may have a very
specific function in Arabidopsis’s chloroplast and that the redox environment of the
chloroplast may have a direct influence on the protease activity
RLR1 and RLR2, two novel Arabidopsis thaliana atypical aspartic proteases involved in primary root development and lateral root formation
No full textTese de doutoramento em Biologia Experimental e Biomedicina, na especialidade de Biologia Molecular, Celular e do Desenvolvimento, apresentada ao Instituto de Investigação Interdisciplinar da Universidade de CoimbraAspartic proteases (APs) represent the second largest class of plant proteases after serine proteases. Members of the pepsin-like family are widely distributed in plants, e.g. 70 APs homologues are found in Arabidopsis thaliana and 166 in Oryza sativa, the vast majority of them having atypical characteristics and properties (atypical APs). This contrasts strikingly with the considerably fewer number of APs encoded in the mammalian genomes (e.g. Homo sapiens has only 17 APs). The overrepresentation of APs in plants suggests potentially important and diverse roles for these proteins. Although plant APs have been much less studied than other protease classes, some functions are starting to be uncovered, with proposed roles in highly regulated processes like resistance to biotic and abiotic stresses, programmed cell death (PCD), plastid homeostasis and reproduction, which is consistent with functional specialization of plant APs and tight activity regulation.
Our major goals in this thesis were to describe the in vivo functions, the biochemical properties, the in vivo substrates and molecular pathways of two putative atypical APs from the model plant Arabidopsis: At2g03200 and At4g30040 gene products. At2g03200 expression is regulated by the MS1 transcription factor whose absence leads to a severe male sterility phenotype. At4g30040 is indirectly regulated by MYB80, also a transcription factor whose absence leads to a severe male sterility phenotype. Because of this, our first working hypothesis was that both of these APs could be involved in pollen development. This hypothesis was further supported by the expression patterns of these proteases in reproductive tissues. Somewhat unexpected, their T-DNA knock-out (KO) mutants displayed no obvious pollen/anther phenotype. Since both proteins were also expressed in root tissues, we next sought to examine whether the absence of these genes affected root growth. Indeed, our results revealed significant reductions in primary root length and in lateral root number in both KO lines, showing that both proteins are involved in primary root development and lateral root formation. Moreover, when evaluated under nutrient limitation (N deprivation), mutant phenotypes suggest that these genes may be involved in two different regulatory mechanisms of lateral root formation. Therefore, these genes were designated Regulator of Lateral Root (RLR) 1 and 2 (At2g03200 and At4g30040, respectively). Moreover, both KO mutants showed no altered response to auxin treatment in terms of primary root growth but showed an increased response in lateral root number, connecting these enzymes to auxin signaling pathways. RLR1 and RLR2 overexpression mutants also showed de-regulation of lateral root formation and primary root growth further strengthening the importance of these atypical APs in both mechanisms.
To determine their biochemical properties, recombinant forms of both proteins were produced using the innovative plant-based expression platform magnICON® in Nicotiana benthamiana leaves, and we have successfully purified recombinant RLR1 (rRLR1). rRLR1 was shown to be glycosylated, active at acidic pHs, not completely inhibited by pepstatin A, and with a distinct specificity pattern determined by Proteomics Identification of Cleavage Sites PICS, suggesting closest resemblance with fungal APs. Redox agents also have a significant inhibitory effect on rRLR1 activity suggesting that this protein might be involved in redox sensing mechanisms. These results clearly demonstrate that RLR1 is an AP with distinct and atypical biochemical properties.
Finally, and in order to identify RLR1 and RLR2 in vivo substrates and molecular pathways, two proteomics techniques were used: quantitative shotgun proteomics and N-TAILS. Due to sample constrains, we were not able to obtain valid N-TAILS results on time to include in this document. However, the shotgun results allowed the identification of several de-regulated proteins in both KO lines, under normal or N-deprived growth conditions. Interestingly, our results anticipate similarities between both KO lines, with several proteins related to auxin signaling/biosynthesis and redox homeostasis being de-regulated in both RLR1 and RLR2 KOs. This global profiling also revealed de-regulated proteins unique for each KO line, and for each condition, which suggest that these proteases may still participate in different pathways.
Our results unveil a new role for two atypical APs in the regulation and adaptation of root development in Arabidopsis, under normal growth conditions as well as under abiotic stress. To our knowledge, this is the first study linking atypical APs to root development and, hopefully, this will pave the way to a better understanding of the role of these enzymes in this important plant process and the molecular mechanism with which they mediate their biological function.A classe das proteases aspárticas (PAs) é a segunda mais representada em plantas e os
membros da família A1 (tipo pepsina) são os mais comuns, encontrando-se em elevado
número em várias espécies de plantas (por exemplo, 70 homólogos de PAs podem ser
encontrados no genoma de Arabidopsis thaliana e 166 no genoma de Oryza sativa). Se
considerarmos que em Homo sapiens só estão presentes 17 PAs, o elevado número de genes
identificados em plantas sugere a sua participação e relevância funcional em diferentes
processos celulares. A maioria destas PAs putativas de plantas possui características atípicas
(PA atípicas) em relação à sua organização de estrutura primária e localização intracelular e,
apesar de terem sido muito pouco estudadas até agora, alguns estudos reportam a sua
importância em processos celulares críticos para o desenvolvimento da planta, tais como
resistência a stresses bióticos e abióticos e morte celular programada durante a
gametogénese, entre outros.
O presente trabalho consistiu no estudo de duas PAs atípicas putativas de A. thaliana,
e teve como objectivos principais o estudo funcional destas duas enzimas, das suas
propriedades bioquímicas, e dos mecanismos moleculares através dos quais elas exercem a
sua função. O produto do gene At2g03200 foi uma das PAs estudadas, tendo sido mostrado
num estudo recente que a sua expressão é regulada pelo factor de transcrição MS1. A segunda
PA estudada foi o produto do gene At4g30040, cuja expressão é regulada pelo factor de
transcrição MYB80. A ausência de cada um destes factores de transcrição provoca alterações
severas no desenvolvimento do pólen, culminando na esterilidade da planta mutada.
Suportados por estas evidências, a nossa hipótese inicial era que estas PAs pudessem estar
envolvidas em processos relacionados com o desenvolvimento de pólen. De facto, esta
hipótese foi reforçada pelo facto de ambas as proteínas serem expressas nos tecidos
reprodutores. No entanto, linhas mutantes onde a expressão de cada PA foi abolida (mutantes
KO) não apresentaram qualquer defeito ao nível do pólen/antera. Já a análise fenotípica do
desenvolvimento de raiz em cada um dos mutantes KO revelou uma redução significativa no
comprimento da raiz primária, bem como no número de raízes laterais em comparação com as
plantas controlo. Para além disso, a análise fenotípica dos mutantes em condições de
crescimento onde a disponibilidade de azoto foi limitada (stress abiótico) sugere que estas
protéases controlam o aparecimento de raízes laterais através de dois mecanismos
moleculares distintos. Em resultado do fenótipo observado no número de raízes laterais, as
duas proteínas foram denominadas de Regulator of Lateral Root (RLR) 1 e 2 (At2g03200 e
At4g30040, respectivamente). Os nossos resultados apontam ainda para a possibilidade destas
duas PAs atípicas fazerem parte de uma das vias de sinalização da hormona auxina. Mais
ainda, os mutantes de sobre-expressão de RLR1 e RLR2 mostraram também desregulação do desenvolvimento da raiz primária e raízes laterais, reforçando a importância destas PAs para
estes processos de raiz.
De modo a estudar as suas propriedades bioquímicas, as protéases RLR1 e RLR2 foram
produzidas na sua forma recombinante em folhas de Nicotiana benthamiana usando o sistema
magnICON®. A forma recombinante da RLR1 foi purificada com sucesso e demonstrámos que
esta PA é glicosilada, activa a pHs acídicos, não é totalmente inibida por pepstatina A mas
significativamente inibida por agentes redox, o que sugere o seu envolvimento em
mecanismos de controlo redox. Mais ainda, o perfil de especificidade desta protéase revelou
preferências distintas quando comparado ao de outras PAs, sendo semelhante a PA de fungos.
Todos estes resultados demonstram claramente que a RLR1 é uma PA com características
bioquímicas atípicas.
Por último, e de modo a identificar os substratos in vivo destas PAs e os seus
mecanismos moleculares, recorremos a duas técnicas de proteómica: quantitative shotgun
proteomics e N-TAILS. Infelizmente, não nos foi possível obter dados de N-TAILS a tempo de
serem incluídos neste documento mas os resultados de shotgun permitiram a identificação de
várias proteínas desreguladas nos dois mutantes KO crescidos em condições normais ou de
stress abiótico. Apesar de revelarem proteínas desreguladas únicas para cada mutante (o que
implica mecanismos moleculares distintos para cada PA), estes resultados também revelaram
alterações comuns em ambos os KOs. Os resultados desta análise de proteómica antecipam a
potencial relevância destas duas protéases em mecanismos de regulação de auxina e/ou de
balanço redox.
Em resumo, os nossos resultados revelam um novo papel para duas PAs atípicas de
Arabidopsis na regulação e adaptação do desenvolvimento de raiz em condições de
crescimento normal ou em resposta a privação de azoto. Este é o primeiro estudo que
demonstra a participação de PAs neste processo fundamental para o desenvolvimento da
planta. Mais ainda, este estudo confirma a diversidade/relevância funcional das PAs atípicas de
plantas, reforçando a necessidade de mais estudos integrados que permitam compreender em
mais detalhe os processos biológicos regulados por este grupo de protéases.German Academic Exchange Service with funding from the Federal Ministry of Education and Research (Project-ID 57128819 to PFH)FCT - (grant: Scientific and Technological Bilateral Agreement 2015/2016 to IS
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
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