1,721,080 research outputs found
BDNF-induced changes in the expression of the translation machinery in hippocampal neurons: protein levels and dendritic mRNA
No full textBDNF plays a key role in neuronal development, in short- and long-term changes in synaptic activity, and in neuronal survival. These effects are mediated, to a great extent, by changes in protein synthesis. We conducted a gel-based proteome profiling of the long-term (12 h) effects of BDNF in cultured hippocampal neurons. BDNF changed the abundance of proteins involved in (i) Nucleobase, nucleoside, nucleotide and nucleic acid metabolism, (ii) protein metabolism, (iii) carbohydrate metabolism, (iv) regulators of apoptosis, and (v) regulators of cell proliferation. A large majority of the identified proteins involved in translation activity were upregulated, but not all changes in the protein content were correlated with alterations in the corresponding mRNA. The upregulation of Seryl-aminoacyl-tRNA-synthetase and Eef2 was sensitive to the mTOR inhibitor rapamycin, as determined by Western blot. Since the mRNAs for proteins involved in translation represent a large fraction of the diversity of dendritic mRNAs, we investigated the effect of BDNF on the distribution of the transcripts in the soma versus neurite compartments. The increase in mRNA for proteins of the translation machinery in the soma was differentially coupled with the upregulation of neurite transcripts. BDNF also downregulated specific mRNAs in neurite compartments suggesting that the neurotrophin may act by regulating mRNA stability and thereby affecting the dendritic protein content
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
Analysis of the hippocampal and cortex proteome of mice exposed to psychotropic medication
No full textTese no âmbito do Programa Doutoral em Biologia Experimental e Biomedicina, ramo de Neurociências e Doença,
apresentada ao Instituto de Investigação Interdisciplinar da Universidade de Coimbra.Psychotic disorders are very serious and costly mental diseases. Throughout the years the understanding of the genetic basis of these illnesses has improved considerably but there is only rudimentary knowledge of its pathophysiology, mostly due to inconsistent results and confounding factors.
In the case of schizophrenia, the disease is characterized by a combination of positive, negative and cognitive symptoms, and throughout the literature several explanations for the origin of the disorder have emerged, such as the dopaminergic, glutamatergic or serotoninergic hypotheses. The treatment of these disorders consists essentially in the chronic treatment with antipsychotics, sometimes accompanied by psychosocial or even electroconvulsive therapy.
The study of alterations triggered by antipsychotic medication is essential to better understand their action, but also to elucidate targets to study psychosis physiology and treatment. Having all this in mind, two antipsychotics were selected for this project: haloperidol and clozapine, first and second-generation antipsychotics, respectively. Haloperidol is believed to act essentially in dopamine D2 receptors, and clozapine, along with softer action in D2 receptors, also has affinity for serotonin and other receptors. Haloperidol is effective against positive symptoms, while clozapine has shown effectiveness in positive symptoms, even in treatment-refractory patients, and is believed to have some efficacy against negative symptoms also.
The main goal of this project was to investigate the differential protein expression in the rodent brain following chronic exposure to commonly prescribed psychotropic medication, in order to distinguish between pharmacological and disease-related changes and correlate expression profiles with drug effects.
A state-of-the-art quantitative proteomics approach, SWATH-MS, was used to quantify the proteome of the prefrontal cortex (PFC) and hippocampus of mice chronically exposed to both antipsychotics. In the case of haloperidol, striatum was also studied.
With this approach thousands of proteins were identified in all experimental groups, enabling the creation of representative protein libraries. With these libraries and using the SWATH approach, more than 1000 proteins were confidently quantified in each tissue, which upon the application of quality and statistical filters some of the proteins were considered as significantly modulated by the drugs. By applying a principal component analysis to these altered proteins, the distinction between control and medicated groups was achieved, giving further validity to the results here presented.
Functional, ontological and correlation analysis were performed leading to the conclusion that many of the altered proteins are related to metabolic processes, suggesting a metabolic shift caused by the two drugs. Also, many of the modulated proteins are involved in several synaptic systems, with special focus to the GABAergic system, and the synaptic vesicle cycle. Interestingly, in the PFC and hippocampus of the mice exposed to haloperidol several protein subunits belonging to the translation machinery were found to be altered.
In summary, this study shows indication about the mechanism of action of long-term antipsychotic exposure, hinting to the fact that similar pathways are modulated by both drugs but in different ways and extents. This study may help to choose possible target molecular pathways for the investigation of psychotic schizophrenia’s physiology and treatment.As doenças psicóticas são doenças muito sérias e economicamente desgastantes. Ao longo dos anos o conhecimento das bases genéticas destas doenças tem melhorado consideravelmente mas o conhecimento da sua patofisiologia continua rudimentar, especialmente devido a resultados inconsistentes e diferentes variáveis na estrutura dos estudos.
No caso da esquizofrenia, a doença é caracterizada pelo combinação de sintomas positivos, negativos e cognitivos, e na literatura é possível encontrar várias explicações para o surgimento da doença, nomeadamente as hipóteses dopaminérgica, glutamatérgica e serotoninérgica. O tratamento destas doenças consiste essencialmente na toma crónica de antipsicóticos, por vezes acompanhada por terapia psicossocial ou até electroconvulsiva.
O estudo das alterações provocadas pela medicação antipsicótica é essencial para melhor compreender o seu mecanismo de ação, mas também para elucidar alvos para o estudo da fisiologia da psicose e seu tratamento. Tendo toda esta informação em conta, 2 antipsicóticos foram selecionados para este projeto: haloperidol e clozapina, antipsicóticos de primeira e segunda geração, respetivamente. O haloperidol é conhecido por atuar essencialmente nos recetores D2 de dopamina, e a clozapina, para além de uma atuação mais suave nestes, tem também afinidade para outros recetores. O haloperidol é eficaz contra os sintomas positivos, enquanto que a clozapina tem mostrado eficácia contra os sintomas positivos, até em doentes refratários, e pensa-se que apresenta também alguma eficácia contra sintomas negativos.
O principal objetivo deste projeto foi investigar a expressão diferencial de proteínas no cerébro de ratinhos após a exposição crónica a medicação antipsicótica comummente prescrita, de forma a distinguir entre as alteração que advém da medicação das que advém da doença e de forma a correlação estes níveis de expressão com os efeitos dos medicamentos.
A abordagem de última geração para a proteómica quantitativa, SWATH-MS, foi utilizada para quantificar o proteoma do córtex pré-frontal e hipocampo de ratinhos que foram expostos a ambos os antipsicóticos. No caso do haloperidol o estriado também foi estudado.
Com esta abordagem foi possível identificar milhares de proteínas em todos os grupos experimentais, permitindo a criação de bibliotecas de proteínas representativas de cada um. Com estas bibliotecas e utilizando a abordagem SWATH, foi possível quantificar com confiança mais de 1000 proteínas em cada tecido em estudo, em que algumas foram consideradas como significativamente moduladas pelos medicamentos após a aplicação de filtros de qualidade e estatísticos. Após a aplicação de uma análise de componentes principais utilizando estas proteínas consideradas alteradas, foi possível atingir uma separação entre grupos experimentais, conferindo maior validade aos resultados apresentados.
Análises funcionais, de ontologia e de correlação foram aplicadas, permitindo concluir que muitas das proteínas alteradas estão relacionadas com processos metabólicos, sugerindo que existe uma alteração metabólica provocada por estes medicamentos. Mais se verificou que muitas das proteínas moduladas estão envolvidas em diferentes sistemas sináticos, especialmente no sistema GABAérgico, e no ciclo das vesiculas sinápticas. Foi interessante observar que, tanto no córtex pré-frontal como do hipocampo dos ratinhos expostos a haloperidol, várias subunidades proteicas pertencentes à maquinaria de tradução foram consideradas alteradas.
Em suma, este estudo mostra indicações de qual o mecanismo de ação da exposição prolongada a antipsicóticos, mostrando evidências para o facto de que vias semelhantes são moduladas por ambos os medicamentos mas de formas e níveis diferentes. Este estudo tem o potencial de ajudar a escolher vias moleculares para continuar o estudo da fisiologia e tratamento da esquizofrenia
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
DJ‐1 neuronal rescue under oxidative stress: implications for Parkinson's disease
No full textTese de doutoramento em Biociências, na especialidade de Biologia Celular e Molecular, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de CoimbraMutações em vários genes, incluindo o gene DJ-1, têm sido referidas como causadores de formas hereditárias da Doença de Parkinson (DP), sendo que a partir do estudo das proteínas codificadas por esses genes já foram obtidas informações relevantes sobre os mecanismos moleculares da degeneração dopaminérgica que caracteriza esta patologia. De entre esses mecanismos o stress oxidativo tem-se evidenciado, sendo apontado como a principal causa da morte dos neurónios dopaminérgicos independentemente dos estímulos iniciais que levam à doença.
A proteína DJ-1, associada a uma das formas hereditárias da DP, tem sido considerada como uma proteína de resposta ao stress oxidativo que apresenta um papel importante na protecção contra este estímulo. Para além disso, diversas funções têm-lhe sido propostas, as quais têm contribuído para aumentar o conhecimento sobre os mecanismos envolvidos na neurodegeneração associada a stress oxidativo, observada em doentes com DP. A proteína DJ-1 é considerada uma proteína multifuncional, contudo os mecanismos pelos quais esta exerce a sua função, assim como os mecanismos responsáveis pela sua regulação, não estão totalmente compreendidos. Assim, será espectável que ao se elucidar a função fisiológica da DJ-1 sejam também alcançados importantes conhecimentos sobre a DP. Além disso, o stress oxidativo é crucial para a actividade da DJ-1, encontrando-se associado à regulação da maioria dos mecanismos de acção e das interacções estabelecidas por esta. Contudo, os mechanismos exactos responsáveis pela regulação da proteína DJ-1 pelo stress oxidativo, assim como muitos dos seus interactores continuam por identificar.
Nesse sentido, com este projecto pretendeu-se elucidar os mecanismos de acção da DJ-1. Mais precisamente pretendeu clarificar os mecanismos pelos quais esta protéina exerce a sua capacidade neuroprotectora a curto e longo prazo, através da identificação dos seus interactores e da dinâmica das interacções durante o stress oxidativo.
Para alcançar os objectivos pretendidos, foi realizado um estudo exaustivo de interactómica que permitiu identificar e quantificar 881 proteínas envolvidas na rede de interacções estabelecidas por intermédio da actividade da DJ-1 em condições de stress oxidativo. Este estudo foi realizado em condições bem definidas associadas à activação de duas importantes vias de sobrevivência centrais para a capacidade neuroprotectora da DJ-1 (as vias da ERK1/2 e PI3-K/Akt), e com importantes implicações para o estudo da DJ-1 no contexto da DP dado que foi observado um impacto evidente na actividade mitocondrial causado pelas condições experimentais usadas.
Com vista a uma melhor caracterização da natureza dinâmica das interacções estabelecidas foi desenvolvida uma estratégia baseada na optimização da análise SWATH-MS que consistiu no uso de um método de digestão em gel com elevada reprodutibilidade e eficiência, designado Short-GeLC, combinado com o uso de proteínas recombinantes como padrão interno. Esta análise permitiu traçar perfis de interacção revelando grupos de proteínas com o mesmo tipo de modulação que também partilhavam actividades biológicas similares, apontado para a implicação da DJ-1 na modulação desses mecanismos e contribuindo assim para clarificar a sua actividade neuroprotectora. A rede de interacções identificada neste trabalho contribuiu em grande parte para aumentar o conhecimento associado às vias envolvidas na resposta da DJ-1 ao stress oxidativo, que podem ser divididas de um modo geral em dois tipos de resposta: um correspondendo a uma resposta mais imediata que está principalmente associada à identificação de proteínas de resposta ao stress tais como oxireductases e fosfatases; e outro mecanismo associado a uma resposta adaptativa e de longo prazo que está associada com a forte representação de proteínas envolvidas em expressão de genes. Para além disso, foi possível confirmar que: i) a proteína DJ-1 para além de actuar em diferentes vias, também controla o mesmo mecanismo a diversos níveis, tal como no caso da regulação da expressão de proteínas e das vias intrínsecas e extrínsecas de apoptose, e ii) apontar pela primeira vez para o envolvimento da DJ-1 com grânulos de stress e na resposta a danos no ADN, cuja importância para a viabilidade celular e implicação nos mecanismos de neurodegeneração, incluindo os envolvidos em DP, tem vindo a aumentar entre a comunidade científica.
Em conclusão, este trabalho resultou na identificação de várias novas proteínas envolvidas na rede de interacções da DJ-1 contribuíndo para a elucidação dos mecanismos de protecção neuronal mediados pela DJ-1 contra o stress oxidativo, e apontando novos mecanismos. Para além disso, muitas da proteínas identificadas estão bem estabelecidas em várias funções celulares implicadas na DP. Deste modo, estes resultados contribuem também para um melhor entendimento da DP e dos vários mecanismos envolvidos no estabelecimento e progressão da doença.
Mutations in several genes, including DJ‐1 gene, have been reported to cause hereditary forms of
Parkinson’s Disease (PD), and in fact the study of these genes has already provided valuable
insights into the molecular mechanisms of dopaminergic degeneration characteristic of this
pathology, from which oxidative stress has been considered the cause of dopaminergic neurons
death in PD independently of the trigger of the disease.
The PD‐ associated protein DJ‐1 has been recognized as a redox response protein with an important
role in the protection against the oxidative stress insults. Moreover, several functions have been
reported for DJ‐1 which have contributed with significant insights into the mechanisms of oxidative
stress‐related neurodegeneration observed in PD patients. Considering all these evidences, DJ‐1 was
considered as a multifunctional protein although its exact mechanism of action and regulation remains
largely unknown. It is therefore expected that by elucidating DJ‐1 physiological function important
insights into PD will also be achieved. Moreover, oxidative stress conditions seem to be crucial for
DJ‐1 activity, being associated with the regulation of the majority of its mechanisms of action and the
interactions established by it. However, all the exact mechanisms behind oxidative stress regulation of
DJ‐1 and many of the interaction partners of DJ‐1 remain unidentified.
Therefore, this project proposed to elucidate the mechanisms of action of DJ‐1. More specifically,
aimed to clarify the mechanisms by which DJ‐1 modulate short‐term and long‐term neuroprotection,
by identifying the binding partners of DJ‐1 during oxidative stress and the dynamics of these
interactions under oxidative stress.
To achieve these goals, a comprehensive interactomic study of DJ‐1 under oxidative stress conditions
was conducted, allowing the identification and quantification of 881 proteins involved in the network
of interactions established via DJ‐1 activity. Moreover, this study was performed in a well‐defined
model associated with the activation of two key survival pathways central for DJ‐1 neuroprotective
function (ERK1/2 and PI3‐K/Akt pathways), and with important implications for the study of DJ‐1 role
in Parkinson’s Disease context, since an evident impact in the mitochondrial activity was also reflected
in the experimental conditions used.
More importantly, the optimized SWATH‐MS based pipeline developed consisting in the use of a high
reproducible and efficient in‐gel digestion methods, designed as short‐GeLC, combined with the use of
recombinant proteins as internal standard, was applied to characterize the dynamic nature of the
interactions. The SWATH‐MS analysis allowed to trace the profiles of interaction revealing clusters of
proteins with the same type of modulation that also shared similar biological activities, pointing out
for an implication of DJ‐1 in those mechanisms and largely contributing to clarify the DJ‐1 neuroprotective activity. In fact, the network of interactors identified in this work largely contributes
to extend the knowledge regarding the pathways involved in DJ‐1 response to oxidative stress, which
can be generically divided in two different types: one corresponding with a more immediate response,
associated with the identification of stress responsive proteins such as oxireductases and
phosphatases; and the other corresponding to an adaptive and long term response associated with the
strong representation of proteins involved in gene expression. Moreover, it was possible to confirmed
that: i) DJ‐1 not only acts at different pathways but also controls the same mechanisms at different
stages, such as in the case of the regulation of protein expression and of the intrinsic and extrinsic
apoptotic pathways, and ii) to point out for the first time, the involvement of DJ‐1 with stress granules
and with the response to DNA damage, which relevance for cell survival and implication in
neurodegeneration (including in PD) is increasing in importance within the scientific community.
In conclusion, this study resulted in the identification of several novel proteins involved in DJ‐1 network
contributing to the elucidation of DJ‐1‐mediated neuronal protection against oxidative stress, and
pointing out some new mechanisms. In addition, many of the proteins identified are well established
in distinct cellular functions implicated in PD. Thus, these results may also contribute to a better
understanding of PD and the distinct pathways involved in the establishment and progression of the disease.Mutations in several genes, including DJ‐1 gene, have been reported to cause hereditary forms of
Parkinson’s Disease (PD), and in fact the study of these genes has already provided valuable
insights into the molecular mechanisms of dopaminergic degeneration characteristic of this
pathology, from which oxidative stress has been considered the cause of dopaminergic neurons
death in PD independently of the trigger of the disease.
The PD‐ associated protein DJ‐1 has been recognized as a redox response protein with an important
role in the protection against the oxidative stress insults. Moreover, several functions have been
reported for DJ‐1 which have contributed with significant insights into the mechanisms of oxidative
stress‐related neurodegeneration observed in PD patients. Considering all these evidences, DJ‐1 was
considered as a multifunctional protein although its exact mechanism of action and regulation remains
largely unknown. It is therefore expected that by elucidating DJ‐1 physiological function important
insights into PD will also be achieved. Moreover, oxidative stress conditions seem to be crucial for
DJ‐1 activity, being associated with the regulation of the majority of its mechanisms of action and the
interactions established by it. However, all the exact mechanisms behind oxidative stress regulation of
DJ‐1 and many of the interaction partners of DJ‐1 remain unidentified.
Therefore, this project proposed to elucidate the mechanisms of action of DJ‐1. More specifically,
aimed to clarify the mechanisms by which DJ‐1 modulate short‐term and long‐term neuroprotection,
by identifying the binding partners of DJ‐1 during oxidative stress and the dynamics of these
interactions under oxidative stress.
To achieve these goals, a comprehensive interactomic study of DJ‐1 under oxidative stress conditions
was conducted, allowing the identification and quantification of 881 proteins involved in the network
of interactions established via DJ‐1 activity. Moreover, this study was performed in a well‐defined
model associated with the activation of two key survival pathways central for DJ‐1 neuroprotective
function (ERK1/2 and PI3‐K/Akt pathways), and with important implications for the study of DJ‐1 role
in Parkinson’s Disease context, since an evident impact in the mitochondrial activity was also reflected
in the experimental conditions used.
More importantly, the optimized SWATH‐MS based pipeline developed consisting in the use of a high
reproducible and efficient in‐gel digestion methods, designed as short‐GeLC, combined with the use of
recombinant proteins as internal standard, was applied to characterize the dynamic nature of the
interactions. The SWATH‐MS analysis allowed to trace the profiles of interaction revealing clusters of
proteins with the same type of modulation that also shared similar biological activities, pointing out
for an implication of DJ‐1 in those mechanisms and largely contributing to clarify the DJ‐1 neuroprotective activity. In fact, the network of interactors identified in this work largely contributes
to extend the knowledge regarding the pathways involved in DJ‐1 response to oxidative stress, which
can be generically divided in two different types: one corresponding with a more immediate response,
associated with the identification of stress responsive proteins such as oxireductases and
phosphatases; and the other corresponding to an adaptive and long term response associated with the
strong representation of proteins involved in gene expression. Moreover, it was possible to confirmed
that: i) DJ‐1 not only acts at different pathways but also controls the same mechanisms at different
stages, such as in the case of the regulation of protein expression and of the intrinsic and extrinsic
apoptotic pathways, and ii) to point out for the first time, the involvement of DJ‐1 with stress granules
and with the response to DNA damage, which relevance for cell survival and implication in
neurodegeneration (including in PD) is increasing in importance within the scientific community.
In conclusion, this study resulted in the identification of several novel proteins involved in DJ‐1 network
contributing to the elucidation of DJ‐1‐mediated neuronal protection against oxidative stress, and
pointing out some new mechanisms. In addition, many of the proteins identified are well established
in distinct cellular functions implicated in PD. Thus, these results may also contribute to a better
understanding of PD and the distinct pathways involved in the establishment and progression of the
disease.Rede Nacional de Espectrometria de Massa (RNEM) - REDE/1506/REM/200
Quantification of glucose turnover and Cori cycling in ATGL-knockout mice by [U- 13C6]glucose infusion and LC-MS/MS analysis of glucose 13C-isotopomers
No full textDissertação de mestrado em Bioquímica, apresentada ao Departamento Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra.A patofisiologia de doenças metabólicas como a diabetes mellitus tipo II (T2DM) é
caracterizada pela perda de sincronia entre a síntese e catabolismo de triacilgliceróis (TAG). A
lipase adiposa de triacilglicerídeos (ATGL), uma enzima envolvida na hidrólise de TAGs, tem sido
alvo de vários estudos de forma a compreender o seu papel na resistência à insulina. A razão
para este aumento de interesse é o fato de os ATGL knock‐outs (ATGL‐/‐) terem demonstrado
maior sensibilidade à insulina e tolerancia à glucose em comparação com ratinhos sem a
delecção (WT). Apesar de eles não recorrerem a ácidos gordos armazenados como fonte de
energia, estas características fazem dos ratinhos ATGL‐/‐ um modelo único para a melhor
compreensão da relação entre o metabolismo de TAGs e a resistência à insulina.
A taxa do turnover de glucose e reciclagem pelo ciclo de Cori é altamente suscetível
tanto à sensibilidade a insulina como à disponibilidade de nutrientes. Sob condições normais de
jejum, a maioria da glucose é reciclada pelo ciclo de Cori. No entanto, a maior dependência de
glucose por parte dos ratinhos ATGL‐/‐, levou‐nos a colocar a hipótese de que estes ratinhos
seriam mais dependentes da produção de glucose endógena (EGP) para fazer face a esta
necessidade e que a glucose seria eliminada principalmente através da oxidação, resultando em
menor reciclagem através do ciclo de Cori.
Para estudar esta hipótese, os enriquecimentos de glucose de ratinhos ATGL‐/‐ e WT,
infundidos com [U‐13C6]glucose, foram quantificados por análise de gotas de sangue (DBS) por
cromatografia líquida acoplada à espectrometria de massa (LC‐MS/MS). Após o
desenvolvimento e validação do método, a quantificação do enriquecimento da [U‐13C6]glucose
precursora permitiu a determinação da taxa de EGP, enquanto a quantificação dos isotopómeros
parcialmente marcados [1,2‐13C2]glucose e [1,2,3‐13C3]glucose, forneceram uma avaliação do
ciclo de Cori.
A análise da EGP e do metabolismo do ciclo de Cori entre ratinhos ATGL‐/‐ e wild‐type,
revelou que as taxas de EGP eram tendencialmente mais baixas para ratinhos ATGL‐/‐, quando
comparados com ratinhos wild‐type e que o ciclo de Cori contribuía quantitativamente para a
EGP em ambos os grupos. Assim, estes dados não confirmam a hipótese de maior oxidação de
glucose e ciclo de Cori reduzido em ratinhos ATGL‐/‐.The pathophysiology of metabolic diseases such as type II diabetes mellitus (T2DM) is
characterized by a loss of synchrony between triacylglycerols (TAG) synthesis and catabolism.
The adipose triacylglyceride lipase (ATGL), an enzyme involved in TAGs hydrolysis, has been the
target of several studies in order to understand its role in insulin resistance. The reason for this
increasing interest is the fact that ATGL knock‐outs (ATGL‐/‐) have demonstrated to be more
insulin sensitive and glucose tolerant in comparison to wild‐types. Even though they do not
resort to stored fatty acids (FAs) as energy fuel, these characteristics make ATGL‐/‐ mice a unique
model for the better understanding of the relationship between TAGs metabolism and insulin
resistance.
The rate of glucose turnover and recycling via the Cori cycle is highly susceptible to both
insulin sensitivity and nutrient availability. Under normal fasting conditions, the majority of
glucose is recycled via the Cori cycle. However, in the case of ATGL‐/‐ mice, since they are more
dependent on glucose, it was hypothesized that these mice would be more dependent on
endogenous glucose production (EGP) to meet this demand and that glucose would be disposed
mainly through oxidation, resulting in less recycling via the Cori cycle.
To study this hypothesis, glucose enrichments from ATGL‐/‐ mice and wild‐type mice,
infused with [U‐13C6]glucose, were quantified by liquid chromatography coupled to tandem mass
spectrometry (LC‐MS/MS) analysis of dried blood spots (DBS). After method development and
validation, the quantification of the parent [U‐13C6]glucose enrichment allowed the
determination of the rate of EGP, while the quantification of the partially labelled isotopomers
[1,2‐13C2]glucose and [1,2,3‐13C3]glucose, provided a measure of the Cori cycle.
Analysis of EGP and Cori cycle metabolism between ATGL‐/‐ and wild‐type mice revealed
that EGP rates tended to be lower for ATGL‐/‐ mice compared to wild‐types and that the Cori
cycle contributed quantitatively to EGP in both groups. Therefore, these data do not support the
hypothesis of higher glucose oxidation and reduced Cori cycling in ATGL‐/‐ mice
Oxidoreductase protein family interaction with DJ-1 and oxidative stress-induced modulation of HADHA interactome
No full textFERREIRA, Andreia Sofia da Costa de Mónica - Oxidoreductase protein family interaction with DJ-1 and oxidative stress-induced modulation of HADHA interactome. Coimbra : [s.n.], 2016A doença de Parkinson (DP) é a segunda doença neurodegenerativa mais comum, sendo
a mais incidente dentro das doenças do movimento, e caracterizada por uma drástica perca de
neurónios dopaminérgicos na região cerebral substantia nigra pars compacta (SNpc), levando à
degeneração dos circuitos dopaminérgicos nigroestriatais. Várias evidências apontam para
diversas disfunções celulares importantes na patogénese da DP, incluindo o stress oxidativo.
Apesar de vários estudos na área terem sido realizados para uma maior elucidação sobre a
doença, a etiologia e patogénese da Doença de Parkinson continuam por estar esclarecidas. No
entanto, é sabido que a causa da maioria dos casos de DP são esporádicos, e que alguns casos
raros têm uma componente genética, hereditária. De facto, diversas mutações em diversos genes
têm sido identificadas como causadoras da forma hereditária da doença, o que contribuiu com
importantes conhecimentos sobre os mecanismos moleculares que levam à neurodegeneração.
O gene dj1, associado às formas genéticas da DP, foi identificado como sendo um gene
autossómico recessivo responsável pela DP familiar, que se desenvolve em idades mais novas, o
que o torna um candidato para ser estudado de forma a fornecer importantes conhecimentos
sobre os mecanismos moleculares da doença. Várias funções foram atribuídas à proteína DJ-1, no
entanto o seu papel de neuroprotecção é o mais relevante, provavelmente devido ao seu
envolvimento na proteção contra o stress oxidativo. Estudos indicam que a proteína DJ-1 opera
em diversos níveis interagindo, direta ou indiretamente, com várias proteínas. Apesar dos
mecanismos através dos quais a proteína exerce a sua função neuroprotetora não serem
completamente conhecidos, esta aparenta ter uma importante função na mediação de vias de
sinalização e na modelação do metabolismo oxidativo. Vários estudos demonstraram que de facto
o stress oxidativo é a condição que despoleta a função neuroprotectiva da DJ-1, alterando a
conformação da proteína para uma forma oxidada, que é considerada a sua forma ativa. Além do
mais, a estabilidade da maioria das interações estudadas entre a DJ-1 e outras proteínas é
influenciada pelo seu estado oxidativo. Tendo isto em mente, o interatoma dinâmico da DJ-1 foi
anteriormente estudado, de forma a fornecer uma caracterização abrangente dos interatores da
proteína em condições do stress oxidativo. Este estudo possibilitou a identificação de diversas
proteínas que interagem com a DJ-1, nomeadamente proteínas pertencentes à família das
oxidoredutases, que estão relacionadas com a resposta celular ao stress oxidativo, como a
NDUFA4, PGDH e a HADHA. No entanto, as interações da DJ-1 com essas proteínas estão ainda
por serem validadas.
Dentro deste contexto, diversas análises complementares foram realizadas de forma a
validar a interação da DJ-1 com as proteínas pertencentes à família das oxidoredutases, incluindo
imunoprecipitação, pull down e imunocitoquímica seguida de uma análise por microscopia
confocal, em condições fisiológicas e de stress oxidativo. através do ensaio de imunocitoquímica
seguido de análise por confocal das células SH-SY5Y, foi possível validar a interação entre as
proteínas in situ, o que parece confirmar que tanto a HADHA como a PGDH são interatores da DJ-
1. Além disso, os resultados obtides através da análise por pull down sugerem uma interação das
proteínas HADHA e PGDH com a proteína DJ-1 recombinante, na sua forma nativa, sendo essa
interação aparentemente modelado pelo stress oxidativo, o que vem corroborar a os esukltados
anteriores.foi utilizado de forma a estudar o interatoma da proteína em células SH-SY5Y em células
expostas ao stress oxidativo ou em condições fisiológicas, de forma a aferir o envolvimento desta
proteína na resposta celular ao stress oxidativa, e para expandir o interactoma dinâmico da DJ-1.
Com esta metodologia, foi possível concluir que a proteína HADHA tem um interatoma dinâmico
que é modelado pelo stress oxidativo. Diversas proteínas foram identificadas como interatores,
pertencendo a diversos grupos funcionais, tais como proteínas do citoesqueleto, proteínas
motoras, proteínas envolvidas na reparação do DNA e expressão de genes, e proteínas envolvidas
em diferentes vias de sinalização. Além disso, muitas das proteínas identificadas como potenciais
interatores da HADHA foram também anteriormente identificadas como interatores da DJ-1. A
generalidade das proteínas quantificadas apresentou um aumento de interação com a HADHA em
condições de stress oxidativo, fornecendo informações fundamentais sobre as suas funções
biológicas associada à resposta celular a essa patologia, apontando para uma nova função da
proteína HADHA relacionada com a proteção neuronal contra o stress oxidativo. Estes resultados
contribuíram também para o aprofundar dos conhecimentos sobre a patogénese da doença de
Parkinson, as potenciais vias envolvidas no despoletar da doença e a sua progressão, podendo
fornecer novos potenciais alvos para o prognóstico, terapia e progressão da DP.Parkinson’s disease (PD) is the second most common neurodegenerative disease and the
most common movement disorder, characterized by massive loss of dopaminergic neurons in the
substantia nigra pars compacta (SNpc), leading to degeneration of nigrostriatal dopaminergic
pathways. Evidence suggest several cellular dysfunctions important for pathogenesis in PD,
including oxidative stress. Even though several efforts have been made to obtain a deeper
knowledge of this disease, the etiology and pathogenesis still remain unclear. Nevertheless, there
is an agreement that the majority of PD cases are sporadic and some rare cases have familial
background. Indeed, mutations in several genes have been reported to cause hereditary forms of
the disease, leading to valuable insights into the molecular mechanisms of neurodegeneration.
Among the genetic forms, Dj1 gene was identified as an autosomal recessive gene
responsible for familial early-onset PD, meaning that deeper knowledge on its physiological
functions will provide important insights into molecular mechanisms of PD. Several functions have
been attributed to DJ-1 protein, being its putative role in neuroprotection the most relevant,
probably due to its involvement in protection against oxidative stress. Evidence indicate that DJ-1
operates at multiple levels and interact, directly or indirectly, with several partners. Although the
mechanisms through which DJ-1 mediates neuroprotection is not fully understood, it seems to
play a pivotal role in the mediation of signaling pathways and modulation of the oxidative
metabolism. Several studies reported that indeed oxidative stress is the trigger condition for DJ-1
neuroprotective function, in a way that conformational changes to an oxidized form were pointed
out as protein’s active form. Moreover, the stability of the majority of the interactions reported is
also influenced by the oxidation state of DJ-1. A dynamic DJ-1 interactome screening was
previously made in order to provide a comprehensive characterization of DJ-1 binding partners
under oxidative stress conditions, leading to the identification of several binding partner. From
those, proteins belonging to oxidoreductase family were identified, which are mainly associated
with cellular response to oxidative stress, such as NDUFA4, PGDH and HADHA. However, such
interactions still require validation.
Based on this, complementary assays were performed to validate DJ-1 interaction with
oxidoreductase proteins, such as immunoprecipitation, pull down assay, and
immunocytochemistry followed by confocal analysis, in normal and oxidative stress conditions.
iImunocytochemistry assay followed by confocal analysis of SH-SY5Y cells revealed that DJ-1
interacts with both PGDH and HADHA proteins in situ, thus confirming they are indeed binding
partners. Moreover, pull down approach results suggested an interaction between HADHA and
PGDH with recombinant WT DJ-1, which is modulated by oxidative stress., thus corroborating
previous assumptions.
After confirming HADHA as a DJ-1 bindings partner, an AP-SWATH approach was used to
study HADHA interactome in SH-SY5Y cells exposed to normal and oxidative stress conditions, to
assess this protein’s involvement in cellular response to oxidative stress, and to expand DJ-1’s
dynamic interactome. With this methodology, it was possible to conclude that HADHA has a
dynamic interactome that it is modulated by oxidative stress conditions. Several proteins were
identified as binding proteins, like cytoskeleton proteins, motor proteins, proteins involved in DNA
repair and gene expression, and in different signaling pathways. Moreover, some of the identified
HADHA putative interactors were previously also identified as DJ-1 interactors. The majority ofproteins quantified showed an increased interaction with HADHA in oxidative stress conditions,
thus providing insights into their biological functions in cellular response to such insult, pointing
to a new HADHA role in neuronal protection against oxidative stress. Such results, also contribute
to deeper knowledge on PD pathogenesis, the distinct pathways involved in the establishment
and progression of the disease, highlighting potential new targets for PD prognosis, therapy and
prevention
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
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