107 research outputs found
The potential of metabolomics in the diagnosis of thyroid cancer
No full textThyroid cancer is the most common endocrine system malignancy. However, there is still a lack of reliable and specific markers for the detection and staging of this disease. Fine needle aspiration biopsy is the current gold standard for diagnosis of thyroid cancer, but drawbacks to this technique include indeterminate results or an inability to discriminate different carcinomas, thereby requiring additional surgical procedures to obtain a final diagnosis. It is, therefore, necessary to seek more reliable markers to complement and improve current methods. “Omics” approaches have gained much attention in the last decade in the field of biomarker discovery for diagnostic and prognostic characterisation of various pathophysiological conditions. Metabolomics, in particular, has the potential to identify molecular markers of thyroid cancer and identify novel metabolic profiles of the disease, which can, in turn, help in the classification of pathological conditions and lead to a more personalised therapy, assisting in the diagnosis and in the prediction of cancer behaviour. This review considers the current results in thyroid cancer biomarker research with a focus on metabolomics
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
El Tlacuache Núm. 444 (2010). 444 Año 10 (2010) noviembre. El Tlacuache
No full textLos Chalchihuites de Tepoztécatl, centro del mundo y gracia del Dios por Jaime Francisco Reséndiz Machón. -Dinámica ceremonial en los días de Muertos al sur del estado por Bertha Martínez Rivera, Leonardo A. Beltrán-Rodríguez, Alfredo Paulo Maya. -Subyacentes Emociones para El Diario de Campo Las últimas manadas del desierto por Fanny Campillo
Bursaphelenchus xylophilus and B. mucronatus secretomes: a comparative proteomic analysis
No full textThe pinewood nematode, Bursaphelenchus xylophilus, recognized as a worldwide major forest pest, is a migratory endoparasitic nematode with capacity to feed on pine tissues and also on fungi colonizing the trees. Bursaphelenchus mucronatus, the closest related species, differs from B. xylophilus on its pathogenicity, making this nematode a good candidate for comparative analyses. Secretome profiles of B. xylophilus and B. mucronatus were obtained and proteomic differences were evaluated by quantitative SWATH-MS. From the 681 proteins initially identified, 422 were quantified and compared between B. xylophilus and B. mucronatus secretomes and from these, 243 proteins were found differentially regulated: 158 and 85 proteins were increased in B. xylophilus and B. mucronatus secretomes, respectively. While increased proteins in B. xylophilus secretome revealed a strong enrichment in proteins with peptidase activity, the increased proteins in B. mucronatus secretome were mainly related to oxidative stress responses. The changes in peptidases were evaluated at the transcription level by RT-qPCR, revealing a correlation between the mRNA levels of four cysteine peptidases with secretion levels. The analysis presented expands our knowledge about molecular basis of B. xylophilus and B. mucronatus hosts interaction and supports the hypothesis of a key role of secreted peptidases in B. xylophilus pathogenicity
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Distribución y aspectos poblacionales del lobo Ibérico (Canis lupus signatus) en las provincias de Pontevedra y A Coruña (Galicia)
No full textPara determinar el área de distribución del lobo en las provincias de Pontevedra y A Coruña se realizaron itinerarios de muestreo y se enviaron cuestionarios a los Agentes de Medio Ambiente de la Xunta de Galicia y a las Sociedades de Cazadores del área de estudio. Los resultados se representaron en cuadrículas U.T.M. 10x10 Km. Se ha confirmado la presencia del lobo en el 67,05% de las cuadrículas del área de estudio. La especie se distribuye de forma continua en ambas provincias, donde estimamos un área de distribución de 6.400 km2 en la provincia de A Coruña y 3.200 km2 en la de Pontevedra, ocupando principalmente el área montañosa de la Dorsal Gallega y el occidente de A Coruña; esta última zona está conectada con el resto del área de distribución del lobo en Galicia a través del norte de la provincia de Pontevedra. Para la localización de las manadas se muestreó el territorio a nivel de cuadrícula U.T.M. 10x10 Km, y en las zonas donde se detectó una mayor concentración de indicios, se realizaron estaciones de escucha y espera con el fin de localizar la presencia de manadas (cachorros y adultos). Se determinó la presencia de 29 manadas (14 en la provincia de A Coruña y 15 en Pontevedra), de las cuales se confirmó la reproducción del 52%. Se ha estimado un tamaño poblacional para la provincia de A Coruña entre 87-129 lobos (1,36-2,02 lobos/100 Km2) y entre 95-140 para la provincia de Pontevedra (2,97-4,38 lobos/100 Km2). Las zonas de mayor densidad se localizaron en la Dorsal Gallega. Se han localizado algunas manadas en áreas muy humanizadas, lo que podría explicar que la principal causa de mortalidad conocida entre enero de 2000 y octubre de 2003 (n=29) sea el atropello (n=21)
Resistência e susceptibilidade à manifestação da dor neuropática
No full textTese de doutoramento em Envelhecimento e Doenças CrónicasA maioria dos indivíduos que desenvolve dor após uma lesão retorna ao estado saudável quando
a lesão dissipa. Por outro lado, numa pequena percentagem de pessoas a dor persiste por longos
períodos e evolui para dor crónica (DC), geralmente acompanhada por comorbidades emocionais e
cognitivas. Nos animais, a DC também é caracterizada pelos mesmas comorbidades. Todavia, os
mecanismos neuronais que intervêm na transição da dor aguda para a DC permanecem amplamente
desconhecidos quer em humanos quer em animais, pelo que a determinação dos fatores que conferem
suscetibilidade e resistência ao desenvolvimento da dor é crucial para perceber esta alteração
disfuncional. Estas questões no campo da dor motivaram-nos a investigar quais são os fatores a esta
transição, antes e após instalação de um modelo de dor crónica em roedores, Spared Nerve Injury (SNI).
Nesta tese, começamos por mostrar que a maioria dos ratos SNI Sprague dawley (SD) apresenta
alodinía mecânica (limiar baixo - LT), porém 13% deles apresentam valores semelhantes aos controlos
(limiar alto - HT). Sem diferenças estruturais nos nervos ciáticos dos animais LT e HT, a lesão no nervo
ciático esquerdo (SNI-L) potenciou ansiedade e uma diminuição do bem-estar em animais lesionados
nesse nervo ciático. Além disso, também descobrimos que o córtex insular (divisão agranular) dos
animais SNI-L e dos animais lesionados no nervo ciático direito (SNI-R) era significativamente menor no
HT do que no LT.
Em seguida, investigamos que fatores comportamentais, celulares e moleculares estão
relacionados com a suscetibilidade e a resistência à dor crónica antes da instalação do SNI. Utilizando o
mesmo modelo animal da última experiência, demonstramos que melhor flexibilidade cognitiva, menor
número de linfócitos T, maior número de linfócitos B e de interleucina-10 (IL-10) estão associados a
condições HT. No entanto, esses valores são observados apenas no estudo com o modelo clássico do
SNI e não no modelo de dor neuropática do SNI modificado. Além disso, o aumento da expressão de IL 10 à priori do SNI num modelo de ratinho foi suficiente para atenuar o fenótipo da dor e a ansiedade um
mês após lesão. Mostramos ainda, através de uma análise proteómica que a expressão de proteínas do
proteossoma 20S (no núcleo accumbens) está aumentada em animais LT.
Em conclusão, neste trabalho, mostramos que: i) a manifestação da dor não é necessária para
promover comorbidades emocionais; ii) aumento do rácio das células B/T, respetivamente, promovem
a não manifestação da dor e que iii) a IL-10 promove a resistência à dor em ratos e ratinhos. Finalmente,
a redução das proteínas do proteossoma 20S é um fator de distinção entre os animais LT e HT.The majority of the individuals who develop pain following a noxious stimulus return to the healthy
state as the injury heals. On the other hand, in a small percentage of people the pain persists for longer
periods and evolve to chronic pain (CP), which is accompanied by emotional and cognitive impairments.
In animals, CP is also characterized by these impairments. However neural mechanisms mediating the
transition from acute to CP remain largely unknown in humans and in animals, whereby determining
susceptible and resistant factors for pain development is crucial to understand this maladaptive
change. These questions in pain field, motivate us to explore the factors associated with painful and
painless, prior and after a chronic neuropathic pain model – the spared nerve injury (SNI).
In this thesis, we first showed that the majority of SNI Sprague-dawley (SD) rats presented
mechanical allodynia (Low threshold - LT), however 13% of them present similar values as controls (High
threshold – HT). Without structural differences in ipsilateral and contralateral sciatic nerves from LT and
HT animals, those lesioned in left sciatic nerve (SNI-L) presented increased anxiety-like behaviors and
decreased well-being. Furthermore, we also found that insular cortex (agranular division) from SNI-L and
right-lesioned (SNI-R) animals was significantly smaller in HT than in LT.
Next, we investigated which behavioral, cellular and molecular factors are related with painless
and painful conditions prior to SNI installation. Using the same animal model as before, we were able to
demonstrated that better behavioral flexibility, decreased number of T lymphocytes, higher number of B
lymphocytes and interleukin-10 (IL-10) were associated with painless conditions (HT for mechanical
allodynia). Also, increased expression of IL-10 prior to SNI in a mouse model is sufficient to attenuate
pain phenotype and anxiety-like behavior. Nonetheless, these values are only observed in SNI experiment
and not in a modified SNI neuropathic pain model. We further show, through a proteomic analysis of
nucleus accumbens (NAc) from rat LT and HT animals, that expression of 20S proteasomal proteins are
increased in LT animals.
In conclusion, with this work we showed that: i) painless also promotes emotional impairments;
ii) lower and higher numbers of T and B cells, respectively, are associated with pain resistance and iii) IL-10 potentiates the pain resistance both in rats and in mice. Finally, reduction of 20S proteasomal proteins
are a distinguish factor between LT and HT animals.O apoio financeiro foi proporcionado por uma bolsa de doutoramento da Fundação para a Ciência e a Tecnologia (FCT - PD/BD/114117/2015) através do Programa Inter-Universitário de Doutoramento em Envelhecimento e Doença Crónicas (PhDOC). Este trabalho também foi financiado pelo IASP Early Career Research Grant 2015, fundos do Fundo Europeu de Desenvolvimento Regional (FEDER) através do Programa de Fatores de Competitividade Operacional – (COMPETE) e fundos nacionais através da FCT no âmbito dos projetos POCI-01-0145-FEDER-007038, PTDC/NEU-SCC/5301/2014 e NORTE-01-0145-ERDF-000023, apoiado pelo Programa Operacional Regional do Norte de Portugal (NORT 2020), no âmbito do Acordo de Parceria PORTUGAL 2020
Abordagens metabolómicas para compreender os transtornos mentais psicóticos
No full textMental illnesses such as Schizophrenia (SCZ) and Bipolar Disorder (BD) can cause major disturbances in the cognitive and emotional control of their patients, which has a great impact on their lives. SCZ and BD are diseases that are difficult to diagnose due to the number of symptoms that overlap between them. Through the study of omics, it is possible not only to better understand these diseases, but also to study possible biomarkers, which could be extremely important to define a diagnosis, and consequently, provide a better treatment and prognosis for these patients. The Liquid Chromatography coupled to Mass Spectrometry (LC-MS) approach was used to study the metabolomics and peptidomics of 69 patients and 15 controls. Two comparisons were analyzed: Controls vs Disease and SCZ/Schizophreniform (SCZFM) vs BD/intermediate diagnosis. From the first comparison, it was possible to detect 4 interesting “features”, 7 endogenous metabolites and 11 statistically altered peptides. The interesting features were 452.7/11.8, 497.2/14.9, 516.3/24.5 and 757.4/8.3. The 7 interesting metabolites were Isoleucine/Leucine, N-Acetylalanine, Ornithine, Palmitoylcarnitine, Phenylalanine, Proline and Valine. Of the 11 peptides, there are 3 from the Fibrinogen Alpha Chain, 2 from the Apolipoprotein A-II protein, 2 from the Complement protein C4-B, 1 from the Complement protein C3, 1 from the Fibrinogen Beta Chain, 1 from the Kininogen-1 and 1 of the Selenocysteine- Specific Elongation Factor. In the second comparison, 4 statistically altered “features” were detected, 377.1/15.6, 448.3/21.9, 452.3/24.8 and 564.2/15.9. In addition to the individual molecules, in the first comparison 3 altered pathways were detected, Bile Acid Biosynthesis, Vitamin E Metabolism and Aminoacyl-tRNA Biosynthesis and in the second comparison no altered pathway was detected as altered. Although it was not possible to propose a potential biomarker or altered pathways that confidently distinguish SCZ from BD, we present the metabolic pathways on which future studies should focus on these two diseases for the improvement of the treatment.As doenças mentais como Esquizofrenia (SCZ) e Transtorno Bipolar (BD) podem causar grandes distúrbios no controle cognitivo e emocional dos seus pacientes, o que causa um grande impacto nas suas vidas. A SCZ e o BD são doenças de difícil diagnóstico devido ao número de sintomas sobrepostos entre ambas. Através do estudo das ómicas, é possível não só compreender melhor estas doenças, como estudar possíveis biomarcadores, que poderão vir a ser de extrema importância para definir um diagnóstico, e consequentemente, providenciar um melhor tratamento e prognóstico a estes pacientes. Foi utilizado uma abordagem usando Cromatografia Liquída acoplada a Espectrometria de Massa (LC-MS) para estudar a metabolómica e peptidómica de 69 pacientes e 15 controlos. Analisaram-se duas comparações: Controlos vs Doença e SCZ/Esquizofreniforme (SCZFM) vs BD/diagnóstico Intermédio. A partir da primeira comparação, foi possível detetar 4 “features” interessantes, 7 metabolitos endógenos e 11 péptidos estatisticamente alterados. As features interessantes foram 452.7/11.8, 497.2/14.9, 516.3/ 24.5 e 757.4/8.3. Os 7 metabolitos interessantes foram Isoleucina/Leucina, N-Acetilalanina, Ornitina, Palmitoilcarnitina, Fenilalanina, Prolina e a Valina. Dos 11 peptídos, são 3 da Cadeia Alfa do Fibrinogénio, 2 da proteína Apolipoproteína A-II, 2 da proteína de Complemento C4-B, 1 da proteína de Complemento C3, 1 da Cadeia Beta do Fibrinogénio, 1 do Cininogénio-1 e 1 do Fator de Elongação Específico da Selenocisteina. Na segunda comparação, foram detetadas 4 “features” estatisticamente alteradas, 377.1/15.6, 448.3/21.9, 452.3/24.8 e 564.2/15.9. Além das moléculas individuais, na primeira comparação foram detetadas 3 vias alteradas, Biossíntese dos Ácidos Biliares, Metabolismo da Vitamina E and Biossíntese dos Aminoacil-tANR e na segunda comparação nenhuma via foi detetada alterada. Apesar de não ter sido possível propor um potencial biomarcador ou vias alteradas que distingam SCZ de BD com confiança, apresentamos as vias metabólicas nos quais futuros estudos deverão incidir nestas duas doenças. Assim, podemos estar um passo mais perto de entender estes transtornos.Mestrado em Bioquímic
Intercellular relationships in the tumor microenvironment: implications for cancer pathophysiology
No full textTese de Doutoramento em Envelhecimento e Doenças CrónicasCancer cells do not manifest the disease alone, but rather recruit and corrupt resident normal
cell types to create the tumor microenvironment (TME). Collaborative interactions between
neoplastic cancer cells and their supporting stroma leads to chronic proliferation and to the
formation of organ-like structures that typify most human tumors. The TME per se is a major
driver of intratumor heterogeneity, malignant progression and development of resistance to
conventional anticancer therapies. Although this was recognized more than a decade ago, the
high complexity of the interactions in the tumor network are far from being fully understood.
In this scenario, understanding the crosstalk between the different cellular compartments of the
TME, to better understand the biology of the tumors, is crucial for the clinical management of
oncologic patients. The research summarized in this thesis focus on the intercellular
relationships in the TME and their implications in cancer, adding to the body of knowledge that
may incrementally contribute to a better understanding of this devastating group of diseases.
The brain has a particular TME, in part because of the specific brain-resident cell types. We
initiated our studies by investigating how the paracrine activity of resident glial cells is
modulated by brain tumor cancer cells, and how this crosstalk can influence the malignant
phenotype of cancer cells. For this, we used glioblastoma (GBM) as a tumor model because it
is the most malignant primary brain tumor. A proteomic analysis of the secretome of primary
mouse glial cells unexposed or exposed (primed) to the secretome of GBM cells was conducted.
The pre-exposure of glial cells to the secretome of cancer cells led to the upregulation of several
proteins related to inflammatory response, cell adhesion and extracellular structure organization
within the secretome of glial cells. Additionally, GBM cells exposed to the secretome of primed
glial cells increased their viability and cell-death resistance, while decreased their migratory
capacity. These effects on GBM cells were accompanied by the activation of intracellular
cancer-related pathways, mainly the MAPK/ERK pathway, a regulator of cell proliferation.
With these results, we showed that glial cells can impact on the pathophysiology of brain
tumors, and that the paracrine interactions between cancer cells and neighbouring glial cells
regulate the “go-or-grow” phenotype of GBM cells.
Besides resident cells, also circulating immune cells that are recruited to the tumor play a crucial
role in the TME, reason why escaping from the immune system is a hallmark of cancer.
Additionally, the immunosuppressive TME can hamper the efficiency of cancer therapies.
Thus, in the second part of this thesis, we explored the potential targeting of an axon-guidance molecule, Plexin A4 (PlxnA4), in the stromal cells to increase anti-tumor immunity. The
constitutive deletion of PlxnA4 in the immune system showed to decelerate the tumor growth
of several tumor models by increasing the infiltration of cytotoxic T lymphocytes (CTLs),
revealing that its expression in immune cells is critical for immunosuppression. Particularly,
the loss of PlxnA4 in CTLs increased their migratory capacity as well as their proliferation,
being sufficient to re-shape the TME towards a more immunopermissive state. Moreover, the
therapeutic use of PlxnA4 deletion in CTLs showed that PlxnA4 targeting can be a potential
new anti-tumor immunotherapy that can be used alone or in combination with other cancer
therapies.
In summary, the work presented in this thesis contributes to a better understanding of the highly
complex interactions at the TME, highlighting its remarkable relevance for cancer
pathophysiology. Particularly, the characterization of the interaction between glial cells and
cancer cells may be relevant to understand brain tumors and metastasis TME, and may be an
important tool for the development of new therapeutic approaches. Additionally, the use of
targeting strategies involving the inactivation of PlxnA4 in CTLs may be important as a new
anti-cancer therapy by re-shaping the immunosuppressive TME.As células cancerígenas não provocam sozinhas a doença, em vez disso, elas recrutam e
modificam células residentes normais para criar o microambiente tumoral. Interações entre as
células neoplásicas e o seu estroma de suporte levam a uma proliferação crónica e à formação
de estruturas tipo-órgão que caracterizam a maioria dos tumores humanos. O microambiente
tumoral per se é um dos maiores impulsionadores da heterogeneidade intratumoral, progressão
maligna e desenvolvimento de resistência às terapias convencionais. Embora isso tenha sido
reconhecido há mais de uma década, a elevada complexidade das interações tumorais está longe
de ser completamente compreendida. Neste cenário, compreender as interações entre os
diferentes compartimentos celulares do microambiente tumoral, para melhor compreender a
biologia dos tumores, é crucial para a gestão clínica dos pacientes oncológicos. A investigação
sumariada nesta tese foca-se nas relações intercelulares no microambiente tumoral e nas suas
implicações para o cancro, acrescentando conhecimento que poderá contribuir para uma melhor
compreensão deste grupo de doenças devastadoras.
O cérebro tem um microambiente tumoral particular, em parte por causa das células que nele
residem. No nosso trabalho, começámos por investigar como é que a atividade parácrina das
células gliais residentes é modulada pelas células cerebrais tumorais, e como é que essa
interação pode influenciar o fenótipo maligno das células tumorais. Para isso, usámos um
modelo tumoral de glioblastoma (GBM) porque este é o tumor primário mais maligno do
cérebro. Foi realizada uma análise proteómica do secretoma de células primárias de glia não
expostas ou expostas (condicionadas) ao secretoma de células de GBM. A pré-exposição das
células da glia ao secretoma das células tumorais levou a um aumento de diversas proteínas
relacionadas com a resposta inflamatória, adesão celular e organização da estrutura extracelular
no secretoma das células da glia. Além disso, células de GBM expostas ao secretoma de células
da glia condicionadas aumentaram a sua viabilidade assim como resistência à morte celular,
enquanto perderam capacidade de migração. Estes efeitos nas células de GBM foram
acompanhados pela ativação de vias intracelulares relacionadas com o cancro, em particular a
via MAPK/ERK, uma via que regula a proliferação celular. Com estes resultados,
demonstrámos que as células da glia podem ter um impacto na patofisiologia dos tumores
cerebrais e que as interações parácrinas entre as células tumorais e as células gliais vizinhas
regulam o fenótipo de “migrar-ou-crescer” das células de GBM. Para além das células residentes, também as células imunes que estão em circulação podem ser
recrutadas para o tumor, onde desempenham um papel no microambiente tumoral, razão pela
qual escapar do sistema imune é uma das características do cancro. Além disso, o
microambiente imunossupressor pode dificultar a eficiência das terapias. Assim, na segunda
parte desta tese, explorámos a potencial manipulação de uma molécula guia de axónios, a
plexina A4 (PlxnA4), nas células do estroma de modo a aumentar a imunidade anti-tumoral. A
deleção constitutiva da PlxnA4 no sistema imune mostrou desacelerar o crescimento tumoral
em vários modelos através do aumento da infiltração de linfócitos T citotóxicos, revelando que
a sua expressão nas células imunes é fundamental para a imunossupressão. Em particular, a
perda da PlxnA4 nas células T citotóxicas levou a um aumento da sua capacidade migratória e
proliferativa, sendo suficiente para alterar o microambiente tumoral para um estado mais
imunopermissivo. Além disso, o uso da PlxnA4 como alvo terapêutico em células T citotóxicas
mostrou ter potencial para ser uma nova imunoterapia que pode ser usada como monoterapia
ou em combinação com outras terapias contra o cancro.
Em suma, o trabalho apresentado nesta tese contribui para um melhor entendimento das
complexas interações no microambiente tumoral, salientando a sua extrema importância para a
patofisiologia do cancro. Em particular, a caracterização da interação entre as células gliais e
as células de glioma pode ser relevante para compreender o microambiente das células tumorais
bem como das metástases cerebrais, podendo ser uma ferramenta importante para o
desenvolvimento de novas abordagens terapêuticas. Além disso, o uso de terapias envolvendo
a inativação da PlxnA4 em células T citotóxicas pode ser importante como uma nova terapia
por alterar o microambiente tumoral imunossupressor.The work presented in this thesis was performed at the Life and Health Sciences Research Institute (ICVS), University of Minho, CNC - Center for Neuroscience and Cell Biology, University of Coimbra and VIB
Center for Cancer Biology, Department of Oncology, University of Leuven. The author was supported by grant SFRH/BD/52287/2013 from Fundação para a Ciência e a Tecnologia (FCT) through the Inter-
University Doctoral Programme in Aging and Chronic Disease (PhDOC). Financial support was provided by grants from FCT (PTDC/SAU-GMG/113795/2009, PTDC/NEU-NMC/0205/2012, PTDC/NEU-SCC/7051/2014, PEst-C/SAU/LA0001/2013–2014 and UID/NEU/04539/2013), Liga Portuguesa Contra o Cancro, Fundação
Calouste Gulbenkian and European Research Council (ERC starting grant – OxyMO, 308459). Project co-financed by ON.2—O Novo Norte; QREN; FEDER funds through the Operational Programme
Competitiveness Factors - COMPETE and National Funds through FCT under the project POCI-01-0145-FEDER-007038; and the project NORTE-01-0145-FEDER-000013, supported by Norte Portugal
Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) and by The National Mass Spectrometry Network (RNEM) under the contract REDE/1506/REM/2005
Changes in the intestinal mucosal proteome of turkeys (Meleagris gallopavo) infected with haemorrhagic enteritis virus
No full textHaemorrhagic enteritis (HE) is a viral disease affecting intestinal integrity and barrier function in turkey (Meleagris gallopavo) and resulting in a significant economic loss. Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra (SWATH-MS) was applied to identify crucial proteins involved in HE infection. A total of 938 proteins were identified and used to generate a reference library for SWATH-MS analysis. In total, 523 proteins were reliably quantified, and 64 proteins were found to be differentially expressed, including 49 up-regulated and 15 down-regulated proteins between healthy and HE-affected intestinal mucosa. Functional analysis suggested that these proteins were involved in the following categories of cellular pathways and metabolisms: 1) energy pathways; 2) intestine lipid and amino acid metabolism; 3) oxidative stress; 4) intestinal immune response. Major findings of this study demonstrated that natural HE infection is related to the changes in abundance of several proteins involved in cell-intrinsic immune defense against viral invasion, systemic inflammation, modulation of excessive inflammation, B and T cell development and function and antigen presentation. mRNA quantitative expression demonstrated that most of the proteins involved in innate immunity that were found to be differentially abundant were produced by intestinal mucosa, suggesting its direct involvement in immune defences against HE infection
Functional and structural characterization of synthetic cardosin B-derived rennet
No full textThe potential of using a synthetic cardosin-based rennet in cheese manufacturing was recently demonstrated with the development and optimization of production of a recombinant form of cardosin B in Kluyveromyces lactis. With the goal of providing a more detailed characterization of this rennet, we herein evaluate the impact of the plant-specific insert (PSI) on cardosin B secretion in this yeast, and provide a thorough analysis of the specificity requirements as well as the biochemical and structural properties of the isolated recombinant protease. We demonstrate that the PSI domain can be substituted by different linker sequences without substantially affecting protein secretion and milk clotting activity. However, the presence of small portions of the PSI results in dramatic reductions of secretion yields in this heterologous system. Kinetic characterization and specificity profiling results clearly suggest that synthetic cardosin B displays lower catalytic efficiency and is more sequence selective than native cardosin B. Elucidation of the structure of synthetic cardosin B confirms the canonical fold of an aspartic protease with the presence of two high mannose-type, N-linked glycan structures; however, there are some differences in the conformation of the flap region when compared to cardosin A. These subtle variations in catalytic properties and the more stringent substrate specificity of synthetic cardosin B help to explain the observed suitability of this rennet for cheese production.This work was funded by Fundo Europeu de
Desenvolvimento Regional (FEDER) Funds through the Operational
Competitiveness Programme (COMPETE) and by National Funds
through Fundação para a Ciência e a Tecnologia (FCT) under projects
PTDC/AGR-ALI/102540/2008 and POCI-01-0145-FEDER-007274.
Support by the BStructured program on bioengineered therapies for infectious diseases and tissue regeneration^ (Norte-01-0145-FEDER-000012),
funded by Norte Portugal Regional Operational Programme (NORTE
2020), under the PORTUGAL 2020 Partnership Agreement, through
FEDER is also acknowledged. The authors would like to acknowledge
the strategic project UID/NEU/04539/2013 and the National Mass
Spectrometry Network (RNEM) under the contract REDE/1506/REM/
2005. Edman sequencing data were obtained by the Analytical
Laboratory, Analytical Services Unit, Instituto de Tecnologia Quıímica
e Biológica, Universidade Nova de Lisboa. We acknowledge the ESRF
for provision of synchrotron radiation facilities and thank the ESRF staff
for the help with data collection. The authors would like to thank Sandra
Isabel Anjo for her inputs on acquisition of MS data
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