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Ensuring Access to Safe and Nutritious Food for All Through the Transformation of Food Systems
Examples of works to practice staccato technique in clarinet instrument
Klarnetin staccato tekniğini güçlendirme aşamaları eser çalışmalarıyla uygulanmıştır. Staccato
geçişlerini hızlandıracak ritim ve nüans çalışmalarına yer verilmiştir. Çalışmanın en önemli amacı
sadece staccato çalışması değil parmak-dilin eş zamanlı uyumunun hassasiyeti üzerinde de
durulmasıdır. Staccato çalışmalarını daha verimli hale getirmek için eser çalışmasının içinde etüt
çalışmasına da yer verilmiştir. Çalışmaların üzerinde titizlikle durulması staccato çalışmasının ilham
verici etkisi ile müzikal kimliğe yeni bir boyut kazandırmıştır. Sekiz özgün eser çalışmasının her
aşaması anlatılmıştır. Her aşamanın bir sonraki performans ve tekniği güçlendirmesi esas alınmıştır.
Bu çalışmada staccato tekniğinin hangi alanlarda kullanıldığı, nasıl sonuçlar elde edildiği bilgisine
yer verilmiştir. Notaların parmak ve dil uyumu ile nasıl şekilleneceği ve nasıl bir çalışma disiplini
içinde gerçekleşeceği planlanmıştır. Kamış-nota-diyafram-parmak-dil-nüans ve disiplin
kavramlarının staccato tekniğinde ayrılmaz bir bütün olduğu saptanmıştır. Araştırmada literatür
taraması yapılarak staccato ile ilgili çalışmalar taranmıştır. Tarama sonucunda klarnet tekniğin de
kullanılan staccato eser çalışmasının az olduğu tespit edilmiştir. Metot taramasında da etüt
çalışmasının daha çok olduğu saptanmıştır. Böylelikle klarnetin staccato tekniğini hızlandırma ve
güçlendirme çalışmaları sunulmuştur. Staccato etüt çalışmaları yapılırken, araya eser çalışmasının
girmesi beyni rahatlattığı ve istekliliği daha arttırdığı gözlemlenmiştir. Staccato çalışmasını yaparken
doğru bir kamış seçimi üzerinde de durulmuştur. Staccato tekniğini doğru çalışmak için doğru bir
kamışın dil hızını arttırdığı saptanmıştır. Doğru bir kamış seçimi kamıştan rahat ses çıkmasına
bağlıdır. Kamış, dil atma gücünü vermiyorsa daha doğru bir kamış seçiminin yapılması gerekliliği
vurgulanmıştır. Staccato çalışmalarında baştan sona bir eseri yorumlamak zor olabilir. Bu açıdan
çalışma, verilen müzikal nüanslara uymanın, dil atış performansını rahatlattığını ortaya koymuştur.
Gelecek nesillere edinilen bilgi ve birikimlerin aktarılması ve geliştirici olması teşvik edilmiştir.
Çıkacak eserlerin nasıl çözüleceği, staccato tekniğinin nasıl üstesinden gelinebileceği anlatılmıştır.
Staccato tekniğinin daha kısa sürede çözüme kavuşturulması amaç edinilmiştir. Parmakların
yerlerini öğrettiğimiz kadar belleğimize de çalışmaların kaydedilmesi önemlidir. Gösterilen azmin ve
sabrın sonucu olarak ortaya çıkan yapıt başarıyı daha da yukarı seviyelere çıkaracaktır
Estudo da remodelagem reversa miocárdica através da análise proteómica do miocárdio e do líquido pericárdico
Valve replacement remains as the standard therapeutic option for aortic
stenosis patients, aiming at abolishing pressure overload and triggering
myocardial reverse remodeling. However, despite the instant hemodynamic
benefit, not all patients show complete regression of myocardial hypertrophy,
being at higher risk for adverse outcomes, such as heart failure. The current
comprehension of the biological mechanisms underlying an incomplete reverse
remodeling is far from complete. Furthermore, definitive prognostic tools and
ancillary therapies to improve the outcome of the patients undergoing valve
replacement are missing. To help abridge these gaps, a combined myocardial
(phospho)proteomics and pericardial fluid proteomics approach was followed,
taking advantage of human biopsies and pericardial fluid collected during
surgery and whose origin anticipated a wealth of molecular information
contained therein.
From over 1800 and 750 proteins identified, respectively, in the myocardium
and in the pericardial fluid of aortic stenosis patients, a total of 90 dysregulated
proteins were detected. Gene annotation and pathway enrichment analyses,
together with discriminant analysis, are compatible with a scenario of increased
pro-hypertrophic gene expression and protein synthesis, defective ubiquitinproteasome system activity, proclivity to cell death (potentially fed by
complement activity and other extrinsic factors, such as death receptor
activators), acute-phase response, immune system activation and fibrosis.
Specific validation of some targets through immunoblot techniques and
correlation with clinical data pointed to complement C3 β chain, Muscle Ring
Finger protein 1 (MuRF1) and the dual-specificity Tyr-phosphorylation
regulated kinase 1A (DYRK1A) as potential markers of an incomplete
response. In addition, kinase prediction from phosphoproteome data suggests
that the modulation of casein kinase 2, the family of IκB kinases, glycogen
synthase kinase 3 and DYRK1A may help improve the outcome of patients
undergoing valve replacement. Particularly, functional studies with DYRK1A+/-
cardiomyocytes show that this kinase may be an important target to treat
cardiac dysfunction, provided that mutant cells presented a different response
to stretch and reduced ability to develop force (active tension).
This study opens many avenues in post-aortic valve replacement reverse
remodeling research. In the future, gain-of-function and/or loss-of-function
studies with isolated cardiomyocytes or with animal models of aortic bandingdebanding will help disclose the efficacy of targeting the surrogate therapeutic
targets. Besides, clinical studies in larger cohorts will bring definitive proof of
complement C3, MuRF1 and DYRK1A prognostic value.A substituição da válvula aórtica continua a ser a opção terapêutica de
referência para doentes com estenose aórtica e visa a eliminação da
sobrecarga de pressão, desencadeando a remodelagem reversa miocárdica.
Contudo, apesar do benefício hemodinâmico imediato, nem todos os pacientes
apresentam regressão completa da hipertrofia do miocárdio, ficando com maior
risco de eventos adversos, como a insuficiência cardíaca. Atualmente, os
mecanismos biológicos subjacentes a uma remodelagem reversa incompleta
ainda não são claros. Além disso, não dispomos de ferramentas de
prognóstico definitivos nem de terapias auxiliares para melhorar a condição
dos pacientes indicados para substituição da válvula. Para ajudar a resolver
estas lacunas, uma abordagem combinada de (fosfo)proteómica e proteómica
para a caracterização, respetivamente, do miocárdio e do líquido pericárdico
foi seguida, tomando partido de biópsias e líquidos pericárdicos recolhidos em
ambiente cirúrgico.
Das mais de 1800 e 750 proteínas identificadas, respetivamente, no miocárdio
e no líquido pericárdico dos pacientes com estenose aórtica, um total de 90
proteínas desreguladas foram detetadas. As análises de anotação de genes,
de enriquecimento de vias celulares e discriminativa corroboram um cenário de
aumento da expressão de genes pro-hipertróficos e de síntese proteica, um
sistema ubiquitina-proteassoma ineficiente, uma tendência para morte celular
(potencialmente acelerada pela atividade do complemento e por outros fatores
extrínsecos que ativam death receptors), com ativação da resposta de fase
aguda e do sistema imune, assim como da fibrose.
A validação de alguns alvos específicos através de immunoblot e correlação
com dados clínicos apontou para a cadeia β do complemento C3, a Muscle
Ring Finger protein 1 (MuRF1) e a dual-specificity Tyr-phosphoylation
regulated kinase 1A (DYRK1A) como potenciais marcadores de uma resposta
incompleta. Por outro lado, a predição de cinases a partir do fosfoproteoma,
sugere que a modulação da caseína cinase 2, a família de cinases do IκB, a
glicogénio sintase cinase 3 e da DYRK1A pode ajudar a melhorar a condição
dos pacientes indicados para intervenção. Em particular, a avaliação funcional
de cardiomiócitos DYRK1A+/- mostraram que esta cinase pode ser um alvo
importante para tratar a disfunção cardíaca, uma vez que os miócitos mutantes
responderam de forma diferente ao estiramento e mostraram uma menor
capacidade para desenvolver força (tensão ativa).
Este estudo levanta várias hipóteses na investigação da remodelagem reversa.
No futuro, estudos de ganho e/ou perda de função realizados em
cardiomiócitos isolados ou em modelos animais de banding-debanding da
aorta ajudarão a testar a eficácia de modular os potenciais alvos terapêuticos
encontrados. Além disso, estudos clínicos em coortes de maior dimensão
trarão conclusões definitivas quanto ao valor de prognóstico do complemento
C3, MuRF1 e DYRK1A.Programa Doutoral em Biomedicin
Terapia fotodinâmica na inativação de bacteriófagos com porfirina e potenciadoresem águas residuais
Pathogenic viruses are frequently introduced into marine and estuarine waters through the discharge of treated and untreated sewage, since current treatments are unable to provide virus-free wastewater (WW) effluents, affecting the receiving waters quality and, consequently, human health. The removal of harmful constituents by the conventional treatments comprises a combination of chemical, physical and biological methods. Usually, WW from urban areas is secondarily, rarely tertiary, treated. Although the secondary effluent contains high concentrations of microorganisms, the effect of water dilution makes it acceptable in terms of quality indicators. In tertiary treatment, chlorination is the most common method used to ensure microbiological safety in tertiarily treated effluents. However, its massive utilization, both in free and combined chlorine forms, may lead to the formation of chemical disinfection by-products though the reaction with organic matter present in the effluents, being those chemicals toxic to aquatic organisms, representing potential health hazards. Unfortunately, these conventional methods are limited and may not be adequate to reach the quality levels specified by the guidelines. Photodynamic therapy (PDT) with porphyrins may be a promising approach for the inactivation of pathogens as they are effective in inactivating microorganisms without the formation of potentially toxic products. Some studies have reported an enhancer effect on antimicrobial photodynamic therapy (aPDT) by the combined used of some photosensitizer (PS) with potassium iodide (KI) and hydrogen peroxide (H2O2). The main objective of this study was to evaluate the aPDT efficacy of a PS based on a low-cost formulation constituted by five cationic porphyrins (Form) and its potentiation effect by KI and H2O2 in the inactivation of a T4-like bacteriophage in WW. The experiments were done in phosphate buffered saline and in filtered and non-filtered contaminated wastewater. The aPDT assays in filtered WW (0.45 μm pore-size) were performed with different concentrations of Form (1.0 to 10 μM). In a second phase was evaluated the effect of KI (100 mM) in the photodynamic action of Form (1.0 to 10 μM). The results of these experiments demonstrated that Form is efficient in filtered WW treatment and that the efficacy of bacteriophage photoinactivation is correlated with the concentration of the used PS. When combined with KI, the Form is clearly less effective to inactivate the bacteriophage. To evaluate if the organic matter present in water influences the efficiency of PS, the WW was filtered using three different pore-sized membranes (0.45, 0.30 and 0.22 μm). The results demonstrated that the increase of organic matter promote a significant decrease in the efficiency of Form. In order to evaluate if the efficiency of aPDT to inactivate bacteriophages is maintained when the treatments are performed in non-filtrated WW, the effect of Form alone (10 μM) and combined with H2O2 (2, 5 and 9%) in non-filtered WW was evaluated. The Form alone proved to be an efficient PS to photoinactivate the bacteriophage in non-filtered WW, but the presence of H2O2 enhanced the photodynamic effect. The FORM can be an effective alternative to control viruses in WW, particularly if combined with H2O2.Os vírus patogénicos são frequentemente introduzidos nas águas marinhas e estuarinas através da descarga de esgoto tratado e não tratado, uma vez que os tratamentos atuais não inativam os vírus presentes nas águas residuais (WW), afetando a qualidade das águas recetoras e, consequentemente, a saúde humana. Nos tratamentos convencionais, a remoção de constituintes nocivos consiste no uso de métodos químicos, físicos e biológicos. Geralmente, a WW de áreas urbanas é tratada secundariamente e não terciariamente. Embora o efluente secundário contenha altas concentrações de microrganismos, o efeito da diluição na água torna-o aceitável em termos de indicadores de qualidade. A cloração é o método mais comum usado para garantir a segurança microbiológica em efluentes tratados terciariamente. No entanto, a sua utilização maciça, tanto na forma de cloro livre como combinada, pode levar à formação de subprodutos químicos como resultado da reação com a matéria orgânica presente nos efluentes, sendo esses produtos químicos tóxicos para os organismos aquáticos, apresentando riscos para a saúde. Os métodos convencionais são limitados e podem não ser adequados para manter os níveis de qualidade especificados nas diretrizes. As porfirinas quando usadas como fotossensibilizadores (PS) na terapia fotodinâmica (PDT) podem ser desinfetantes promissores para a inativação de microrganismos patógenicos, pois são eficazes na inativação de microrganismos sem formação de produtos tóxicos. Alguns estudos mostraram efeito potenciador de alguns PS usados em terapia fotodinâmica antimicrobiana (aPDT) quando estes são usados em combinação com iodeto de potássio (KI) e peróxido de hidrogénio (H2O2). O principal objetivo deste estudo foi avaliar a eficácia da aPDT de um PS baseado numa formulação de baixo custo constituída por cinco porfirinas catiónicas (Form) e o seu efeito potenciador por KI e H2O2 na inativação de um bacteriófago tipo T4. As experiências foram realizadas em solução salina tamponada com fosfato e em água residual contaminada filtrada e não filtrada. Os ensaios de aPDT em WW filtrada (tamanho do poro de 0,45 μm) foram realizados com diferentes concentrações de Form (1,0 a 10 μM). Numa segunda fase foi avaliado o efeito do KI (100 mM) na ação fotodinâmica da FORM (1,0 a 10 μM). Os resultados dessas experiências demonstraram que a Form é eficiente no tratamento de WW filtrada e que a eficácia da fotoinativação de bacteriófagos está correlacionada com a concentração do PS usado. Quando combinada com o KI, a Form é claramente menos eficaz na inativação do bacteriófago. Para avaliar se a matéria orgânica presente na água influencia a eficiência do PS, a WW foi filtrada usando três membranas com tamanho de poros diferentes (0,45, 0,30 e 0,22 μm). Os resultados mostraram que o aumento da matéria orgânica promove uma diminuição significativa na eficiência da Form. Para avaliar se a eficiência da aPDT para inativar bacteriófagos é mantida quando os tratamentos são realizados em WW não filtrada, o efeito da Form sozinha (10 μM) e combinado com H2O2 (2, 5 e 9%) em WW não filtrada foi avaliado. A Form por si só provou ser um PS eficiente para fotoinativar o bacteriófago em WW não filtrada, mas a presença de H2O2 aumentou significativamente o efeito fotodinâmico. A Form pode ser uma alternativa eficaz para controlar vírus na WW, principalmente se combinada com H2O2.This work was supported by funding FEDER through COMPETE – Programa Operacional Factores de Competitividade, and by National funding through Fundação para a Ciência e Tecnologia (FCT) and Marine Studies (CESAM).Mestrado em Biologia Molecular e Celula
Mathematical models to evaluate the impact of increasing serotype coverage in pneumococcal conjugate vaccines
Of over 100 serotypes of Streptococcus pneumoniae, only 7 were included in the first pneumo- coccal conjugate vaccine (PCV). While PCV reduced the disease incidence, in part because of a herd immunity effect, a replacement effect was observed whereby disease was increasingly caused by serotypes not included in the vaccine. Dynamic transmission models can account for these effects to describe post-vaccination scenarios, whereas economic evaluations can enable decision-makers to compare vaccines of increasing valency for implementation. This thesis has four aims. First, to explore the limitations and assumptions of published pneu- mococcal models and the implications for future vaccine formulation and policy. Second, to conduct a trend analysis assembling all the available evidence for serotype replacement in Europe, North America and Australia to characterise invasive pneumococcal disease (IPD) caused by vaccine-type (VT) and non-vaccine-types (NVT) serotypes. The motivation behind this is to assess the patterns of relative abundance in IPD cases pre- and post-vaccination, to examine country-level differences in relation to the vaccines employed over time since introduction, and to assess the growth of the replacement serotypes in comparison with the serotypes targeted by the vaccine. The third aim is to use a Bayesian framework to estimate serotype-specific invasiveness, i.e. the rate of invasive disease given carriage. This is useful for dynamic transmission modelling, as transmission is through carriage but a majority of serotype-specific pneumococcal data lies in active disease surveillance. This is also helpful to address whether serotype replacement reflects serotypes that are more invasive or whether serotypes in a specific location are equally more invasive than in other locations. Finally, the last aim of this thesis is to estimate the epidemiological and economic impact of increas- ing serotype coverage in PCVs using a dynamic transmission model. Together, the results highlight that though there are key parameter uncertainties that merit further exploration, divergence in serotype replacement and inconsistencies in invasiveness on a country-level may make a universal PCV suboptimal.Open Acces
Understanding interactions between Ramularia collo-cygni and barley leaf physiology to target improvements in host resistance and disease control strategy
Ramularia Leaf Spot (RLS) is an increasingly problematic disease of barley.
Control options are limited as the causal fungus, Ramularia collo-cygni, has
developed resistance to several of the major fungicide groups. Developing
new methods for controlling this disease is therefore a priority. R. collo-cygni
can grow systemically in barley plants from infected seed, without inducing
visible symptoms. In the field, visible symptoms normally only appear after
flowering. The relative contribution of the latent and symptomatic stages of
the fungal lifecycle to reduction in barley yield is not currently known with any
certainty. Two possibilities are that the effect of asymptomatic infection on
pre-flowering photosynthetic activity, and the development of grain sink
capacity, plays an important role; or that reduction in photosynthetic activity
during grain filling, resulting from lesion development and loss of green leaf
area, is the predominant factor. This research aimed to increase our
understanding of the impact of different phases of the fungal lifecycle on
barley photosynthesis and yield formation, to better target host resistance
and disease control strategies.
Controlled environment and field experiments were used to determine the
relative effects of asymptomatic and symptom-expressing phases of R. collo-cygni infection on photosynthesis and yield formation in spring barley. In
controlled environment experiments leaf photosynthetic activity was
measured in seedlings inoculated with suspensions of R. collo-cygni mycelia.
Measurements were made before and after visible symptom development
using Infra-Red Gas Analysis (IRGA), chlorophyll fluorescence analysis and
chlorophyll fluorescence imaging. No reduction in photosynthetic activity was
observed in leaves infected with R. collo-cygni, compared to those of non-
infected leaves, during the latent phase of infection. After the appearance of
visible symptoms, photosynthetic activity within lesions reduced as the
lesions developed. However, this did not lead to reductions in photosynthetic
activity when measured across the whole leaf area, suggesting that for there
to be a significant effect of disease on whole leaf photosynthetic activity,
visible symptoms must develop into mature lesions and coalesce to cover
larger areas of the leaf surface.
In field experiments plots were treated with a full fungicide regime, left
untreated, or inoculated with R. collo-cygni and treated with fungicide to
which R. collo-cygni is resistant (the latter as a precaution against lack of
natural RLS disease that year and/or other diseases developing on untreated
plots). RLS was the only disease of significance that developed in untreated
or inoculated plots. Symptoms first appeared after flowering, around Zadoks
Growth Stage 72. Fungicide-treated plots remained free of disease.
Chlorophyll fluorescence analysis of field plants showed no effect of infection
on the maximum quantum efficiency of Photosystem II (Fv/Fm) before visible
symptom development, consistent with results from controlled environment
experiments. Grain yield of untreated and fungicide-treated plots was
predicted from fixed common values of radiation use efficiency (RUE) and
utilisation of soluble sugar reserves, and measured values of post-flowering
healthy (green) leaf area light interception. Grain yields predicted from the
difference in post-flowering light interception between fungicide-treated plants
and untreated or inoculated plants displaying symptoms of RLS were
comparable with the measured yield response to fungicide. This suggests
that yield loss to RLS is primarily associated with a reduction in light capture
during grain filling, resulting from lesion development and loss of green leaf
area.
Results from controlled environment and field experiments suggested that
symptom expression was associated with leaf senescence. Further controlled
environment experiments tested this relationship by using treatments to vary
the onset and rate of leaf senescence. Seedlings that were treated with
cytokinin to delay senescence after inoculation with suspensions of R. collo-cygni mycelia developed fewer lesions than control plants. Fungal growth, as
measured by quantification of R. collo-cygni DNA in leaves, was also
restricted in plants treated with cytokinin.
Collectively these results suggest that prevention of visible symptom
development, rather than prevention of asymptomatic growth, is the most
important target for management of this disease. Control methods targeted at
delaying senescence could be a useful avenue for further investigation
Desenvolvimento de testes genéticos por PCR em tempo-real para diagnóstico rápido de LHON e surdez
Mitochondrial cytopathies are a set of diseases caused by a disturbance in the cell energy production. Mitochondrial dysfunction impairs efficiency of the mitochondrial respiratory chain (MRC) and ATP production, affecting the organism’s energetic equilibrium. Pathogenic sequence variants in mitochondrial DNA (mtDNA) that lead to these pathologies are more frequent in tissues that need higher energy levels to function.
The presented work looks into two such diseases: Leber’s Hereditary Optic Neuropathy and mitochondrial non-syndromic Hearing Loss (MNSHL).
LHON is characterized by presence of genetic alterations in mtDNA, with three main primary pathogenic sequence variants existing, which represent 90-95% of LHON cases with an identified genetic cause: m.3460G>A, in ND1 subunit gene; m.11778G>A, in ND4 subunit gene; and m.14484T>C, in ND6 subunit gene. All of these are subunits of the MRC’s complex I. These mtDNA variations lead to mitochondrial dysfunction in complex I, creating ATP depletion, reactive oxygen species (ROS) increase and oxidative stress.
LHON is commonly characterized by a sequential vision loss and, within 1 year of symptoms starting, 97% of patients with vision loss in one eye develop loss in the second. Therapy administration yields good outcomes, if done in a short-time span after first vision loss. It is essential to quickly and reliably scan for pathogenic sequence variants, in order to act timely and rescue function.
Mitochondrial non-syndromic hearing loss and deafness (MNSHL) is characterized by sensorineural hearing loss (SNHL). This type of hearing loss, particularly when induced by aminoglycosides, has also three primary pathogenic sequence variants associated with ototoxicity: m.1494C>T and m.1555A>G, both in the MTRNR1 gene, and m.7445A>G, in the MTCO1 and MTTS1 genes. These are responsible for ATP depletion, an increase of ROS and oxidative stress, due to alterations in the mitochondrial ribosome or tRNA.
In MNSHL, the cochlea is the affected tissue. With this disorder the principal modifier factor is the administration of aminoglycosides, a type of antibiotics, which trigger a cascade, that leads the individual permanently deaf. The best course of action is prevention, and to ensure clinical action is not dramatically slowed down, results that show whether administration is safe or not need to be quick.
The aim of this work, for both diseases, is the development of a screening method characterized by fast and reliable approach for genetic assessment, to be used for clinical guidance, particularly in therapeutics.
For LHON, the screening method is based on real-time PCR with High-Resolution Melting (HRM) analysis, for detection of the TOP-3 pathogenic sequence variants, by assessing the amplicon’s Tm. In this case, 94 samples were analyzed, including LHON suspected patients, relatives, other mitochondrial disease patients and healthy controls. All samples were previously
classified by another method, having then been blinded before the performance of this work.
For analysis, Real-Time PCR was run in triplicates, to allow for a more robust HRM analysis. The software had the ability to classify samples as different variants, wild-type or mutant; information which was then crossed with the previous classification of the sample to assess the success of the software classification. Samples were correctly assigned.
This approach provides results in a quick fashion that guides clinical action in a timely fashion. The presence of other polymorphisms in the amplicons might be a hindrance to the robustness of the results provided by this technique and their effect on variant classification needs to be considered. For this, a predictive in-silico analysis was performed, regarding all described variants’ presence in the sequences in analysis. Accordingly, an additional complementary method may be necessary for assurance of result’s specificity.
For MNSHL, the screening method was also real-time PCR based, but this one was performed with Amplification-Refractory Mutation System (ARMS) primers, designed for the pathogenic sequence variants previously associated in literature for the MNSHL. Discrimination of results was done based on amplification in positive cases and lack of it in negative cases. This approach analyzed 32 samples, including MNSHL suspected patients, their relatives, other mitochondrial disease patients and healthy controls, but only results concerning the m.1555A>G were obtained timely. All samples were previously classified by another method, having then been blinded before performance of this work.
For optimization, Real-Time PCR was run in duplicates, to increase robustness of analysis. The Real-Time software showed if samples amplified as wild-type or mutant, with classification following. This data was crossed with previous known classification of the samples to assess the success of the approach. All analyzed samples were correctly identified with this approach. However, two of the three pathogenic sequence variants did not achieve implementation within the timeframe necessary for their inclusion, namely m.1494C>T and m.7445A>G. The optimization of their screening was not possible and further work is necessary to optimize and implement the approach concerning the analysis for these variants.
In conclusion, it was possible to implement an analysis method for LHON’s TOP-3 pathogenic sequence variants within 24h, which represents a big step in precision medicine for diagnosis of this disease.
On the other hand, although the implementation was not concluded, a similar approach was started for MNSHL – that, when concluded, will have an enormous impact in preventing aminoglycoside induced HL.
This work represents a high impact scientific contribution in reverse translational research.As citopatias mitocondriais são um conjunto de doenças causadas por um distúrbio na produção de energia celular. A disfunção mitocondrial prejudica a eficiência da cadeia respiratória mitocondrial (CRM) e a produção de ATP, afetando o equilíbrio energético do organismo. As variações de sequência patogénicas no DNA mitocondrial (mtDNA) que levam a estas patologias são mais frequentes em tecidos que necessitam de maiores níveis de energia para funcionar.
O presente trabalho explora duas dessas doenças: Neuropatia ótica hereditária de Leber (LHON) e Surdez mitocondrial induzida por aminoglicosídeos.
A LHON é caracterizada pela presença de alterações genéticas do mtDNA, existindo três variações de sequência patogénicas primárias principais, que representam 90-95% de casos de LHON com identificação da causa genética: m.3460G>A, no gene que codifica a subunidade ND1; m.11778G>A, no gene que codifica a subunidade ND4; e m.14484T>C, no gene que codifica a subunidade ND6. Todas estas subunidades pertencem ao complexo I da CRM. Estas alterações no mtDNA levam a disfunção mitocondrial no complexo I, criando depleção de ATP, aumento de espécies reativas de oxigénio (ROS) e stresse oxidativo.
A LHON é comummente caracterizada pela perda sequencial de visão e, 1 ano após o início dos sintomas, 97% dos casos com perda de visão num olho desenvolvem perda de visão no segundo. A administração de terapia produz bons resultados, quando realizada num curto período de tempo após a primeira perda de visão. Assim, é essencial pesquisar variações de sequência patogénicas genéticas de forma rápida e fiável, para atuar rapidamente e recuperar a função visual.
A Surdez mitocondrial não-sindrómica (MNSHL), em particular a induzida por aminoglicosídeos, tem também três mutações principais associadas à perda de audição: m.1494C>T e m.1555A>G, ambas no gene MTRNR1, e m.7445A>G, nos genes MTCO1 e MTTS1. Estas são responsáveis pela depleção de ATP, aumento de ROS e stresse oxidativo, devido a alterações no ribossoma ou no tRNA mitocondrial.
Aqui, o tecido afetado é a cóclea. Nesta doença, o fator modificador em destaque é a administração de antibióticos de tipo aminoglicosídeos, que despoletam uma cascata de acontecimentos, levando à surdez permanente. A melhor estratégia passa pela prevenção, enquanto ao mesmo tempo se garante que a ação clínica não sofre atrasos. Desta forma, são necessários resultados rápidos, que demonstrem se a administração será segura ou não.
O objetivo deste trabalho, para ambas as doenças, é o desenvolvimento de um método de screening, caracterizado por uma abordagem rápida e fiável, usado para guiar a decisão clínica, particularmente na terapêutica.
Para a LHON, o método de screening é baseado em PCR em tempo-real com análise de High-Resolution Melting (HRM), para deteção das variantes
patogénicas TOP-3, avaliando as Tm dos amplicons. Neste caso, foram analisadas 94 amostras, incluindo doentes com suspeita de LHON, familiares, outros doentes com suspeita de outra doença mitocondrial e controlos saudáveis. Todas as amostras foram previamente classificadas por outro método, tendo sido sujeitas a anonimização antes da realização do trabalho.
Para a análise, a PCR em tempo-real foi realizada em triplicados, para permitir uma análise de HRM mais robusta. O software teve a capacidade de classificar amostras como diferentes variantes, ou seja, normal ou mutante. Esta informação foi cruzada com as classificações previamente existentes para avaliar o sucesso da classificação pelo software. As amostras foram corretamente classificadas.
Esta abordagem fornece resultados de forma rápida, podendo guiar a ação clínica em tempo útil. A presença de outros polimorfismos nos amplicons poderão obstruir a robustez dos resultados fornecidos por esta técnica e o seu efeito na classificação de variantes precisa de ser considerado. Por esta razão, foi realizada uma análise de previsão in-silico, considerando a presença de todas as variantes descritas. Nesse sentido, pode ser necessário um método complementar de análise para assegurar a especificidade dos resultados.
Para a Surdez mitocondrial não-sindrómica, o método de screening baseou-se também na PCR em tempo-real, mas foi realizada com primers de Amplification-Refractory mutation system (ARMS), desenhados para as variantes de sequência patogénicas associadas à MNSHL induzida por aminoglicosídeos, previamente descritas na literatura para esta doença. A discriminação de resultados foi feita com base na presença/ausência de amplificação para cada variante. Foram analisadas 32 amostras com esta abordagem, incluindo doentes com suspeita de MNSHL, seus familiares, doentes com suspeita de outra doença mitocondrial e controlos saudáveis, mas apenas foram obtidos resultados em tempo útil para a m.1555A>G. Todas as amostras tinham sido previamente classificadas por outro método, tendo sido anonimizadas antes da realização do trabalho.
Para a otimização, a PCR em tempo-real foi realizada em duplicados, aumentando a robustez da análise. O software de tempo-real mostrou quais as amostras que amplificaram como normais ou mutantes, permitindo a classificação das mesmas. Os dados foram comparados com as classificações previamente conhecidas, para avaliar o sucesso da abordagem em estudo. Todas as amostras em análise foram corretamente identificadas. No entanto, duas das três variantes patogénicas não foram implementadas em tempo útil para inclusão neste trabalho. Para a m.1494C>T e a m.7445A>G, a otimização não foi possível, e será necessário trabalho adicional no futuro, para a implementação da análise destas variantes.
Em conclusão, foi possível implementar um método da análise das variantes genéticas TOP-3 da LHON em 24h, o que representa um grande passo na medicina de precisão para diagnóstico desta doença.
Por outro lado, apesar de não ter sido concluída a implementação, iniciou-se uma abordagem semelhante para a MNSHL – que, quando for concluída, terá um enorme impacto para evitar a perda auditiva por exposição a aminoglicosídeos.
Este trabalho representa uma contribuição científica de alto impacto na investigação translacional reversa.O Laboratório de Biomedicina Mitocondrial e Teranóstica recebeu apoio financeiro da Santhera Pharmaceuticals que permitiu implementação do projeto nacional “Investigação Translacional Epidemiológica, Bigenómica e Funcional nas Atrofias Ópticas” (IP Professora Doutora Manuela Grazina).
Apoio financeiro do CNC.IBILI no âmbito do Plano Estratégico UID/NEU/04539/2019.Mestrado em Biologia Aplicad
Investigation of a Histidine-Based Probe for the Exploration of Proteomes
Leishmaniasis is a neglected tropical disease which affects 0.7-1 million people per year. Current chemotherapies for leishmaniasis are toxic with long treatment times and reports of increasing resistance, which stresses the importance of this research area. Inositol phosphorylceramide synthase is a membrane bound enzyme that has no direct human homologue, which converts ceramide to inositol phosphorylceramide through the action of a highly conserved HHD catalytic triad. An ideal method to study this enzyme further would be through activity-based protein profiling, however, there are currently no activity-based probes reported that reacts with this type of active site. Therefore, an activity-based probe was designed based on the structure of diethyl pyrocarbonate, a compound known to bind covalently to active site histidine residues. The synthesised activity-based probe was shown to inhibit Leishmania major inositol phosphorylceramide synthase in a simple assay. In addition, the probe was shown to selectively bind to the active site histidine residue in two pure enzyme models; one of which has the same catalytic triad as inositol phosphorylceramide synthase, and the other was an acid base active site histidine residue. Further, this activity-based probe was able to isolate an overexpressed enzyme in the lysate of Escherichia coli as well as bind to intrinsic proteins. Following the function validation of the activity-based probe, preliminary work was started in Leishmania to isolate proteins identify expressed enzymes
Investigating the mechanism of human beta defensin-2-mediated protection of skin barrier in vitro
The human skin barrier is a biological imperative. Chronic inflammatory skin diseases, such as Atopic Dermatitis (AD), are characterised by a reduction in skin barrier function and an increased number of secondary infections. Staphyloccocus aureus (S. aureus) has an increased presence on AD lesional skin and contributes significantly to AD pathology. It was previously demonstrated that the damage induced by a virulence factor of S. aureus, V8 protease, which causes further breakdown in skin barrier function, can be reduced by induction of human β- defensin (HBD)2 (by IL-1β) or exogenous HBD2 application. Induction of this defensin is impaired in AD skin. This thesis examines the mechanism of HBD2-mediated barrier protection in vitro; demonstrating that in this system, HBD2 was not providing protection through direct protease inhibition, nor was it altering keratinocyte proliferation or migration, or exhibiting specific localisation within the monolayer. Proteomics data demonstrated that HBD2 did not induce expression of known antiproteases but suggested that HBD2 stimulation may function by modulating expression of extracellular matrix proteins, specifically collagen- IVα2 and Laminin-β-1. Alternative pathways of protection initiated by IL-1β and TNFα stimulation were also investigated, as well as their influence over generalised wound healing. Finally, novel 3D human skin epidermal models were used to better recapitulate the structure of human epidermis and examine alterations to skin barrier function in a more physiological system. These data validate the barrier-protective properties of HBD2 and extended our knowledge of the consequences of exposure to this peptide in this context
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