Functional characterization of mitochondrial suspensions by cytofluorimetric techniques towards the screening of mitochondrial target drugs

Dissertação de mestrado em Bioquímica AplicadaBesides its role in respiration, mitochondria control other key metabolic pathways, cell proliferation and regulated cell death, with a crucial role in the regulation of the intrinsic apoptotic pathway. Recent studies have focused on the screen for anti-cancer agents that induce apoptosis through permeabilization of the mitochondrial outer membrane, leading to the cytosolic release of pro-apoptotic proteins and to the activation of the caspase cascade and consequently to cell dismantling. In the present study, staining protocols were developed and optimized aiming at the structural and functional characterization of isolated mitochondrial populations from yeast cells and rat liver by flow cytometry. Mitotracker Green and Nonyl Acridine Orange were used to measure changes in mitochondrial mass, 3,3'- Dihexyloxacarbocyanine Iodide and Mitotracker Red CMXROS to monitor the electrical potential of the inner membrane (Δψm), Mitotracker Red CM-H2XROS and Dihydroethidium to evaluate the accumulation of ROS, and 2',7'-Bis-(2-Carboxyethyl)- 5-(and-6)-Carboxyfluorescein and Acetoxymethyl Ester (BCECF-AM) to measure the pH of the mitochondrial matrix. Subsequent flow cytometry studies were designed to evaluate the effect in mitochondria function of two well-known apoptosis inducers in both mammalian cell lines and yeast cells, namely bovine lactoferrin (bLf) and acetate, and to understand whether or not they act directly on mitochondria independently of upstream cellular pathways. The results showed that both bLf and acetate promoted changes in mitochondrial mass, matrix mitochondrial pH, Δψm and ROS levels of rat liver mitochondria. Moreover, the assessment of the activity of the complexes of electron transport chain (ETC) showed that bLf and acetate affect their activity and trigger mitochondrial swelling. These results are particularly interesting since the two compounds appear to induce in isolated mitochondria mainly the same alterations as those occurring in whole cells, and which have been proposed to be involved in the induction of cell death. Altogether, this study show that flow cytometry is a valuable tool to study key mitochondrial functional parameters and to screen for mitochondriatargeted drugs, including the anticancer agents, such as acetate and bLf.Para além do seu papel na respiração celular, as mitocôndrias controlam outros aspetos chaves do metabolismo, bem como a proliferação celular e a morte celular regulada, tendo um papel crucial na regulação da via apoptótica intrínseca. Estudos recentes têm-se focado na procura de agentes anticancerígenos capazes de induzir apoptose mediada pela permeabilização da membrana mitocondrial externa, conduzindo à libertação de proteínas pró-apoptóticas e à ativação da cascata de caspases e consequente desmantelamento celular. No presente estudo, foram desenvolvidos e otimizados protocolos de marcação com vista à caracterização estrutural e funcional por citometria de fluxo de populações de mitocôndrias isoladas de células de levedura e de hepatócitos de rato. As sondas Mitotracker Green e laranja de nonil-acridina foram usadas para medir variações da massa mitocondrial, o iodeto de 3,3'-Dihexiloxacarbocianina e o Mitotracker Red CMXROS para medir o potencial eléctrico da membrana mitocondrial interna (Δψm), o Mitotracker Red CM-H2XROS e dihidroetídeo para avaliar a acumulação de ROS e o acetoximetil éster de (2',7'-Bis-(2- Carboxietil)-5-(and-6)-carboxifluoresceína e(BCECF-AM) para medir o pH da matriz mitocondrial. Os estudos subsequentes de citometria de fluxo foram desenhados para avaliar o efeito de diferentes drogas na função mitocondrial, em particular da lactoferrina bovina (bLf) e do acetato, dois reconhecidos indutores de apoptose, no sentido de se compreender se atuam diretamente na mitocôndria independentemente de outras vias celulares a montante. Os resultados mostraram que tanto a bLf como o acetato promovem alterações na massa mitocondrial, pH da matriz mitocondrial, Δψm e nos níveis de ROS em mitocôndrias isoladas de fígado de rato. Além disso, a avaliação da atividade dos complexos da cadeia de transportadora de eletrões (ETC) mostrou que a bLf e o acetato afetam a sua atividade e desencadeiam a tumefação mitocondrial. Estes resultados são particularmente interessantes uma vez que os dois compostos parecem induzir, em mitocôndrias isoladas as mesmas alterações que ocorrem em células inteiras, e que têm sido propostas como estando envolvidas na indução da morte celular. No seu conjunto, este estudo mostra que a citometria de fluxo é uma ferramenta valiosa para o estudo da funcionalidade mitocondrial e para o rastreio de drogas que têm como alvo a mitocôndria, incluindo agentes anticancerígenos, tais como a bLf e o acetato

Cultivation systems of Coelastrella sp. isolated from lichens collected in the north of Portugal

There is a growing interest towards finding new bioactive compounds from natural, safe and sustainable sources and extremophilic microalgae have emerged as one of the most promising options. Lichens are constituted by microalgae/cyanobacteria cells associated with a fungus that are subjected to especially extreme conditions, resulting in the production of a great variety of specific biologically active metabolites. However, lichens have not been considered so far as a potential commercial source of bioactive compounds mostly due to their extremely low growth rates. In this work microalgae of the genus Coelastrella sp were isolated from lichens collected in the northern region of Portugal and the optimal conditions for biomass productivity were identified: temperature of 18 ºC, pH 7 and light intensity of 250 m. Three cultivation systems were evaluated, namely the bubble column (BC), flat panel (FP) and split cylinder airlift (SCA) photobioreactors, in order to evaluate and compare their influence on the growth and productivity of microalgae. The BC photobioreactor showed the lowest biomass productivity (Pmax) and specific growth rate (max), and the biomass concentration reached by FP and SCA was, respectively, 14.4 and 35.4% higher than that of BC. The SCA presented the highest Pmax of 0.113 ± 0.019 g.L-1.d-1 and max of 0.187 ± 0.00913 d-1. These values are a very significant advance in otherwise very slow-growing cultures, and point at the possibility of using these strains in larger scale cultivation systems in the near future.info:eu-repo/semantics/publishedVersio

Improving extraction of biomolecules from Coelastrella sp biomass using ohmic heating

Coelastrella sp is recognized by its double-layered cell wall and ability to produce high levels of pigments and interesting amounts of proteins and lipids. These molecules have potential to be applied in functional food and feed arena, but also in other biotechnological fields (e.g. nutraceuticals and cosmetics). However, most of the metabolites resulting from microalgaes metabolism are intracellular, which limits their accessibility during physical or biological processes. Currently, there are several traditional downstream processing methodologies aiming cells disruption to extract compounds of interest to the extracellular environment, envisaging production of bioactive-rich extracts. An emerging and sustainable extraction approach consists in the utilization of Ohmic Heating (OH). OH brings an unique synergy between thermal and electric field effects as a way to permeabilize microalgae cell wall and thus increase aqueous extraction yield. In this work Coelastrella sp was pre-treated using OH by applying electro-thermal protocols of different electrical field intensities (from 12 to 217 V/cm), treatment temperatures (50 to 100 ºC) and total treatment times (from 7 seconds to 10 minutes). OH and its moderate electric fields (MEF) contributed to an increased aqueous extraction of macronutrients with bioactive properties, allowing a recovery of 50 % and 20 % of the total chlorophylls and proteins, respectively, that were initially enclosed in the cells. Cytometry analysis confirmed disruption effects induced by OH treatments, while fluorescence measurements confirmed the presence of chlorophyl a and aromatic amino acids (such as tryptophan) in the obtained extracts. OH can be used to weaken microalgae cell wall structure facilitating subsequent aqueous and solvent extraction. These results also bring new insights regarding bioaccessibility of important biocompounds during gastrointestinal digestion of microalgae biomass.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit; Project Algavalor (POCI-01-0247-FEDER-035234; LISBOA-01-0247-FEDER-035234; ALG-01-0247-FEDER-035234) funded by FEDER funds through COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI); AgriFood XXI R & D & I project, operation number NORTE-01-0145-FEDER-000041, co-financed by the European Regional Development Fund (FEDER) through NORTE 2020 (Northern Regional Operational Program 2014/2020); Vitor Sousa acknowledges the Foundation for Science and Technology (FCT) for their fellowship UI/BD/151238/2021; Ricardo N. Pereira acknowledge FCT for its Assistant Research contract obtained under CEEC Individual 2017info:eu-repo/semantics/publishedVersio

Nanostructures as conveyors of functionality in foods: the case of cannabidiol-based nanostructured lipid carriers

Nanostructures incorporating functional compounds have earned their place as a very efficient means of conveying functionality in foods. They may be used to tackle malnutrition, reduce calorie density, reduce food digestibility, increase micronutrient bioavailability, control gut health, allow personalized nutrition and provide appropriate food for the elderly, among other potential uses. Building such nanostructures, particularly when considering that they need to be edible, is a challenging task. This keynote will address the latest developments made by our research group towards tackling some of these challenges, together with our vision on what still needs to be done and which partnerships are important to lead us to further improve their performance. The incorporation of cannabidiol (CBD) into nanostructured lipid carriers (NLC) will be given as an example of a potential solution to mitigate its low bioavailability, which represents a big challenge for the development of CBD-products. Some details regarding their development (e.g., using innovative emulsifiers) will be provided and the consequences in the stability of CBD will be analysed.info:eu-repo/semantics/publishedVersio

Development and characterization of nanostructured lipid carriers for cannabidiol delivery

Supplementary data to this article can be found online at https://doi.org/10.1016/j.foodchem.2023.138295.This study evaluated the physicochemical characteristics of nanostructured lipid carriers (NLCs) as a potential vehicle for cannabidiol (CBD), a lipophilic molecule with great potential to promote health benefits. NLCs were produced using hemp seed oil and fully-hydrogenated soybean oil at different proportions. The emulsifiers evaluated were soybean lecithin (SL), Tween 80 (T80) and a mixture of SL:T80 (50:50). CBD was tested in the form of CBD-rich extract or isolate CBD, to verify if it affects the NLCs characteristics. Based on particle size and polydispersity, SL was considered the most suitable emulsifier to produce the NLCs. All lipid proportions evaluated had no remarkable effect on the physicochemical characteristics of NLCs, resulting in CBD-loaded NLCs with particle size below 250 nm, high CBD entrapment efficiency and CBD retention rate of 100% for 30 days, demonstrating that NLCs are a suitable vehicle for both CBD-rich extract or isolate CBD.This study received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie SkłodowskaCurie grant agreement No 101062938 and was supported by Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit, and by LABBELS – Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems, LA/P/0029/2020. Fernanda L. Lüdtke thanks Iceland Liechtenstein Norway Grants (PT-INNOVATION-0105) for the fellowship

Assessment of cell disruption efficiency of Microcystis aeruginosa using different mechanical techniques

This research work was supported by the grant SFRH/BPD/98694/2013 (Bruno Fernandes) and SFRH/BD/52335/2013 (Pedro Geada) from Fundação para a Ciência e a Tecnologia (Portugal). Luís Loureiro is recipient of a fellowship supported by a doctoral advanced training (call NORTE-69-2015-15) funded by the European Social Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684), Project UID/Multi/04423/2013, Project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462), FCT Strategic Project of UID/BIO/04469/2013 unit, by the project NOVELMAR (reference NORTE-01-0145-FEDER-000035), co-financed by the North Portugal Regional Operational Programme (Norte 2020) under the National Strategic Reference Framework (NSRF), through the ERDF, and by BioTecNorte operation (NORTE-01-0145-FEDER000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

TÉCNICA DE CRISS-CROSS NO TRATAMENTO DE TROMBOSE VENOSA PROFUNDA DO MEMBRO INFERIOR — A PROPÓSITO DE UM CASO CLÍNICO

Introdução: A técnica criss-cross combina acesso vascular anterógrado e retrógrado da veia poplítea, de modo a obter recanalização venosa em doentes com trombose venosa profunda (TVP) ileofemoral associada a trombose das veias poplítea e gemelares.  Caso clínico: Doente do sexo feminino de 57 anos, com antecedentes de histerectomia radical e linfadenectomia de nódulos linfáticos pélvicos em 2013, devido a neoplasia uterina, com consequente linfedema crónico do membro inferior direito. Admitida na urgência por edema grave do membro inferior direito, com 7 dias de evolução. À observação, a paciente apresentava edema da coxa e perna, cianose da perna, dor gemelar, extremidades quentes e pulsos distais palpáveis. Estudo com ecodoppler a objetivar trombose dos eixos venosos do membro inferior direito. Estudo com TC contrastado excluiu extensão proximal do trombo para a veia ilíaca comum direita ou veia cava; excluiu tromboembolismo pulmonar e evidenciou clips cirúrgicos em proximidade com a veia ilíaca externa direita, causando uma respetiva redução luminal superior a 50%. A doente foi submetida a trombectomia e fibrinólise dirigida por cateter. Obteve-se acesso vascular através de punção ecoguiada da veia poplítea. Após colocação de introdutores em sentido retrógrado e anterógrado, realizou-se trombectomia das veias gemelares até à veia femoral comum, com recurso a aspiração por cateter. Instituiu-se perfusão de alteplase e heparina não fracionada por via periférica, mantidas durante 72 horas, com controlo angiográfico a cada 24h. Após recanalização satisfatória, realizou-se angioplastia com balão 14x40mm, seguida de stent 14x80mm na veia ilíaca externa. Não ocorreram complicações hemorrágicas major. O tratamento invasivo foi complementado com drenagem postural e meia elástica compressiva, permitindo melhoria clínica imediata e progressiva. Após dois dias, a doente recebeu alta medicada com heparina de baixo peso molecular. Em consulta de follow-up após um mês, constatou-se melhoria clínica progressiva e alterada hipocoagulação para rivaroxabano ad aeternum.  Conclusão: A técnica de criss-cross permite recanalização do inflow e outflow, restaurando patência venosa e mantendo a função valvular, aspetos com eventual papel crítico nos resultados após TVP ileofemoral extensa.

MAMMALS IN PORTUGAL : A data set of terrestrial, volant, and marine mammal occurrences in P ortugal

Mammals are threatened worldwide, with 26% of all species being includedin the IUCN threatened categories. This overall pattern is primarily associatedwith habitat loss or degradation, and human persecution for terrestrial mam-mals, and pollution, open net fishing, climate change, and prey depletion formarine mammals. Mammals play a key role in maintaining ecosystems func-tionality and resilience, and therefore information on their distribution is cru-cial to delineate and support conservation actions. MAMMALS INPORTUGAL is a publicly available data set compiling unpublishedgeoreferenced occurrence records of 92 terrestrial, volant, and marine mam-mals in mainland Portugal and archipelagos of the Azores and Madeira thatincludes 105,026 data entries between 1873 and 2021 (72% of the data occur-ring in 2000 and 2021). The methods used to collect the data were: live obser-vations/captures (43%), sign surveys (35%), camera trapping (16%),bioacoustics surveys (4%) and radiotracking, and inquiries that represent lessthan 1% of the records. The data set includes 13 types of records: (1) burrowsjsoil moundsjtunnel, (2) capture, (3) colony, (4) dead animaljhairjskullsjjaws, (5) genetic confirmation, (6) inquiries, (7) observation of live animal (8),observation in shelters, (9) photo trappingjvideo, (10) predators dietjpelletsjpine cones/nuts, (11) scatjtrackjditch, (12) telemetry and (13) vocalizationjecholocation. The spatial uncertainty of most records ranges between 0 and100 m (76%). Rodentia (n=31,573) has the highest number of records followedby Chiroptera (n=18,857), Carnivora (n=18,594), Lagomorpha (n=17,496),Cetartiodactyla (n=11,568) and Eulipotyphla (n=7008). The data setincludes records of species classified by the IUCN as threatened(e.g.,Oryctolagus cuniculus[n=12,159],Monachus monachus[n=1,512],andLynx pardinus[n=197]). We believe that this data set may stimulate thepublication of other European countries data sets that would certainly contrib-ute to ecology and conservation-related research, and therefore assisting onthe development of more accurate and tailored conservation managementstrategies for each species. There are no copyright restrictions; please cite thisdata paper when the data are used in publications.info:eu-repo/semantics/publishedVersio

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear un derstanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5–7 vast areas of the tropics remain understudied.8–11 In the American tropics, Amazonia stands out as the world’s most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepre sented in biodiversity databases.13–15 To worsen this situation, human-induced modifications16,17 may elim inate pieces of the Amazon’s biodiversity puzzle before we can use them to understand how ecological com munities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple or ganism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region’s vulnerability to environmental change. 15%–18% of the most ne glected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lostinfo:eu-repo/semantics/publishedVersio