CHAPTER 8
APPENDICES
English Summary
Nederlandse Samenvatting
Sumário em Português
Acknowledgements/Agradecimentos
Curriculum vitae
List of publications
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English Summary
Multiple sclerosis
Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disease of the central nervous system characterized by damage to the myelin, which is the isolating layer of neurons. It affects mainly young adults and is three times more predominant in women than in men. MS can be divided in four sub-types: relapsing-remitting MS (RRMS) which is characterized by attacks which can last from a few days to weeks, followed by a period of partial or full recovery from the symptoms. Secondary-progressive MS (SPMS) is characterized by initial attacks followed by a slow deterioration of brain function. Primary-progressive MS (PPMS) is characterized by the lack of attacks, with increased brain deterioration from the start of the disease. Finally, primary-relapsing MS (PRMS) is characterized by a continuous deterioration of brain function from the start of the disease, but in later stages patients suffer attacks, hence these two last forms cannot be distinguished in early stages of the disease. MS is usually more common in areas around the northern hemisphere and less around the equator which suggests a strong influence of the environment. However, the exact cause of the disease is currently unknown. Studies performed using brain tissue from MS patients have shown the presence of cells from the immune system in the brain. It is believed that these immune cells attack the myelin sheet, consequently reducing the transmission of signals through neurons. This immune cell entry initiates inflammation in the brain, also known as MS lesions, leading to cell and tissue damage. Therefore, this autoimmune response against myelin accounts for the neurological symptoms associated with the disease. Symptoms of MS can include visual disturbance, muscle weakness, difficulties in coordination and balance, numbness or tingling, memory problems, or changes in bowel and bladder function. Other symptoms include cognitive changes, fatigue and mood alterations.
Blood-brain barrier function in health and disease
The study of the brain vasculature emerged in the 19th century with the studies of the
German scientist Paul Ehrlich. He observed that after injection of dyes into the body of animals, all organs of the body became stained, except the brain. However, Edwin Goldmann, one of Ehrlich students, observed that when he injected the dye directly into the brain, only the brain was stained but the remaining organs of the animal were not. Goldmann could, therefore, illustrate the separation between the brain and the rest of
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the body. This separation is possible due to the presence of specific blood vessels in the brain. These blood vessels from the brain maintain a barrier between the brain and the immune system. Therefore, they are known as the blood-brain barrier, acting as a gate-keeper of the brain. The blood-brain barrier is composed by specialized cells that communicate with each other via specific proteins. This close interaction between cells provides a tight barrier, avoiding the entrance of molecules and immune cells from the blood to the brain. Therefore, the BBB guarantees proper neuronal and brain function. In MS, inflammatory processes lead to an altered barrier function of the blood-brain barrier. This altered function of the brain vessels has a negative effect in MS patients. Therefore, the aim of this thesis was to understand how inflammation alters the function of the blood-brain barrier and how this affects the entrance of immune cells from the blood to the brain.
The function of blood vessels is determined by specific mechanisms that take place during the development of the central nervous system. One of these mechanisms is the Notch signaling. This signaling pathway occurs between neighboring cells and it starts when Notch receptors contact Notch ligands. This pathway has been shown essential for proper development of the blood vessels. However, the role of Notch signaling in blood-brain barrier function, in heath and disease has not been studied. We show in chapter 2 that Notch signaling is affected by inflammation of the blood-brain barrier. This phenomenon has a negative effect in the barrier function of the blood vessels of the brain. This study highlights the importance of understanding how blood-brain barrier function is regulated in health and disease.
Infiltration of immune cells into the brain
In healthy conditions, the blood vessels in the brain form a barrier and no immune cells can access the brain. However, during diseases such as multiple sclerosis, the cells of the immune system enter the brain because the blood-brain barrier does not function properly. This dysfunction of the blood vessels is mainly due to the inflammation caused by these immune cells that entered the brain, leading to tissue damage.
Importantly, for successful entry into the brain, the cells from the immune system contact the blood-brain barrier via specific proteins that are present in the surface of the blood vessels. These proteins provide the first point of contact for the immune cells, allowing them to attach to and subsequently cross the blood vessels making their way into the brain. Therefore, understanding how this important process occurs in MS is of extreme importance for the development of new therapeutics that reduce the entry of immune cells into the brain of MS patients.
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We show in chapter 3 that Notch ligands also play a role during the entry of immune cells into the brain. In healthy conditions, the blood-brain barrier shows a low amount of these ligands in its surface. However, in inflammatory conditions, like seen in MS, the blood vessels increase these surface proteins, helping the immune cells entry into the brain.
In MS, it is still not known why immune cells enter the brain. Some researchers believe that in MS, only the immune cells that recognize brain proteins, like myelin, can enter the brain. However, what regulates the entry of these specific immune cells to the inflamed areas of the brain is still unclear. Therefore, we wanted to investigate if the blood-brain barrier can show these specific proteins to the cells of the immune system. In chapter 4 we show that the blood vessels of the brain can present brain-specific proteins to immune cells. When these immune cells recognize these proteins, they enter the brain more easily than immune cells that do not recognize these brain proteins. This study highlights the important immune regulatory function of the brain vessels in disease initiation.
In MS, not only proteins are involved in the progression of the disease but also lipids. It has been shown that the balance of lipids is also deregulated in MS due to the inflammation in the brain. In chapter 5, we show that some lipids play an important role in the gate-keeping function of the blood-brain barrier. We show that some enzymes, that create lipids in the blood vessels, are produced in high quantities by inflamed blood vessels. As a result, the cells from the immune system enter the brain more easily, since the barrier function of the blood vessels is lost. Interestingly, in inflammatory conditions the blood-brain barrier can secrete these enzymes. In chapter 6 we investigated the potential use of these enzymes as a biomarker for MS. Our preliminary results suggest that the levels of these enzymes are increased in the serum of MS patients with blood-brain barrier dysfunction and decreased in patients that were taking medicines against MS. The development of better biomarkers in MS is essential for better diagnosis, prognosis and treatment options. However, due to the different types of symptoms and different disease progression between patients, biomarker discovery in MS still represents a great challenge.
Future perspectives and concluding remarks
In this thesis, we provide new evidence on how inflammation alters the function of the blood-brain barrier and its consequences for the entry of immune cells into the brain in MS. Our results further demonstrate that understanding the barrier function of brain vessels is still evolving. We demonstrate that different proteins and mechanisms are
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important and possibly interconnected in regulating immune cell entry across the brain vessels. This might shed new light in understanding MS. Although current therapies in MS mainly target the entry of immune cells into the brain, patients often suffer from side-effects associated with decreased function of the immune system. Therefore, understanding the complex mechanisms that regulate the entry of immune cells into the brain in MS might lead to the discovery and development of better therapies to reduce barrier dysfunction and the negative immune cell entry to the brain.
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Nederlandse Samenvatting
Multiple sclerose
Multiple sclerose (MS) is een chronische ontstekingsziekte van het centrale zenuwstelsel dat gekenmerkt wordt door beschadigingen van het myeline, de isolatielaag om de zenuwen. Hierdoor wordt de prikkelgeleiding door de zenuwen verminderd en ontstaan er verlammings- en uitvalsverschijnselen. De ziekte treft vooral jong volwassenen en komt drie keer vaker voor bij vrouwen dan bij mannen. MS kan opgedeeld worden in vier subtypen: relapsing-remitting MS (RRMS) dat wordt gekenmerkt door aanvallen die kunnen variëren van een paar dagen tot weken, gevolgd door een periode van gedeeltelijk of volledig herstel van de symptomen. Secundair-progressieve MS (SPMS) wordt gekenmerkt door initiële aanvallen, gevolgd door langzame achteruitgang van de hersenfunctie. Primair progressieve MS (PPMS) wordt gekenmerkt door het ontbreken van aanvallen, maar wel met verhoogde achteruitgang vanaf het begin van de ziekte. Tenslotte, primaire relapsing MS (PRMS) wordt gekenmerkt door een continue achteruitgang van de hersenfunctie vanaf het begin van de ziekte, maar in later stadia lijdt de patiënt aan aanvallen, waardoor deze laatste twee vormen van MS niet te onderscheiden zijn van elkaar in de vroege stadia van de ziekte. MS komt vaker voor in gebieden rond het noordelijk halfrond en minder vaak voor rond de evenaar, wat aangeeft dat de omgeving een invloedrijke rol heeft. De exacte oorzaak van de ziekte is onbekend. Studies die zijn uitgevoerd met hersenweefsel van MS patienten tonen de aanwezigheid van cellen van het immuunsysteem in de hersenen aan. Er wordt aangenomen dat deze immuuncellen de myelineschede, wat leidt tot het onstaan van onstekingshaarden, oftewel MS laesies, en uiteindelijk tot cel- en weefselschade. Deze auto-immuunreactie tegen myeline verklaart de neurologische symptomen geassocieerd met de ziekte. De symptomen van MS zijn; visuele stoornissen, spierzwakte, problemen met coördinatie en evenwicht, gevoelloosheid of tintelingen, geheugenproblemen, of veranderingen in de darmen en blaasfunctie. Andere symptomen zijn onder meer cognitieve veranderingen, vermoeidheid en stemmingswisselingen.
Bloed-hersenbarrière functie in gezondheid en ziekte
De studie van de hersenvasculatuur is ontstaan in de 19e eeuw met de studies van de Duitse wetenschapper Paul Ehrlich. Hij merkte op dat na injectie van kleurstoffen in het lichaam van dieren, alle organen van het lichaam werden gekleurd, behalve de hersenen. Echter, Edwin Goldmann, één van Ehrlichs studenten, ontdekte dat wanneer hij de
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kleurstof direct injecteerde in de hersenen, alleen de hersenen bevlekt werden, maar de overige organen van het dier niet. Goldmann illustreerde hiermee de scheiding tussen de hersenen en de rest van het lichaam. Deze scheiding is mogelijk dankzij de aanwezigheid van specifieke bloedvaten in de hersenen. Deze bloedvaten van de hersenen, handhaven een barrière tussen de hersenen en het immuunsysteem. Daarom zijn ze bekend als de hersenbarrière, die functioneert als een poortwachter van de hersenen. De bloed-hersenbarrière bestaat uit gespecialiseerde cellen die met elkaar communiceren via specifieke eiwitten. Deze nauwe interactie tussen cellen zorgt voor een barrière, en voorkomt de toegang van moleculen en immuuncellen uit het bloed naar de hersenen. Hiermee garandeert de bloed-hersenbarrière optimale neuronale- en hersenfunctie. Bij MS leiden ontstekingsprocessen tot een verandering in barrièrefunctie van de bloed-hersenbarrière, wat leidt tot inflitratie van immuuncellen en het onstaan van laesies bij MS-patiënten. Het doel van dit proefschrift was dan ook om te begrijpen hoe ontsteking de functie van de bloed-hersenbarrière verandert en hoe dit de toegang van immuuncellen uit het bloed naar de hersenen beïnvloedt.
De functie van bloedvaten wordt bepaald door specifieke mechanismen die plaatsvinden tijdens de ontwikkeling van het centrale zenuwstelsel. Eén van deze mechanismen is de Notch-signaleringsroute. Deze signaleringsroute treedt op tussen naburige cellen en begint wanneer Notch-receptoren contact maken met Notch-liganden. Dit mechanisme is aangetoond essentieel te zijn voor een goede ontwikkeling van de bloedvaten. Echter, de rol van Notch-signalering in de bloed-hersenbarrièrefunctie in gezondheid en ziekte, is nog niet onderzocht. In hoofdstuk 2 tonen we aan dat Notch-signalering wordt beïnvloed door ontsteking van de bloed-hersenbarrière. Dit verschijnsel heeft een negatief effect op de barrièrefunctie van de bloedvaten van de hersenen. Deze studie benadrukt het belang om te begrijpen hoe de bloed-hersenbarrière functie wordt geregeld in gezondheid en ziekte.
Infiltratie van immuuncellen in de hersenen
In gezonde toestand, vormen de bloedvaten in de hersenen een barrière waardoor immuuncellen niet kunnen binnendringen. Tijdens ziekten zoals multiple sclerose, hebben cellen van het immuunsysteem toegang tot de hersenen, omdat de bloed-hersenbarrière niet goed functioneert. De dysfunctie van de hersenbloedvaten in MS is grotendeels te wijten aan ontsteking van de hersenen. Deze ontsteking wordt voornamelijk veroorzaakt door de immuuncellen die de hersenen zijn binnengedrongen,
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Voor een succesvolle toegang tot de hersenen, maken de cellen van het immuunsysteem contact met de bloed-hersenbarrière via specifieke eiwitten die aanwezig zijn aan het oppervlak van de bloedvaten. Deze eiwitten vormen het eerste aanspreekpunt voor de immuuncellen, waaraan ze zich hechten, alvorens ze door de hersenbloedvaten migreren, waarna ze hun weg vervolgen naar de hersenen. Begrijpen hoe dit belangrijke proces gebeurt in MS, is uitermate belangrijk voor de ontwikkeling van nieuwe therapieën die het binnendringen van immuuncellen in de hersenen van MS patiënten kunnen verminderen.
We tonen in hoofdstuk 3 dat Notch-liganden een rol spelen tijdens het binnendringen van immuuncellen in de hersenen. In gezonde omstandigheden, brengt de bloed-hersenbarrière een lage hoeveelheid van deze liganden tot expressie aan het oppervlak. Echter, in ontstekingen, zoals in MS, worden deze oppervlakte-eiwitten verhoogd tot expressie gebracht in de hersenbloedvaten, wat bijdraagt aan het binnendringen van immuuncellen in de hersenen.
In MS is het niet bekend waarom immuuncellen de hersenen binnendringen. Sommige onderzoekers zijn van mening dat in MS, alleen de immuuncellen die herseneiwitten herkennen, zoals myeline, de hersenen binnen kunnen komen. Maar wat de invoer van deze specifieke immuuncellen naar de ontstoken gebieden van de hersenen regelt, is nog onduidelijk. Daarom wilden we onderzoeken of de bloed-hersenbarrière deze specifieke eiwitten kunnen presenteren aan de cellen van het immuunsysteem. In hoofdstuk 4 laten we zien dat de bloedvaten van de hersenen, hersenspecifieke eiwitten kunnen presenteren aan immuuncellen. Wanneer immuuncellen deze eiwitten herkennen, komen ze gemakkelijker in de hersenen, dan wanneer ze deze hersen-eiwitten niet herkennen. Deze studie benadrukt de belangrijke immuunregulerende functie van de hersenenbloedvaten bij het ontstaan van de ziekte.
Bij MS zijn niet alleen eiwitten betrokken bij de progressie van de ziekte, maar ook lipiden. Het is aangetoond dat in MS, de verhouding van lipiden ook ontregeld is, als gevolg van de ontsteking in de hersenen. In hoofdstuk 5 laten we zien dat sommige lipiden een belangrijke rol spelen in de poortwachterfunctie van de bloed-hersenbarrière. We tonen aan dat sommige enzymen die lipiden in de bloedvaten creëren, in grote hoeveelheden worden geproduceerd door ontstoken bloedvaten. Als gevolg, kunnen de cellen van het immuunsysteem gemakkelijker de hersenen binnenkomen, doordat de barrièrefunctie van de bloedvaten verloren is gegaan. Interessant is, dat zelfs in ontstoken toestand de bloed-hersenbarrière deze enzymen kan produceren. In hoofdstuk 6 onderzochten we de mogelijke toepassing van deze enzymen als biomarker voor MS. Onze bevindingen suggereren dat de gehaltes van deze
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enzymen verhoogd zijn in het serum van MS-patiënten met bloedhersenbarrière dysfunctie, en verminderd zijn bij patiënten die geneesmiddelen namen voor de behandeling van MS. De ontwikkeling van betere biomerkers in MS is essentieel voor betere diagnose, prognose en behandeling. Aangezien symptomen en ziekteprogressie kunnen verschillen tussen MS patiënten, is biomarker-ontdekking in MS nog altijd een grote uitdaging.
Toekomstperspectieven en conclusie
In dit proefschrift leveren we nieuw bewijs voor hoe ontsteking de functie van de bloed-hersenbarrière kan veranderen, en over de gevolgen daarvan voor het binnendringen van immuuncellen in de hersenen bij MS. Daarnaast demonstreren onze resultaten dat het begrip van de barrièrefunctie van de hersenvaten nog in ontwikkeling is. We tonen aan dat verschillende eiwitten en mechanismen belangrijk zijn en eventueel met elkaar zijn verbonden bij de regulatie van infiltratie van immuuncellen via de hersenbloedvaten. Dit kan nieuw licht werpen op het ontstaan en behandelen van MS. Hoewel de huidige therapieën in MS zich voornamelijk richten op het verminderen van het binnendringen van immuuncellen in de hersenen, hebben patiënten vaak last van bijwerkingen die geassocieerd zijn met verminderde functie van het immuunsysteem. Daarom kan een beter begrip van de complexe mechanismen die betrokken zijn bij de infiltratie van immuuncellen in de hersenen in MS, leiden tot de ontdekking en ontwikkeling van betere therapieën om barrière dysfunctie en infiltratie van immuuncellen in de hersenen te verminderen.
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Sumário Português
Esclerose Múltipla
A esclerose múltipla é uma doença inflamatória crónica e degenerativa do sistema nervoso central caracterizada por danos na mielina, a camada isoladora dos neurónios. A esclerose múltipla afecta principalmente jovens adultos e é três vezes mais frequente em mulheres do que em homens. Esta doença pode ser dividida em quatro sub-tipos: Esclerose Múltipla Recidivante Remitente (EMRR) é caracterizada por surtos (episódios agudos de manifestações sintomáticas) que podem durar de alguns dias a semanas, seguidas por um período de recuperação parcial ou completa dos sintomas. Esclerose Múltipla Secundária Progressiva (EMSP) é caracterizada inicialmente por surtos, seguida de uma deterioração lenta da função cerebral. Esclerose Múltipla Primária Progressiva (EMPP) é caracterizada pela ausência de surtos, com o aumento da deterioração da função cerebral desde o início da doença. Finalmente, Esclerose Múltipla Primária Recidivante (EMPR) é caracterizada por uma deterioração contínua da função cerebral desde o início da doença, mas em fases posteriores os pacientes sofrem de surtos, por conseguinte, estas duas últimas formas não podem ser distinguidas nas fases iniciais da doença.
A esclerose múltipla é geralmente mais comum em áreas ao redor do hemisfério norte e menos em torno do equador, fenómeno este que demonstra uma forte influência do meio ambiente. No entanto, a causa exacta da doença é actualmente desconhecida. Estudos realizados com o tecido cerebral de doentes com esclerose múltipla mostraram a presença de células do sistema imunitário no cérebro. Acredita-se que estas células atacam a baínha de mielina, consequentemente, reduzindo a transmissão de sinais através dos neurónios. Esta entrada das células do sistema imunitário inicia a inflamação no cérebro, também conhecido como lesões, conduzindo a danos nas células e tecidos. Portanto, esta resposta auto-imune contra a mielina é responsável pelos sintomas neurológicos associados à doença. Os sintomas da esclerose múltipla podem incluir distúrbios visuais, fraqueza muscular, dificuldades de coordenação e equilíbrio, dormência ou formigueiro, problemas de memória, ou alterações na função do intestino e da bexiga. Outros sintomas incluem alterações cognitivas, fadiga e alterações de humor.
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Funções da barreira hemato-encefálica na saúde e na doença
O estudo da vasculatura cerebral surgiu no século 19, com os estudos do cientista alemão Paul Ehrlich. Ele observou que, após a injecção de corantes na circulação sanguínea de animais, todos os órgãos do corpo ficavam corados, com excepção do cérebro. No entanto, Edwin Goldmann, um dos alunos Ehrlich, observou-se que quando o corante era directamente injectado no cérebro, apenas o cérebro ficava corado, deixando os restantes órgãos intactos. Goldmann conseguiu assim demonstrar que existe uma separação entre o cérebro e o resto do corpo. Esta separação é possível devido à presença de vasos sanguíneos específicos no cérebro. Estes vasos sanguíneos do cérebro formam uma barreira entre o cérebro e o sistema imunitário. Por conseguinte, eles são conhecidos como a barreira hemato-encefálica , agindo como um porteiro do cérebro. A barreira hemato-encefálica é composta por células especializadas que comunicam entre si através de proteínas específicas. Esta interacção restrita entre células proporciona uma barreira apertada, evitando a entrada de moléculas e células do sistema imunitário do sangue para o cérebro. Portanto, a barreira hemato-encefálica garante uma função cerebral adequada. No entanto, na esclerose múltipla, os processos inflamatórios que ocorrem no cérebro levam à alteração da função da barreira hemato-encefálica. Esta função alterada dos vasos sanguíneos cerebrais tem um efeito negativo em pacientes com esclerose múltipla. Por conseguinte, o objectivo desta tese foi compreender como é que a inflamação altera a função da barreira hemato-encefálica e como isso afecta a entrada de células do sistema imunitário para o cérebro.
A função dos vasos sanguíneos é determinada por mecanismos específicos que ocorrem durante o desenvolvimento do sistema nervoso central. Um desses mecanismos é a sinalização Notch. Esta via de sinalização ocorre entre células vizinhas e começa quando receptores Notch contactam com os respectivos ligandos. Esta via é essencial para o desenvolvimento adequado dos vasos sanguíneos. No entanto, o papel da sinalização Notch na função de barreira hemato-encefálica nunca foi estudado. No capitulo 2 desta tese mostramos que a sinalização Notch é afectada pela inflamação dos vasos sanguíneos cerebrais. Este fenómeno tem um efeito negativo na função de barreira dos vasos sanguíneos do cérebro. Este estudo destaca a importância de compreender como a função da barreira hemato-encefálica é regulada na saúde e na doença.
A infiltração de células do sistema imunitário para o cérebro
Em condições saudáveis, os vasos sanguíneos do cérebro formam uma barreira de tal forma que as células do sistema imunitário não conseguem penetrar no cérebro. No entanto, em doenças neuro-inflamatórias, tais como a esclerose múltipla, as células do
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sistema imunitário infiltram-se no cérebro, porque a barreira hemato-encefálica não funciona adequadamente. Esta disfunção dos vasos sanguíneos é principalmente devida à inflamação do cérebro provocada pelas células do sistema imunitário que acederam ao cérebro, provocando danos no tecido cerebral.
Para entrarem no cérebro, as células do sistema imunitário iniciam o contacto com os vasos sanguíneos através de proteínas específicas que estão presentes na sua superfície. Estas proteínas fornecem o primeiro ponto de contacto para as células do sistema imunitário, permitindo-lhes posteriormente atravessar os vasos sanguíneos e assim entrar no cérebro. Portanto, compreender este processo é de extrema importância para o desenvolvimento de novas terapias que reduzam a entrada de células do sistema imunitário no cérebro dos pacientes com esclerose múltipla.
Mostramos no capítulo 3, que os ligandos dos receptores Notch desempenham também um papel importante durante a migração das células do sistema imunitário para o cérebro. Em condições saudáveis, a barreira hemato-encefálica produz uma baixa quantidade destes ligandos na sua superfície. No entanto, durante processos inflamatórios, como os que ocorrem na esclerose múltipla, os vasos sanguíneos aumentam a produção destas proteínas na sua superfície, o que facilita a infiltração das células do sistema imunitário para o cérebro.
Na esclerose múltipla, ainda não é conhecida a razão pela qual células do sistema imunitário migram para o cérebro. Alguns investigadores acreditam que apenas as células do sistema imunitárias que reconhecem certas proteínas cerebrais, como a mielina, podem entrar no cérebro. No entanto, o que regula a entrada destas células imunes específicas para as áreas inflamadas do cérebro ainda não é claro. Por isso, quisemos perceber se a barreira hemato-encefálica tem a capacidade de mostrar estas proteínas específicas para as células do sistema imunitário. No capítulo 4 mostramos que os vasos sanguíneos do cérebro podem, de facto, apresentar proteínas específicas do cérebro às células do sistema imunitário. Quando estas reconhecem as proteínas cerebrais expostas nos vasos sanguíneos, elas entram no cérebro mais facilmente do que as células imunitárias que não reconhecem estas proteínas cerebrais. Este estudo destaca a importante função reguladora da barreira na iniciação da doença.
Na esclerose múltipla, não são apenas proteínas que estão envolvidas na progressão da doença, mas também lípidos. Demonstrou-se que o equilíbrio de lípidos está também desregulado no cérebro de pacientes com esclerose múltipla devido a processos inflamatórios. No capítulo 5, mostramos que alguns lipidos desempenham um papel importante na função da barreira hemato-encefálica . Mostramos que algumas enzimas, que produzem lípidos nos vasos sanguíneos, são produzidas em quantidades elevadas
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por vasos sanguíneos inflamados. Como resultado, as células do sistema imunitário entram no cérebro mais facilmente, uma vez que a função de barreira dos vasos sanguíneos é perdida. Curiosamente, as células inflamadas da barreira hemato-encefálica podem secretar estas enzimas. No capítulo 6, investigámos o potencial uso destas enzimas como um potencial biomarcador para a essclerose múltipla. Os nossos resultados preliminares sugerem que os níveis destas enzimas estão aumentados no sangue de pacientes com esclerose múltipla que apresentam disfunção da barreira hemato-encefálica e estão diminuidos em doentes que tomam medicamentos contra a doença. O desenvolvimento de melhores biomarcadores é essencial para melhores opções de diagnóstico, prognóstico e tratamento da esclerose múltipla. No entanto, devido aos diferentes tipos de sintomas e progressão da doença entre pacientes, a descoberta de biomarcadores para a esclerose múltipla ainda representa um grande desafio.
Perspectivas futuras e conclusões finais
Nesta tese, investigámos a forma como a inflamação altera a função da barreira hemato-encefálica e suas consequências para a entrada de células do sistema imunitário para o cérebro. Os nossos resultados demonstram que a compreensão da função de barreira dos vasos sanguíneos cerebrais ainda está a evoluir. Demonstramos que diferentes proteínas e mecanismos são importantes na regulação da entrada de células do sistema imunitário pelos dos vasos cerebrais o que poderá ajudar na compreensão da causa da esclerose múltipla. Embora as terapias actuais se destinam principalmente a reduzir a entrada das células do sistema imunitário para o cérebro, os pacientes muitas vezes sofrem de efeitos secundários associados à diminuição da função do sistema imunitário. Portanto, compreender os mecanismos complexos que regulam esta entrada na esclerose múltipla pode levar à descoberta e ao futuro desenvolvimento de melhores terapias que reduzam a disfunção da barreira hemato-encefálica e a entrada descontrolada de células do sistema imunitário para o cérebro.
ACKNOWLEDGEMENTS
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And this is how a PhD ends up, with all your work put together in a fancy little book! Of course, none of this would have been possible without some important people. Hereby my acknowledgements to:
Elga: my promotor, for accepting me as your PhD student in the blood-brain barrier
research group. Elga, I would like to thank you for always keeping a positive attitude and for making me look at the bright side in moments of frustration. For always being open to new ideas and for all the freedom I had to performing my research. I did grow a lot due to your openness. Thank you for always keeping me on track when I would focus in the tiny details and for teaching me the importance of the “helicopter view” of the research. Finally, thank you for always being available for scientific and non-scientific discussions and for always having a door open. I really enjoyed working with you and doing my PhD in your group!
Wendy: It was really nice that you could be part of this exciting journey. Thank you for
sharing your knowledge and expertise. You made a significant impact in the projects you were involved with and I think we all learned a lot from each other. Thank you for all the super organized brain-storming sessions and for always being so positive. Thank you for always being so enthusiastic and driven. You are truly an inspiring researcher. It was a pleasure working with you!
Jaap: thanks for embracing our project as your own, for always being so enthusiastic
and for always giving valuable input. I learned a lot in your lab at the Sanquin. It was a long process but it ended up in a really nice paper published in the JI! And of course I would like to thank Mark H. for always being available and for helping me with the experiments at the Sanquin and to Jeffrey K. for your help with the rebuttal of the paper and the amazing pictures!
Arie: Unfortunately, you had to leave but I really missed you and our brain storming
sessions! Thanks for your help in the first year of my PhD.
Group blue: Mark M., Wouter, Philip, Jack, Maarten and Jamie thanks for all your
input, for sharing your enthusiasm and knowledge during our blue group meetings.
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“new” PhD students Ananya, Cláudio, Alwin and Nienke I wish you all the luck to finish you thesis. A special thanks to Alwin for helping me with some figures!
I would like to thank the technicians that helped me during my PhD. Joost and Kim L. for teaching me a lot of techniques in the beginning of my PhD. Suus thanks for all the IHC stainings. Laura thanks for all your help during the last year of my PhD. I really appreciate your help and readiness. A special thanks to Bert for always being available especially in the last months of my project! I definitely owe you a zak of drop!
MCBI: Thank you all for the scientific discussions and for always being available to help
a colleague. A big thank you to the borrel comissie for organizing drinks and all the social activities. I had a great time at the department!
People from room H-269: from the “old” colleagues António, Evelina, Marieke B., Johanneke and Judith to the “new” ones Cláudio, Dieke and Marieke H. Thanks for all
the moments of laughter, coffee breaks, advice and frustrations shared. It made the PhD journey so much easier.
Lunch group: thanks to all the internationals (and some dutchies) that joined at the
lunch table namely António, Jamie, Esil, Evelina, Ananya, Andreia, Cláudio and Mark
V. Thank you for all the shared stories and frustrations, cultural discussions and alike.
For the coffee breaks after lunch, gossips and all the laughter. Thank you for being there.
Lab girls: Jamie and Esil (my paranymphs!), Evelina, Andreia and Ananya. Thank you
for all your support during this period. For all the lunches and dinners, movie nights and girls night out or afternoons at the spa. Thank you for being my friends. And Jamie, thanks for being there for me since the very beginning.
Leiden group: Sara, Olga, Kate, Yasmine, Elisa, Ricardo, Thanos and Sven thank you
all for the amazing moments we shared from our master studies until now. For all the crazy parties, dinners and brunches. Thank you girls for being there for me when I needed.
Family van Vugt: Ron, Bep, Jeff and Bob. Thank you so much for welcoming me in your
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Michael: my partner and friend. Thanks for always being there for me, for helping me
overcome my frustrations and stressful moments. For all your support and strength. For your love.
++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ E é assim que termina um doutoramento, com todo o esforço e empenho representado num pequeno livro!
Claro que nada disto teria sido possível sem a ajuda de algumas pessoas. Aqui vão os meus agradecimentos:
À Margarida, Joana P., Joana R. e Zelia por me acompanharem desde os tempos da faculdade, da licenciatura ao mestrado. Embora longe, queria agradecer-vos pelo vosso apoio e pela vossa amizade.
À Inês, Sofia e Cristiana por sempre estarem disponíveis aquando das minhas visitas a Marinhais. Pelo jantares e idas ao café. Por todas as memorias que partilhamos. Pelas confissões e conselhos. É sempre tão bom rever-vos. Obrigada pelo vosso apoio e amizade desde sempre.
À vizinha Adelaide, que é como se fosse da família. Obrigada pelo apoio e afeto. Por estar sempre disponível durante as minhas curtas visitas a Marinhais, pelas almoçaradas à portuguesa e por se preocupar comigo.
À Sandrina pelo teu apoio, por estares presente nos momentos difíceis. Por partilharmos tantas histórias, alegrias e tristezas. Por seres minha irmã e amiga. Por mesmo estando longe permanecermos unidas. Obrigada por tudo. E claro obrigada tambem ao Luís, Simão e Marta. Por fazerem com que as minhas idas a Portugal sejam sempre repletas de aventuras, risos e alegria.
Aos meus pais for fazerem de mim quem eu sou. Mãe obrigada por estares presente, pelo teu apoio e carinho. Por nao me deixares desistir e por teres sempre uma palavra amiga. Pai obrigada por teres sempre acreditado em mim e por teres apoiado os meus sonhos. Como não estás presente, esta tese é dedicada a ti.
CURRICULUM VITAE
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Curriculum vitae
After finishing high school, Melissa started the bachelor program in Molecular and Cell Biology at the New University of Lisbon in 2004. She did an internship in the Lymphocyte physiology lab in the Gulbenkian Institute of Science in Lisbon where she investigated the role of bacterial infections in the generation of regulatory T cells. Afterwards, she started her master studies in Molecular Biology and Genetics at the University of Lisbon in 2008. In 2009, she was awarded an ERASMUS scholarship, which allowed her to perform her master’s internship abroad. She joined the Tumor immunology group in the Leiden University Medical Center, in the Netherlands, where she investigated the differences in gene expression patterns between 'poised' and fully activated CD8+ T cells. In 2010, she joined the Blood-brain barrier research group at the
VU University Medical Center in Amsterdam where she studied the influence of inflammatory processes in blood-brain barrier function and how it contributes to T cell migration into the brain. The results of this research are described in this thesis. Since 2015, she works as a junior scientist at Kiadis Pharma B.V., a company developing cell-based therapies for patients with leukemia.
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Depois de terminar a escola secundária, a Melissa decidiu prosseguir os estudos na Universidade Nova de Lisboa onde iniciou a sua licenciatura em Biologia Celular e Molecular em 2004. Passou pelo Instituto Gulbenkian de Ciência em Lisboa onde fez um estágio no laboratorio de Fisiologia de Linfócitos onde estudou o papel de infecções bacterianas no desenvolvimento de células T reguladoras. Em 2008, iniciou o programa de mestrado em Biologia Molecular e Genética na Universidade de Lisboa. Em 2009 ganhou uma bolsa de estudos ERASMUS, possibilitando a realizaçõo do seu estágio curricular no laboratorio de Imunologia Tumoral no Hospital Universitario de Leiden, na Holanda. Durante este estágio estudou as differenças de expressão genética durante a activação de linfócitos T CD8+. Em 2010, iniciou o seu doutoramento no grupo de
investigação da barreira hemato-encefálica na Universidade Livre de Amsterdão onde investigou o papel dos processos inflamatórios nas propriedades desta barreira e as suas consequências para a migração de linfócitos T para o cérebro. Os resultados desta investigação estão descritos nesta tese. Desde 2015 trabalha como investigadora na
LIST OF PUBLICATIONS
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List of publications
Lopes Pinheiro MA, Kroon J, Hoogenboezem M, Geerts D, van het Hof B, van de Pol SM, van Buul JD, de Vries HE. Acid sphingomyelinase-derived ceramide regulates ICAM-1 function during leukocyte transmigration across brain endothelial cells.
J Immunol 196, 72-9 (2016)
Lopes Pinheiro MA, Kooij G, Mizee MR, Kamermans A, Enzmann G, Lyck R, Schwaninger M, Engelhardt B, de Vries HE. Immune cell trafficking across the barriers of the central nervous system in multiple sclerosis and stroke.
Biochim Biophys Acta 1862, 461-71 (2016)
van Doorn R, Lopes Pinheiro MA, Kooij G, Lakeman K, van het Hof B, van der Pol SM, Geerts D, van Horssen J, van der Valk P, van der Kam E, Ronken E, Reijerkerk A, de Vries HE. Sphingosine 1-phosphate receptor 5 mediates the immune quiescence of the human brain endothelial barrier.
J Neuroinflammation 20, 133-147 (2012)
van Doorn R, Nijland PG, Dekker N, Witte ME, Lopes Pinheiro MA, van het Hof B, Kooij G, Reijerkerk A, Dijkstra C, van van der Valk P, van Horssen J, de Vries HE. Fingolimod attenuates ceramide-induced blood-brain barrier dysfunction in multiple sclerosis by targeting reactive astrocytes.
Acta Neuropathol 124, 397-410 (2012)
