chapter 8 - VU-dare

CHAPTER 8
APPENDICES
English Summary
Nederlandse Samenvatting
Sumário em Português
Acknowledgements/Agradecimentos
Curriculum vitae
List of publications
ENGLISH SUMMARY
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. Secondaryprogressive 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
150
ENGLISH SUMMARY
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 gatekeeper 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 bloodbrain 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.
151
8
ENGLISH SUMMARY
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 bloodbrain 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
152
ENGLISH SUMMARY
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.
8
153
NEDERLANDSE SAMENVATTING
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
154
NEDERLANDSE SAMENVATTING
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
bloed-hersenbarrière, die functioneert als een poortwachter van de hersenen. De bloedhersenbarrière 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 bloedhersenbarrière, 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 bloedhersenbarrière 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,
wat leidt tot weefselbeschadiging.
155
8
NEDERLANDSE SAMENVATTING
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 bloedhersenbarrière 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 bloedhersenbarrière. 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
156
NEDERLANDSE SAMENVATTING
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 bloedhersenbarrière 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.
8
157
SUMÁRIO PORTUGUÊS
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 sub-dividida em quatro subtipos: 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.
158
SUMÁRIO PORTUGUÊS
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 hematoencefálica. 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
159
8
SUMÁRIO PORTUGUÊS
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
160
SUMÁRIO PORTUGUÊS
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 hematoencefálica 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 hematoencefálica 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.
8
161
ACKNOWLEDGEMENTS
162
ACKNOWLEDGEMENTS
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.
Thanks for always being available whenever I needed a question answered. To all the
163
8
ACKNOWLEDGEMENTS
“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
family. I really appreciate all your kindness and support. Alvast bedankt!
164
ACKNOWLEDGEMENTS
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.
8
165
166
CURRICULUM VITAE
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 cellbased 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
Kiadis Pharma B.V, uma empresa a desenvolver terapias para a leucemia.
167
8
168
LIST OF PUBLICATIONS
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)
8
169
170