The field of proteomics studies the structure and function of proteins in a large scale and high throughput manner. My work in the field of proteomics focuses on identifying interactions between proteins and discovering novel interactions. The identification of these interactions provides new information on metabolic and disease pathways and the working proteome of a cell. Cells are lysed and purified using antibody based affinity purification followed by digestion and identification using an HPLC coupled to a mass spectrometer. In my studies, I looked at the interaction networks of several AD related genes (Apolipoprotein E, Clusterin variant 1 and 2, Low-density lipoprotein receptor, Phosphatidylinositol binding clathrin assembly protein, Alpha-synuclein and Platelet-activating factor receptor) and an endosomal recycling pathway involved in cholesterol metabolism (Eps15 homology domain 1,2 and 4, Proprotein convertase subtilisin/kexin type 9 and Low-density lipoprotein receptor). Several novel and existing interactors were identified and these interactions were validated using co-immunopurification, which could be the basis for future research.

Objetivo: O objetivo desse estudo foi avaliar a expressão gênica e a determinação proteica de receptores de LDL (LDL-R e LRP-1) na lesão de endometriose profunda e comparar com o endométrio de mulheres com e sem endometriose, assim como determinar o perfil lipídico de pacientes com e sem endometriose profunda. Pacientes e métodos: Realizamos estudo transversal, caso-controle, exploratório com 39 pacientes, sendo 20 pacientes com diagnóstico histológico de endometriose profunda com comprometimento intestinal e 19 pacientes sem endometriose submetidas à laqueadura tubária laparoscópica. Foram coletadas amostras de sangue periférico no dia do procedimento cirúrgico para análise do perfil lipídico por meio da determinação de Colesterol total, HDL, LDL, VLDL, Triglicérides, APO A I e APO B 100. Foram também coletadas amostras de tecido endometrial com cureta de Pipelle e lesão de endometriose como parte do tratamento cirúrgico laparoscópico. Todas as amostras foram enviadas para análise histológica e submetidas à pesquisa de expressão gênica por PCR Real Time e à determinação proteica por imunoistoquímica dos receptores de LDL (LDL-R e LRP-1). A fase do ciclo menstrual foi determinada no momento do procedimento cirúrgico. Resultados: As pacientes com endometriose profunda apresentaram níveis séricos de LDL-c significativamente inferiores às pacientes sem a doença (119 ± 23 vs 156 ± 35; p=0,001). O mesmo não foi observado com o colesterol total (187 ± 27 vs 194 ± 37; p=0,562), HDL-c (42 ± 9 vs 43 ± 14; p=0,792), Triglicérides (130 ± 40 vs 119 ± 55; p=0,486), APO A I (128,1 ± 33,3 vs 136,5 ± 19,9; p=0,373) e APO B 100 (76 ± 20,9 vs 91,7 ± 30,8; p=0,085). A análise de expressão gênica por PCR Real Time dos receptores de LDL revelou que o LDL-R foi significativamente mais expresso na lesão de endometriose comparado ao endométrio da mesma paciente, mas não ao endométrio de mulheres sem endometriose (0,012 ± 0,009 vs 0,019 ± 0,01 vs 0,027 ± 0,022; p < 0,001) e o LRP-1 foi significativamente mais expresso na lesão de endometriose tanto quando comparado ao endométrio da mesma paciente, quanto quando comparado ao endométrio das pacientes sem a doença (0,089 ± 0,076 vs 0,126 ± 0,072 vs 0,307 ± 0,207; p < 0,001). A análise de determinação proteica por imunoistoquímica dos mesmos receptores revelou que o endométrio de mulheres sem a doença apresentou score de intensidade de marcação de LDL-R significativamente maior que o endométrio e a lesão de mulheres com endometriose (15 ± 78,9 vs 9 ± 45 vs 2 ± 10; p=0,026), porém a marcação para o receptor LRP-1 não apresentou diferença estatisticamente significativa (9 ± 47,4 vs 6 ± 30 vs 0 ± 0; p=0,073). O estudo também demonstrou que houve expressão significativamente maior de RNAm do receptor LDL-R (p=0,001) na fase secretora do ciclo menstrual e o mesmo pode ser observado com relação à expressão de RNAm do receptor LRP-1, que foi superexpresso (p=0,008) no endométrio de mulheres sem a doença. Conclusões: De acordo com os resultados de nossa pesquisa, concluímos que há redução dos níveis séricos de LDL em pacientes com endometriose profunda. Além disso, observamos maior expressão gênica de receptores de LDL em membrana celular de focos de endometriose profunda, comparado ao endométrio, tanto de mulheres sem endometriose quanto de mulheres com endometriose, achado não similar ao observado com a determinação proteica. Houve maior expressão de receptores de LDL em endométrio na fase secretora do ciclo. Este estudo abre oportunidade para viabilização de nanoemulsões lipídicas para acoplamento e direcionamento de drogas antiproliferativas no tratamento da endometriose profunda / Objective: The objective of this study was to evaluate the gene expression and protein determination of LDL receptor (LDL-R and LRP-1) in deep endometriosis lesions and compare with the endometrium of women with and without endometriosis, as well as to profile lipid patients with and without deep endometriosis. Methods: We conducted an transversal, exploratory, case-control study with 39 patients: 20 patients with a histological diagnosis of deep endometriosis with intestinal involvement and 19 women without endometriosis who underwent laparoscopic tubal ligation. Peripheral blood samples were collected on the day of surgery for analysis of lipid profile by determining total cholesterol, HDL, LDL, VLDL, triglycerides, APO AI and APO B 100. Specimens of endometrial tissue were collected using a Pipelle curette and endometriosis lesion specimens were obtains during therapeutic laparoscopic surgery. All samples were sent for histological evaluation and gene expression analysis by Real Time PCR and protein determination by immunohistochemistry of the LDL receptor (LDL-R and LRP-1). The phase of the menstrual cycle was determined at the time of surgery. Results: Patients with severe endometriosis had serum LDL-C levels significantly lower than the patients without the disease (119 ± 23 vs 156 ± 35; p = 0.001). The same was not observed with total cholesterol (187 ± 27 vs 194 ± 37, p = 0.562), HDL-C (42 ± 9 vs 43 ± 14, p = 0.792), triglycerides (130 ± 40 vs 119 ± 55; p = 0.486), APO AI (128.1 ± 33.3 vs 136.5 ± 19.9; p = 0.373) and APO B 100 (76 ± 20.9 vs 91.7 ± 30.8, p = 0.085). The analysis of gene expression by Real Time PCR of LDL receptors revealed that there was significantly greater expression of LDL-R in endometriosis lesions as compared to the endometrium of the same patient, but not when compared to the endometrium of women without endometriosis (0.012 ± 0.009 vs 0.019 ± 0, 01 vs 0.027 ± 0.022, p &lt;0.001). LRP-1 was significantly expressed in endometriotic lesions both when compared to the endometrium of the same patient as compared to the endometrium of patients without the disease (0.089 ± 0.076 vs 0.126 ± 0.072 vs 0.307 ± 0.207, p &lt;0.001). Protein determination by immunohistochemistry of the same receptors revealed that the endometrium of women without endometriosis had a significantly higher staining intensity score than the endometrium and the lesions of women with endometriosis (15 ± 78.9 vs 9 ± 2 vs 45 ± 10, p = 0.026) but the measurement for the LRP-1 receptor showed no statistically significant difference (9 ± 47.4 vs 6 ± 30 vs 0 ± 0; p = 0.073). The study also demonstrated that there was significantly higher mRNA expression of the LDL-R receptor (p = 0.001) in the secretory phase of the menstrual cycle, and the same can be observed with respect to the mRNA expression of LRP-1 receptor, which was overexpressed (p = 0.008) in the endometrium of women without the disease. Conclusion: Based on the findings of our research, we concluded that there is a reduction of serum LDL levels in patients with deep endometriosis. Moreover, we observed higher gene expression of LDL receptors on the cell membrane of foci of deep endometriosis, compared to the endometrium, both in women without endometriosis and women with endometriosis, a finding unlike that observed with the protein determination. There was greater expression of LDL receptors in the endometrium during the secretory phase of the cycle. These findings suggest the feasibility of using lipid nanoemulsions for coupling and targeted delivery of antiproliferative drugs in the treatment of deep endometriosis

Objetivo: O objetivo desse estudo foi avaliar a expressão gênica e a determinação proteica de receptores de LDL (LDL-R e LRP-1) na lesão de endometriose profunda e comparar com o endométrio de mulheres com e sem endometriose, assim como determinar o perfil lipídico de pacientes com e sem endometriose profunda. Pacientes e métodos: Realizamos estudo transversal, caso-controle, exploratório com 39 pacientes, sendo 20 pacientes com diagnóstico histológico de endometriose profunda com comprometimento intestinal e 19 pacientes sem endometriose submetidas à laqueadura tubária laparoscópica. Foram coletadas amostras de sangue periférico no dia do procedimento cirúrgico para análise do perfil lipídico por meio da determinação de Colesterol total, HDL, LDL, VLDL, Triglicérides, APO A I e APO B 100. Foram também coletadas amostras de tecido endometrial com cureta de Pipelle e lesão de endometriose como parte do tratamento cirúrgico laparoscópico. Todas as amostras foram enviadas para análise histológica e submetidas à pesquisa de expressão gênica por PCR Real Time e à determinação proteica por imunoistoquímica dos receptores de LDL (LDL-R e LRP-1). A fase do ciclo menstrual foi determinada no momento do procedimento cirúrgico. Resultados: As pacientes com endometriose profunda apresentaram níveis séricos de LDL-c significativamente inferiores às pacientes sem a doença (119 ± 23 vs 156 ± 35; p=0,001). O mesmo não foi observado com o colesterol total (187 ± 27 vs 194 ± 37; p=0,562), HDL-c (42 ± 9 vs 43 ± 14; p=0,792), Triglicérides (130 ± 40 vs 119 ± 55; p=0,486), APO A I (128,1 ± 33,3 vs 136,5 ± 19,9; p=0,373) e APO B 100 (76 ± 20,9 vs 91,7 ± 30,8; p=0,085). A análise de expressão gênica por PCR Real Time dos receptores de LDL revelou que o LDL-R foi significativamente mais expresso na lesão de endometriose comparado ao endométrio da mesma paciente, mas não ao endométrio de mulheres sem endometriose (0,012 ± 0,009 vs 0,019 ± 0,01 vs 0,027 ± 0,022; p < 0,001) e o LRP-1 foi significativamente mais expresso na lesão de endometriose tanto quando comparado ao endométrio da mesma paciente, quanto quando comparado ao endométrio das pacientes sem a doença (0,089 ± 0,076 vs 0,126 ± 0,072 vs 0,307 ± 0,207; p < 0,001). A análise de determinação proteica por imunoistoquímica dos mesmos receptores revelou que o endométrio de mulheres sem a doença apresentou score de intensidade de marcação de LDL-R significativamente maior que o endométrio e a lesão de mulheres com endometriose (15 ± 78,9 vs 9 ± 45 vs 2 ± 10; p=0,026), porém a marcação para o receptor LRP-1 não apresentou diferença estatisticamente significativa (9 ± 47,4 vs 6 ± 30 vs 0 ± 0; p=0,073). O estudo também demonstrou que houve expressão significativamente maior de RNAm do receptor LDL-R (p=0,001) na fase secretora do ciclo menstrual e o mesmo pode ser observado com relação à expressão de RNAm do receptor LRP-1, que foi superexpresso (p=0,008) no endométrio de mulheres sem a doença. Conclusões: De acordo com os resultados de nossa pesquisa, concluímos que há redução dos níveis séricos de LDL em pacientes com endometriose profunda. Além disso, observamos maior expressão gênica de receptores de LDL em membrana celular de focos de endometriose profunda, comparado ao endométrio, tanto de mulheres sem endometriose quanto de mulheres com endometriose, achado não similar ao observado com a determinação proteica. Houve maior expressão de receptores de LDL em endométrio na fase secretora do ciclo. Este estudo abre oportunidade para viabilização de nanoemulsões lipídicas para acoplamento e direcionamento de drogas antiproliferativas no tratamento da endometriose profunda / Objective: The objective of this study was to evaluate the gene expression and protein determination of LDL receptor (LDL-R and LRP-1) in deep endometriosis lesions and compare with the endometrium of women with and without endometriosis, as well as to profile lipid patients with and without deep endometriosis. Methods: We conducted an transversal, exploratory, case-control study with 39 patients: 20 patients with a histological diagnosis of deep endometriosis with intestinal involvement and 19 women without endometriosis who underwent laparoscopic tubal ligation. Peripheral blood samples were collected on the day of surgery for analysis of lipid profile by determining total cholesterol, HDL, LDL, VLDL, triglycerides, APO AI and APO B 100. Specimens of endometrial tissue were collected using a Pipelle curette and endometriosis lesion specimens were obtains during therapeutic laparoscopic surgery. All samples were sent for histological evaluation and gene expression analysis by Real Time PCR and protein determination by immunohistochemistry of the LDL receptor (LDL-R and LRP-1). The phase of the menstrual cycle was determined at the time of surgery. Results: Patients with severe endometriosis had serum LDL-C levels significantly lower than the patients without the disease (119 ± 23 vs 156 ± 35; p = 0.001). The same was not observed with total cholesterol (187 ± 27 vs 194 ± 37, p = 0.562), HDL-C (42 ± 9 vs 43 ± 14, p = 0.792), triglycerides (130 ± 40 vs 119 ± 55; p = 0.486), APO AI (128.1 ± 33.3 vs 136.5 ± 19.9; p = 0.373) and APO B 100 (76 ± 20.9 vs 91.7 ± 30.8, p = 0.085). The analysis of gene expression by Real Time PCR of LDL receptors revealed that there was significantly greater expression of LDL-R in endometriosis lesions as compared to the endometrium of the same patient, but not when compared to the endometrium of women without endometriosis (0.012 ± 0.009 vs 0.019 ± 0, 01 vs 0.027 ± 0.022, p &lt;0.001). LRP-1 was significantly expressed in endometriotic lesions both when compared to the endometrium of the same patient as compared to the endometrium of patients without the disease (0.089 ± 0.076 vs 0.126 ± 0.072 vs 0.307 ± 0.207, p &lt;0.001). Protein determination by immunohistochemistry of the same receptors revealed that the endometrium of women without endometriosis had a significantly higher staining intensity score than the endometrium and the lesions of women with endometriosis (15 ± 78.9 vs 9 ± 2 vs 45 ± 10, p = 0.026) but the measurement for the LRP-1 receptor showed no statistically significant difference (9 ± 47.4 vs 6 ± 30 vs 0 ± 0; p = 0.073). The study also demonstrated that there was significantly higher mRNA expression of the LDL-R receptor (p = 0.001) in the secretory phase of the menstrual cycle, and the same can be observed with respect to the mRNA expression of LRP-1 receptor, which was overexpressed (p = 0.008) in the endometrium of women without the disease. Conclusion: Based on the findings of our research, we concluded that there is a reduction of serum LDL levels in patients with deep endometriosis. Moreover, we observed higher gene expression of LDL receptors on the cell membrane of foci of deep endometriosis, compared to the endometrium, both in women without endometriosis and women with endometriosis, a finding unlike that observed with the protein determination. There was greater expression of LDL receptors in the endometrium during the secretory phase of the cycle. These findings suggest the feasibility of using lipid nanoemulsions for coupling and targeted delivery of antiproliferative drugs in the treatment of deep endometriosis

Bakgrund: Deposition av fibrillär amyloid beta 1-42 (Aβ) i hjärnan är ett välkänt kännetecken för den neurodegenerativa sjukdomen Alzheimer’s (AD). Dessa ansamlingar påverkar pericyter, en celltyp involverad i blodkärlsfunktion och upprätthållande av blodhjärnbarriären (BBB). Pericyter uttrycker både receptorn low density lipoprotein receptor related protein 1 (LRP-1) till vilken Aβ1-42 binder, och proteoglykanet NG2. NG2 har stor betydelse för pericyters samspel med endotelceller och i sin lösliga form (sNG2) främjar den angiogenes. Tidigare studier har visat att mängden NG2 som klyvs från pericyter förändras när de stimuleras med Aβ1-42. Syfte: Att undersöka om Aβ1-42 påverkar NG2 klyvning via LRP-1 Metod: Human brain vascular pericytes (HBVP) stimulerades med monomera, oligomera och fibrillära Aβ1-42 preparationer. Uttrycket av LRP-1 tystades med small interfering (si) LRP-1 och knockdown efficiency analyserades med Western Blot (WB). Även Aβ1-42 preparationer undersöktes med WB. Cellviabilitet mättes med laktatdehydrogenas (LDH) test och proteininnehåll med Bradford analys. Slutligen mättes mängden sNG2 i pericytmedium med hjälp av enzyme-linked immunosorbant assay (ELISA) baserad på electrochemiluminescence (Mesoscale). Resultat: Preparationerna med monomer och oligomer Aβ1-42 ökade NG2 klyvning. Denna Aβ1-42 inducerade ökning försvann när cellernas LRP-1 tystats. Aβ1-42 fibrillpreparationerna inhiberade däremot NG2 klyvningen oavsett närvaro av LRP-1. Aβ1-42 monomerpreparationer inducerade celldöd hos HBVP med LRP-1 men inte hos de HBVP där LRP-1 tystats, och cellviabiliteten hos HBVP ökade hos celler som stimulerats med Aβ1-42 fibrillpreparation och där LRP-1 tystats. Konklusion: Resultaten visar att Aβ1-42 monomer och oligomer påverkar NG2 klyvning via LRP-1. Däremot verkar Aβ1-42 fibrill istället påverka NG2 klyvning via en annan signalväg. Studien belyser hur Aβ1-42 kan påverka pericyter, vilket kan föreligga vaskulära förändringar kopplade till AD patologi. / Background: The deposition of fibrillar amyloid beta 1-42 (Aβ) in the brain is a well-known characteristic for the neurodegenerative Alzheimer’s disease (AD). These accumulations affect pericytes, a cell type implicated in vessel function and maintenance of the blood-brain barrier (BBB). Pericytes express both the receptor low-density lipoprotein receptor related protein 1 (LRP-1), to which Aβ1-42 binds, and the proteoglycan NG2. NG2 is important for the interaction between pericytes and endothelial cells and in its soluble form (sNG2) it promotes angiogenesis. Earlier studies have shown that the amount of NG2 shed from pericytes is altered when stimulated with Aβ1-42. Purpose: To investigate whether the Aβ1-42 influence on NG2 shedding is mediated via LRP-1. Method: Human brain vascular pericytes (HBVP) were stimulated with monomeric, oligomeric and fibrillar preparations of Aβ1-42. Expression of LRP-1 was knocked down by small interfering (si) LRP-1 silencing and knockdown efficiency was analysed with Western blot (WB). Aβ1-42 preparations were also analysed with WB. Cell viability was measured with lactate dehydrogenase (LDH) test and protein concentrations were determined with Bradford assay. Finally the amount of sNG2 in pericytemedium was measured with enzyme-linked immunosorbant assay (ELISA) baserad på electrochemiluminescence (Mesoscale) Results: Monomer and oligomer Aβ1-42 increased NG2 shedding, this Aβ1-42 induced increase was not found in HBVP with a silenced LRP-1. In contrast, fibrillar Aβ1-42 inhibited NG2 shedding regardless of LRP-1 presence. Monomer Aβ1-42 preparations induced cell death of HBVP with LRP-1 but not of HBVP without LRP-1, and cell viability of HBVP lacking LRP-1 was increased after fibrillar Aβ1-42 exposure. Conclusion: The results indicate a monomeric and oligomeric Aβ1-42 induced impact on NG2 shedding via LRP-1. However it appears as if fibrillar Aβ1-42 doesn’t affect NG2 shedding via LRP-1 but rather inhibits the process via another unknown receptor. The study highlights how Aβ1-42 can affect pericytes, which may underlie the vascular changes linked to AD pathology.

No description available.

Les maladies cardiovasculaires (MCV) demeurent au tournant de ce siècle la principale cause de mortalité dans le monde. Parmi les facteurs de risque, l’hypercholestérolémie et l’obésité abdominale sont directement liées au développement précoce de l’athérosclérose. L’hypercholestérolémie familiale, communément associée à une déficience des récepteurs des lipoprotéines de basse densité (LDLR), est connue comme cause de maladie précoce d’athérosclérose et de calcification aortique chez l’humain. La subtilisine convertase proprotéine/kexine du type 9 (PCSK9), membre de la famille des proprotéines convertases, est trouvée indirectement associée aux MCV par son implication dans la dégradation du LDLR. Chez l'humain, des mutations du gène PCSK9 conduisent soit à une hypercholestérolémie familiale, soit à une hypocholestérolémie, selon que la mutation entraîne un gain ou une perte de fonction, respectivement. Il demeure incertain si les individus porteurs de mutations causant un gain de fonction de la PCSK9 développeront une calcification aortique ou si des mutations entraînant une perte de fonction provoqueront une obésité abdominale. Dans cette étude, nous avons examiné : 1) l’effet d’une surexpression de PCSK9 dans le foie de souris sur la calcification aortique ; 2) les conséquences d’une déficience en PCSK9 (Pcsk9 KO), mimant une inhibition pharmacologique, sur le tissu graisseux. Nous avons utilisé un modèle de souris transgénique (Tg) surexprimant le cDNA de PCSK9 de souris dans les hépatocytes de souris et démontrons par tomographie calculée qu’une calcification survient de façon moins étendue chez les souris PCSK9 Tg que chez les souris déficientes en LDLR. Alors que le PCSK9 Tg et la déficience en LDLR causaient tous deux une hypercholestérolémie familiale, les niveaux seuls de cholestérol circulant ne parvenaient pas à prédire le degré de calcification aortique. Dans une seconde étude, nous utilisions des souris génétiquement manipulées dépourvues de PSCK9 et démontrons que l’accumulation de graisses viscérales (adipogenèse) apparaît régulée par la PCSK9 circulante. Ainsi, en l’absence de PCSK9, l’adipogenèse viscérale augmente vraisemblablement par régulation post-traductionnelle des récepteurs à lipoprotéines de très basse densité (VLDLR) dans le tissu adipeux. Ces deux modèles mettent en évidence un équilibre dynamique de la PCSK9 dans des voies métaboliques différentes, réalisant un élément clé dans la santé cardiovasculaire. Par conséquent, les essais d’investigations et d’altérations biologiques de la PCSK9 devraient être pris en compte dans un modèle animal valide utilisant une méthode sensible et en portant une attention prudente aux effets secondaires de toute intervention. / Cardiovascular disease (CVD) is the leading cause of death in the 21st century. Among risk factors, hypercholesterolemia and abdominal obesity are directly linked to premature development of atherosclerosis. Familial hypercholesterolemia, commonly due to low-density lipoprotein receptor (LDLR) deficiency, is known to cause premature atherosclerosis and aortic calcification in humans. Proprotein convertase subtilisin/kexin 9 (PCSK9), a member of the proprotein convertase family, is indirectly associated with CVD through enhanced LDLR degradation. Mutations in the human PCSK9 gene lead to either familial hypercholesterolemia or hypocholesterolemia, depending on whether the mutation causes a gain or a loss of function, respectively. It is uncertain if individuals carrying mutations causing a gain-of-function of PCSK9 will develop aortic calcification or whether loss-of-function mutations will lead to abdominal obesity. In this thesis, we investigated: 1) the effect of PCSK9 overexpression on aortic calcification; 2) the consequences of PSCK9 deficiency, mimicking pharmacological inhibition of PCSK9 on fat tissue. We employed a transgenic (Tg) mouse model overexpressing mouse PCSK9 and illustrated by micro-computerized tomography that calcification occurs to a lesser extent in PCSK9 Tg mice than in LDLR-deficient mice. While both PCSK9 Tg and LDLR deficiency caused familial hypercholesterolemia, circulating cholesterol levels alone could not dictate the degree of aortic calcification. In another study, we used genetically modified mice lacking PCSK9 and demonstrated that visceral fat accumulation (adipogenesis) is regulated by circulating PCSK9. Thus in the absence of PCSK9, visceral adipogenesis increases likely via post-translational regulation of very-low-density lipoproteins receptors (VLDLR) in the adipose tissue. In conclusion, these two studies highlight the dynamic balance of PCSK9 in different metabolic pathways, making it a key element in cardiovascular health. Consequently, attempts to survey and/or alter PCSK9 biology should be performed in a valid animal model using sensitive methods and with careful attention to side effects of any given intervention.

Les maladies cardiovasculaires (MCV) demeurent au tournant de ce siècle la principale cause de mortalité dans le monde. Parmi les facteurs de risque, l’hypercholestérolémie et l’obésité abdominale sont directement liées au développement précoce de l’athérosclérose. L’hypercholestérolémie familiale, communément associée à une déficience des récepteurs des lipoprotéines de basse densité (LDLR), est connue comme cause de maladie précoce d’athérosclérose et de calcification aortique chez l’humain. La subtilisine convertase proprotéine/kexine du type 9 (PCSK9), membre de la famille des proprotéines convertases, est trouvée indirectement associée aux MCV par son implication dans la dégradation du LDLR. Chez l'humain, des mutations du gène PCSK9 conduisent soit à une hypercholestérolémie familiale, soit à une hypocholestérolémie, selon que la mutation entraîne un gain ou une perte de fonction, respectivement. Il demeure incertain si les individus porteurs de mutations causant un gain de fonction de la PCSK9 développeront une calcification aortique ou si des mutations entraînant une perte de fonction provoqueront une obésité abdominale. Dans cette étude, nous avons examiné : 1) l’effet d’une surexpression de PCSK9 dans le foie de souris sur la calcification aortique ; 2) les conséquences d’une déficience en PCSK9 (Pcsk9 KO), mimant une inhibition pharmacologique, sur le tissu graisseux. Nous avons utilisé un modèle de souris transgénique (Tg) surexprimant le cDNA de PCSK9 de souris dans les hépatocytes de souris et démontrons par tomographie calculée qu’une calcification survient de façon moins étendue chez les souris PCSK9 Tg que chez les souris déficientes en LDLR. Alors que le PCSK9 Tg et la déficience en LDLR causaient tous deux une hypercholestérolémie familiale, les niveaux seuls de cholestérol circulant ne parvenaient pas à prédire le degré de calcification aortique. Dans une seconde étude, nous utilisions des souris génétiquement manipulées dépourvues de PSCK9 et démontrons que l’accumulation de graisses viscérales (adipogenèse) apparaît régulée par la PCSK9 circulante. Ainsi, en l’absence de PCSK9, l’adipogenèse viscérale augmente vraisemblablement par régulation post-traductionnelle des récepteurs à lipoprotéines de très basse densité (VLDLR) dans le tissu adipeux. Ces deux modèles mettent en évidence un équilibre dynamique de la PCSK9 dans des voies métaboliques différentes, réalisant un élément clé dans la santé cardiovasculaire. Par conséquent, les essais d’investigations et d’altérations biologiques de la PCSK9 devraient être pris en compte dans un modèle animal valide utilisant une méthode sensible et en portant une attention prudente aux effets secondaires de toute intervention. / Cardiovascular disease (CVD) is the leading cause of death in the 21st century. Among risk factors, hypercholesterolemia and abdominal obesity are directly linked to premature development of atherosclerosis. Familial hypercholesterolemia, commonly due to low-density lipoprotein receptor (LDLR) deficiency, is known to cause premature atherosclerosis and aortic calcification in humans. Proprotein convertase subtilisin/kexin 9 (PCSK9), a member of the proprotein convertase family, is indirectly associated with CVD through enhanced LDLR degradation. Mutations in the human PCSK9 gene lead to either familial hypercholesterolemia or hypocholesterolemia, depending on whether the mutation causes a gain or a loss of function, respectively. It is uncertain if individuals carrying mutations causing a gain-of-function of PCSK9 will develop aortic calcification or whether loss-of-function mutations will lead to abdominal obesity. In this thesis, we investigated: 1) the effect of PCSK9 overexpression on aortic calcification; 2) the consequences of PSCK9 deficiency, mimicking pharmacological inhibition of PCSK9 on fat tissue. We employed a transgenic (Tg) mouse model overexpressing mouse PCSK9 and illustrated by micro-computerized tomography that calcification occurs to a lesser extent in PCSK9 Tg mice than in LDLR-deficient mice. While both PCSK9 Tg and LDLR deficiency caused familial hypercholesterolemia, circulating cholesterol levels alone could not dictate the degree of aortic calcification. In another study, we used genetically modified mice lacking PCSK9 and demonstrated that visceral fat accumulation (adipogenesis) is regulated by circulating PCSK9. Thus in the absence of PCSK9, visceral adipogenesis increases likely via post-translational regulation of very-low-density lipoproteins receptors (VLDLR) in the adipose tissue. In conclusion, these two studies highlight the dynamic balance of PCSK9 in different metabolic pathways, making it a key element in cardiovascular health. Consequently, attempts to survey and/or alter PCSK9 biology should be performed in a valid animal model using sensitive methods and with careful attention to side effects of any given intervention.

Doctor of Philosophy / Department of Human Nutrition / Denis M. Medeiros / Laman Mamedova / The internalization of aggregated low-­density lipoprotein (agLDL) may involve the actin cytoskeleton in ways that differ from the endocytosis of soluble LDL. Based on previous findings the P2Y[subscript]2 receptor (P2Y[subscript]2R) mediates these effects through interaction with filamin‐A (FLN‐A), an actin binding protein. Our findings also showed that uridine 5’‐ triphosphate (UTP), a preferential agonist of the P2Y[subscript]2R, stimulates the uptake of agLDL, and increases expression of low‐density lipoprotein receptor related protein 1 (LRP 1) in cultured mouse vascular smooth muscle cells (SMCs). The strategy of this research was to define novel mechanisms of LDL uptake through the modulation of the actin cytoskeleton in order to identify molecular targets involved in foam cell formation in vascular SMCs. For this project, we isolated aortic SMCs from wild type (WT) and P2Y[subscript]2R‐/‐ mice to investigate whether UTP and the P2Y[subscript]2R modulate expression of LRP 1 and low‐density lipoprotein receptor (LDLR). We also investigated the effects of UTP on uptake of DiI‐labeled agLDL in WT and P2Y[subscript]2R‐/‐ vascular SMCs. For LRP1 expression, cells were stimulated in the presence or absence of 10 [mu]M UTP. To determine LDLR mRNA expression, and for agLDL uptake, cells were transiently transfected for 24 h with cDNA encoding hemagglutinin-­tagged (HA-­tagged) WT P2Y[subscript]2R or a mutant P2Y[subscript]2R that does not bind FLN‐A, and afterwards treated with 10 [mu]M UTP. Total RNA was isolated, reversed transcribed to cDNA, and mRNA relative abundance determined by RT-­PCR using the delta-­delta Ct method with GAPDH as control gene. Results show SMCs expressing the mutant P2Y[subscript]2R that lacks the FLN‐A binding domain exhibit 3‐fold lower LDLR expression than SMCs expressing the WT P2Y[subscript]2R. There was also decrease in LRP1 mRNA expression in response to UTP in P2Y[subscript]2R‐/‐ SMCs compared to WT. Actinomycin‐D (20 [mu]g/ml) significantly reduced UTP-­induced LRP1 mRNA expression in P2Y[subscript]2R‐/‐ SMCs (P < 0.05). Compared to cells transfected with mutant P2Y[subscript]2R, cells transfected with WT P2Y[subscript]2R showed greater agLDL uptake in both WT VSMC and P2Y[subscript]2R-­/-­ cells. Together these results show that both LRP 1 and LDLR expressions are dependent on an intact P2Y[subscript]2R, and P2Y[subscript]2R/ FLN‐ A interaction is necessary for agLDL uptake.

La thérapie cellulaire pourrait représenter une alternative à la transplantation hépatique dans certaines pathologies comme les maladies métaboliques sévères. Toutefois, la pénurie de donneurs d’organes implique la nécessité de trouver de nouvelles sources de cellules hépatiques comme les cellules souches pluripotentes qui peuvent être amplifiées extensivement et différenciées en tout type cellulaire. Les cellules souches embryonnaires humaines (hESC) et les cellules souches pluripotentes induites humaines (hiPSC) générées à partir des cellules somatiques de patients puis différenciées en hépatocytes représentent une source potentielle d’hépatocytes. Ces cellules permettent en outre d’envisager la transplantation d’hépatocytes autologues génétiquement modifiés comme alternative à la transplantation hépatique pour le traitement de certaines maladies génétiques du foie. L’hypercholestérolémie familiale (HF) est une maladie autosomale dominante due à des mutations dans le gène codant le Récepteur aux Low Density Lipoproteins (RLDL) qui est à l’origine d’un taux élevé de cholestérol sanguin de patients HF. Les patients homozygotes doivent épurer leur sérum par LDL-aphérèse en moyenne deux fois par mois dès le plus jeune âge pour éviter les infarctus mortels survenant dès l’enfance. Les hépatocytes différenciées à partir des iPSC de patients et leur correction in vitro, permettent d'évaluer la faisabilité de la transplantation d'hépatocytes autologues génétiquement modifiés pour le traitement de l’hypercholestérolémie familiale.Au cours du développement du foie, des hépatocytes et des cholangiocytes, les deux types de cellules épithéliales hépatiques, dérivent de progéniteurs hépatiques bipotents (les hépatoblastes). Bien que les cholangiocytes formant les canaux biliaires intrahépatiques ne représentent qu'une petite fraction de la population cellulaire totale du foie (3%), ces cellules régulent activement la composition de la bile par réabsorption des acides biliaires, un processus qui est important dans des maladies choléstatiques du foie. Dans la première partie de cette étude nous avons mis au point une approche de différenciation des cellules souches pluripotentes (hESC et hiPSC) en cholangiocytes fonctionnels. Ces cellules serviront à la modélisation des maladies génétiques touchant les cholangiocytes formant des canaux biliaires. Dans la deuxième partie, nous avons généré des iPSC spécifiques de patients HF (HF-iPSC), différenciées en hépatocytes et corrigé le défaut phénotypique par transfert lentiviral de l’ADNc codant le LDLR dans les HF-iPSC. / Cell therapy can be an alternative to liver transplantation in some cases such as severe metabolic diseases. However, the shortage of organ donors implies the need to find new sources of liver cells such as hepatocytes derived from pluripotent stem cells that can be amplified and differentiated extensively into any cell type. Human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) generated from somatic cells of patients and then differentiated into hepatocytes represent a potential source of transplantable hepatocytes. These cells now make it possible to consider the transplantation of genetically modified autologous hepatocytes as an alternative to liver transplantation for the treatment of genetic diseases of the liver.Familial hypercholesterolemia (FH) is an autosomal dominant disorder caused by mutations in the gene encoding the receptor for Low Density Lipoproteins (LDLR), which is the cause of high blood cholesterol in these patients. Homozygous patients should purify their serum LDL-apheresis on average twice a month starting at a young age to avoid fatal myocardial infarction occurring in childhood.Human hepatocytes differentiated from patient’s induced pluripotent stem cells (iPSCs) allow assessing the feasibility to transplant genetically modified autologous hepatocytes as treatment of familial hypercholesterolemia.During the liver development, hepatocytes and cholangiocytes, the two types of hepatic epithelial cells, derive from bipotent hepatic progenitors (hepatoblasts). Although cholangiocytes, forming intrahepatic bile ducts, represent a small fraction of the total liver cell population (3%), they actively regulate bile composition by secretion and reabsorption of bile acids, a process that is important in cholestatic liver diseases. In the first part of this study we developed an approach to differentiate pluripotent stem cells (hESC and hiPSC) into functional cholangiocytes. These cells could be used for the modeling of genetic biliary diseases. In the second part, we generated FH patient specific iPSCs (HF-iPSC), differentiated them into hepatocytes and tried to correct the disease phenotype by lentiviral introduction of LDLR cDNA cassette in HF-iPSC.

Humans have two plasminogen activators (PAs), tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), which generate plasmin to breakdown fibrin and other barriers to cell migration. Both PAs are used as pharmaceuticals but their efficacies are limited by their rapid clearance from the circulation, predominantly by parenchymal cells of the liver. At the commencement of the work presented here, the hepatic receptors responsible for mediating the catabolism of the PAs were little understood. tPA degradation by hepatic cell lines was known to depend on the formation of binary complexes with the major PA inhibitor, plasminogen activator inhibitor type-1 (PAI-1). Initial studies presented here established that uPA was catabolised in a fashion similar to tPA by the hepatoma cell line, HepG2. Other laboratories around this time found that the major receptor mediating the binding and endocytosis of the PAs is Low Density Lipoprotein Receptor-related Protein (LRP1). LRP1 is a giant 600 kDa protein that binds a range of structurally and functionally diverse ligands including, activated α2 macroglobulin, apolipoproteins, β amyloid precursor protein, and a number of serpin-enzymes complexes, including PA??PAI-1 complexes. Further studies for the work presented here centred on this receptor. By using radiolabelled binding assays, ligand blots, and Western blots on cultured cells, the major findings are that: (1) basal LRP1 expression on HepG2 is low compared to a clone termed, HepG2a16, but appears to increase in long term culture; (2) a soluble form of LRP1, which retains ligand-binding capacity, is present in human circulation; (3) soluble LRP1 is also present in cerebral spinal fluid where its role in neurological disorders such as Alzheimer??s disease is a developing area of interest; and (4) the release of LRP1 is a mechanism conserved in evolution, possibly as distantly as molluscs. The discovery, identification, and characterisation of soluble LRP1 introduces this protein in the human circulation, and presents a possible further level of regulation for its associated receptor system.