Dissertations / Theses on the topic 'Soluble factor'

The role of a parasite-produced or -induced soluble immunosuppressor in experimental kala azar was examined. It was found that in vivo infections with Leishmania donovani in the hamster (Mesocricetus auratus) produce a soluble immunosuppressor, which appears in the serum of the host and which reduces the proliferation of responding populations of murine splenocytes in a one-way mixed leukocyte reaction (MLR). The production in vitro infections of murine splenic macrophages from C57BL/6J ($Lsh sp{ rm s}$), C57L/J ($Lsh sp{ rm R}$) and BALB/c strains, the suppressive activity was not contained in either parasite-conditioned culture medium or in parasite extracts or from macrophages which have internalized killed parasites or inert particles and it is not blocked by the action of 2-mercaptoethanol or indomethacin in the culture medium. The suppressor was found to be able to selectively inhibit or reduce the proliferation of splenocytes of both the C57BL/6J and C57LN strains in a one way MLR, with the level of suppression being significantly greater upon splenocytes of the susceptible $Lsh sp{ rm s}$ strain. The suppression was dependent upon the genotype of the macrophages present in the responding population. The suppressor was also able to significantly inhibit the processing of human serum albumin by macrophages, to reduce the number of Ia ligands on the surfaces of macrophages and the production by these cells of IL-1 upon silica stimulation.
Significant reduction was also seen in the production of IL2 and in the expression of its receptor by PHA-stimulated T cells exposed to the suppressor. Partial purification and identification of the suppressor demonstrated that the suppressive activity was present in fractions between 30 and 50 kDa in size; the suppressor was also heat labile and freeze-thaw sensitive. The suppressive molecule(s) may therefore play a significant role in the establishment and pathology of L. donovani infections.

Synaptic dysfunction likely contributes to abnormal brain function in schizophrenia. Patient symptoms indicate that the striatum is involved in this disease. The three neuronal soluble-NSF-attachment receptor (SNARE) proteins (SNAP-25, syntaxin-1 and VAMP) interact at the presynaptic neuronal membrane to facilitate neurotransmission, and are thus key players in synaptic function. SNARE abnormalities have already been reported in cortical and hippocampal brain regions in schizophrenia. Their involvement in striatal dysfunction has not been investigated. Normal synaptic function requires the SNAREs to physically interact with each other, but little is known about how altered SNARE protein levels in schizophrenia relate to SNARE protein interactions. Multiple isoforms of each SNARE exist in the brain, may affect SNARE protein interactions and synaptic transmission differently, and may diverge functionally. SNARE isoform expression in schizophrenia is unknown. Thus, abnormalities in SNARE protein expression or function may underlie or contribute to brain dysfunction and disease. In this thesis, SNARE protein levels were measured in human post mortem brain samples of schizophrenia subjects for the first time in the striatum. The functional consequences of SNARE alterations were investigated by developing a novel ELISA assay to measure SNARE protein interactions. The possible confounding effects of medications were addressed in several ways, including the use of striatal tissue from animals exposed to antipsychotic medications. Alterations in SNAP-25 and syntaxin-1 protein levels were further dissected by measuring protein isoforms. Syntaxin-1 isoforms were assayed by quantitative immunoblotting. A mass-spectrometry based assay was developed and used to measure SNAP-25 protein isoform levels. The results of these investigations suggest that SNARE protein alterations in schizophrenia are restricted to distinct functional regions of the striatum, perturb SNARE protein interactions, involve specific protein isoforms, and may occur independent of patient treatment with antipsychotic medications. Furthermore, these studies contribute a new, high-throughput method for measuring SNARE protein interactions, and for the first time, a means of detecting and quantifying SNAP-25 isoforms in brain tissue.

Computational group theory deals with the design, analysis and computer implementation of algorithms for solving computational problems involving groups, and with the applications of the programs produced to interesting questions in group theory, in other branches of mathematics, and in other areas of science. This thesis describes an implementation of a proposal for a Soluble Quotient Algorithm, i.e. a description of the algorithms used and a report on the findings of an empirical study of the behaviour of the programs, and gives an account of an application of the programs. The programs were used for the construction of soluble groups with interesting properties, e.g. for the construction of soluble groups of large derived length which seem to be candidates for groups having efficient presentations. New finite soluble groups of derived length six with trivial Schur multiplier and efficient presentations are described. The methods for finding efficient presentations proved to be only practicable for groups of moderate order. Therefore, for a given derived length soluble groups of small order are of interest. The minimal soluble groups of derived length less than or equal to six are classified.

Mesenchymal stem cells (MSCs) are bone marrow derived multipotent cells with the ability to self-renew and differentiate into multiple connective cell lineages. In vivo, MSCs travel from the bone-marrow to the inflammatory sites and actively participate in remodeling and regeneration process under the influence of soluble growth factors. Due to these inherent properties, MSCs have emerged as an ideal candidate for diverse regenerative therapeutic applications. The development of MSC-based therapies requires in vitro expansion of MSCs; however, MSC expansion results in phenotypical changes that have limited its efficacy upon reintroduction in vivo. In order to increase the efficacy of MSC-based therapeutics, it is critical for us to improve the current understanding of MSC interactions with its niche specific factors and explore new methods to enhance MSC function in vivo. We used tumor conditioned media, which contains soluble factors secreted by tumor cells in culture (TCM), and inflammatory niche-specific soluble factors, such as platelet derived growth factor (PDGF) and transforming growth factor-β1 (TGF-β1), to characterize the mechanical response of MSCs. The intracellular mechanical properties of MSCs were dramatically altered in response to soluble factors and MSCs displayed cytosolic stiffening in response to TCM and TGF-β1. Although PDGF treated cells did not elicit any mechanical response, blocking PDGF signaling with a small molecule inhibitor reversed the stiffening response in TGF-β1 treated cells, indicating crosstalk between these two pathways is essential in TGF-β1 mediated cell stiffening. Furthermore, a genome-wide microarray analysis revealed TGF-β1 dependent regulation of cytoskeletal actin-binding protein (ABP) genes. Actin crosslinking and bundling protein genes, which regulate cytosolic rheology through changes in semiflexible actin polymer meshworks, were upregulated with TGF-β1 treatment. Since TGF-β1 treatment profoundly altered the MSC phenotype after relatively short exposure times, we sought to understand if pretreated cells could sustain these enhanced characteristics leading to higher efficacy in vivo. We found that MSCs pretreated with TGF-β1 displayed enhanced adhesive properties while maintaining the expression profile of surface adhesion molecules even after removal of stimulus. Additionally, pretreated MSCs exposed to lineage specific induction media, demonstrated superior differentiation potential along multiple lineages. Based on the large number of sustained changes, TGF-β1 pretreated cells were used to treat full thickness skin wounds for in vivo wound healing model to determine their therapeutic efficacy. TGF-β1 pretreated MSCs increased wound closure rate and displayed enhanced migration of MSCs towards the center of the wound compared to the control cells. In conclusion, soluble factor pretreated MSCs with altered mechanical properties displayed significantly improved cell functions leading to highly efficient tissue regeneration in vivo. Mechanical priming of MSCs with niche specific factors prior to transplantation can become a viable strategy to maximize their therapeutic potential.

Angiogenesis is the formation of new blood vessels from existing vasculature. Vascular Endothelial Growth Factor (VEGF), a known angiogenic protein, stimulates endothelial cell proliferation and migration via interactions with its receptors, KDR and Flt-1. A secreted form of Flt-1 (sFlt-1), derived from alternatively-spliced RNA, can inhibit actions of VEGF in vivo. It has been suggested that alterations in sFlt-1 expression could significantly change the angiogenic VEGF activity. This project focuses on characterizing intronic elements that regulate Flt-1 mRNA splicing. A "wild-type" construct (pFIN13), containing the first 13 exons, intron 13 and exons 14-30 of mouse Flt-1, was shown to produce both forms of Flt-1 mRNA after transfection into HEK293 cells. To gauge the strength of the native splicing signals in intron 13 of Flt-1, a series of point mutations were made in the polypyrimidine tract using pFIN13. After transient transfection, the levels of Flt-1 and sFlt-1 protein and mRNA were compared using quantitative PCR, RNA hybridization analysis, and protein immunoblotting. Results from quantitative PCR showed that purine substitutions were associated with 120 to 350 fold decreases in Flt-1 mRNA (normalized against neor), consistent with less efficient splicing. These large decreases in Flt-1 mRNA were accompanied by increases in sFlt-1 mRNA. Modest (20 to 100%) increases in Flt-1 mRNA, reflecting improved splicing, resulted from increasing the uridine complement in the polypyrimidine tract. These results suggest that the wild-type polypyrimidine tract is of intermediate strength and may be a regulatory locus for modulating Flt-1: sFlt-1 ratios.
Master of Science

L’implication d’un facteur circulant et des dysfonctions du système immunitaire entrainant les altérations de la barrière de filtration glomérulaire a été suggérée dans la pathogénèse de la hyalinose segmentaire et focal récidivante. Nous avons identifié par spectrométrie de masse la présence de la protéine CASK dans des sérums de patients après immunoadsoroption sur une colonne de protéine A. CASK recombinante est capable d'induire des modifications de l’architecture des podocytes in vitro, tels qu’une redistribution de la protéine de diaphragme de fente ZO-1 et de la protéine régulatrice d’actine synaptopodine, et une perte de fibres de stress d’actine. Ces podocytes acquièrent ainsi un phénotype motile et une perméabilité accrue à l’albumine en présence de CASK recombinante in vitro. L’injection de CASK chez des souris entraine une protéinurie et l’effacement des pédicelles de podocytes. L’interaction entre CASK et son récepteur CD98 dans les podocytes a été mise en évidence par l’expérience de pontage covalent et co-immunoprécipitation. L’inhibition de l’expression de CD98 par ARNi a permis de préserver l’architecture des podocytes en présence de CASK. Nous avons remarqué la surexpression de CASK dans les monocytes chez les patients atteints de la HSF récidivante par rapport aux témoins. In vitro, CASK est surexprimée dans les macrophages ayant une polarité M2 et est retrouvée dans le surnageant de la culture de ces cellules. La sécrétion de CASK est associée aux exosomes qui sont des microvésicules d’origine endosomale. Dans les cellules, CASK est partiellement co-distribuée avec ALIX, un marqueur exosomal, et leur interaction a été mise en évidence par co-immunoprécipitation. CASK est fortement exprimée dans les exosomes de patients atteints de HSF récidivante comparé aux donneurs sains. Le traitement des podocytes par des exosomes issus des macrophages de type M2 induit des altérations du cytosquelette et augmente la motilité des podocytes comme cela avait été observé en présence de CASK recombinante. Pour conclure, nous avons identifié CASK comme nouveau facteur soluble qui pourrait jouer un rôle au cours de la HSF récidivante après transplantation rénale. Ces découvertes ouvrent de nouvelles orientations pour le traitement des malades atteints de SNI récidivant
Focal segmental glomerulosclerosis (FSGS) is often associated with a high rate of progression to end-stage renal disease. The idiopathic form has a high recurrence rate (rFSGS) after transplantation suggesting the presence of a systemic circulating factor that causes the glomerular permeability. This factor can be removed by plasmapheresis or immunoadsorption using protein-A columns. We used mass spectrometry to analyze the proteins eluted from protein-A columns, taken from patients with rFSGS after immunoadsorption. A serum form of calcium/calmodulin-dependent serine/threonine kinase (CASK) was identified in rFSGS patients but not in controls. In cultured podocytes, recombinant CASK induced reorganization of the actin cytoskeleton. We also demonstrated the interaction of CASK with CD98 at the cell surface. Injection of recombinant CASK in mice induced proteinuria and foot process effacement on podocytes. We identified that CASK is produced by monocytes in patients with rFSGS. CASK is also expressed and secreted by M2 polarized macrophages but not by M1 subset. CASK was associated with exosomes produced by these cells. CASK has a partial codistribution with ALIX, an exosomal component involved in their development. We’ve also demonstrated that CASK interacts with ALIX in M2 macrophages. Moreover exosomes derived from M2 macrophages cause podocytes cytoskeleton alterations and increase of podocyte motility as observed previously with recombinant CASK. In conclusion, a serum form of CASK secreted by macrophages acts as a permeability factor in patients with rFSGS suggesting its involvement in the physiopathology of rFSGS

Le but de cette these est d'etudier la faisabilite de l'utilisation de protheses osseuses modifiees par des facteurs de croissance osteogeniques et angiogenique. L'angiogenese et l'invasion vasculaire sont des conditions necessaires pour initier la formation osseuse, le remodelage et la reparation de fractures. Notre hypothese de travail a ete d'utiliser des systemes de culture de cellules osseuses pour definir de nouvelles molecules d'interet therapeutique potentiel. Nous avons ainsi pu montrer que le vegf (vascular endothelial growth factor) facteur initialement connu comme electif des cellules endotheliales vasculaires induisait une differenciation d'amplitude equivalente a celle obtenue avec le prototype de facteur de croissance osseux qu'est la bmp2 a des doses 100 fois moindres. Ayant demontre l'affinite du vegf pour l'hydroxyapatite, nous avons evalue l'adsorption du vegf sur des phosphates de calcium d'interet biologique, prealablement caracterises, en vue d'application

Après le vieillissement, le diabète Mellitus de type 2 (DMT2) est le facteur de risque le plus important pour développer la maladie d'Alzheimer (MA). Le DMT2 est une maladie métabolique caractérisée par une hyperglycémie et une résistance à l’insuline qui se développe vers la cinquantaine et est fortement favorisée par l’obésité. Nous avons exploré l’impact potentiel du DMT2 sur le développement de la MA chez le Rat. Pour cela, nous avons utilisé un régime alimentaire cafétéria (RC) couplé à des injections de faibles doses de Streptozotocine (STZ) (STZ-CD). Les rats STZ-CD montrent des signes classiques de DMT2 et des déficits légers de consolidation en mémoire de reconnaissance spatiale. Afin d'imiter le développement des stades précoces de la MA, la moitié des rats reçoivent une infusion intracérébrale de peptides β-amyloïdes solubles (Aβ) qui ne conduisent pas à des déficits mnésiques durables. Par contre, le phénotype DMT2 chez les rats STZ-CD exacerbe les déficits mnésiques observés avec le peptide Aβ en les prolongeant dans le temps. L’enrichissement environnemental pendant une période critique de 2 semaines après l’infusion d’Aβ est capable de compenser les déficits mnésiques induits par le peptide Aβ et/ou le traitement STZ-CD ; mais d’une manière limitée dans le temps. Des analyses biochimiques dans l’aire CA1 de l’hippocampe ont été effectuées pour explorer de possibles altérations de la voie PI3K, de marqueurs de la cascade amyloïde et du DMT2. Le peptide Aβ seul induit peu de changements durables ; le phénotype DMT2 seul est associé à des changements pour quelques protéines-clé, largement en liaison avec le régime cafétéria. Par contre, la majorité des modifications dysfonctionnelles de protéines est observée chez les rats montrant un phénotype de type DMT2 et recevant le peptide Aβ. Ces altérations, similaires à celles rapportées chez des patients atteints de la MA et chez des modèles animaux de la MA, concernent notamment des protéines de la voie PI3K-Akt impliquée dans des fonctions comme l’autophagie et l’inflammation et des marqueurs de la MA. L’altération de ces protéines pourrait contribuer aux déficits mnésiques durables observés et mettre en lumière des mécanismes moléculaires induits par le DMT2 et promouvant un milieu neuronal favorisant le développement d’un stade précoce de la maladie d'Alzheimer
Following aging, type 2 Diabetes Mellitus (T2DM) is the most important risk factor of developing Alzheimer’s disease (AD). It is a metabolic disorder characterised by hyperglycemia and insulin resistance that develops in middle age and is promoted largely by obesity. In this study, we used a T2DM rat model to assess the potential impact T2DM may have on the development of AD. Rats were fed cafeteria-style diet (CD) coupled with low dose injections of Streptozotocin (STZ)(STZ-CD). We found that STZ-CD treated rats showed classic signs of T2DM and a modest deficit in consolidation of spatial recognition memory. In order to mimic the development of early stage AD, half of the rats were infused with a soluble oligomeric amyloid beta (Aβ), which alone was not sufficient to induce long-lasting memory deficits. Interestingly, the T2DM phenotype exacerbated the memory deficits induced by Aβ infusion by prolonging these deficits. Environmental enrichment during a critical two-week period following infusion of Aβ rescued memory deficits induced by Aβ and/or STZ-CD treatment; however, this was time-limited. Biochemical analyses were conducted mainly in proteins involved in the PI3K-Akt signalling pathway and markers of AD and T2DM in CA1 of the hippocampus. Aβ alone induced few long-lasting changes; T2DM phenotype alone induced some changes that were largely mediated by CD treatment alone; however, the majority of dysfunctional regulation of proteins was observed in rats showing a T2DM phenotype that were infused with Aβ. More importantly, many of these changes are similar to those reported in brains of AD patients or rodent models of the disease; notably key proteins in the PI3K-Akt signaling pathway that mediate functions such as autophagy, inflammation and markers of AD. Dysregulation of these proteins may contribute to the long-lasting memory deficits seen in this model, which may provide evidence of molecular mechanisms induced by T2DM that could promote a dysfunctional neuronal environment favouring the development of early stages of Alzheimer’s disease

L'effet de différents extraits de cerveau de mammifères contenant des activités lectines sur des cultures de cerveau de souris embryonnaires (e 14,5) et sur des cellules de cerveau de souris dissociées et maintenues en suspension (stades e 14,5, e17,5, p0, p4, p14, p23). Nous avons exploré les propriètés agglutinantes et agrégeantes de ces extraits et montre leur interaction spécifique avec des structures saccharidiques en particulier avec les residus de type béta -d-galactosyle. Ces molécules sont ubiquitaires, leur taux est régulé par le développement. Les propriétés physico-chimiques, la spécificité saccharidique et les différentes caracteristiques rattachent ces molecules aux galaptines, lectines solubles decrites pour d'autres tissus et d'autres especes. Une deuxieme activite lectine, specifique de l'heparine est extraite du cerveau en même temps que la galaptine. La caractérisation de la galaptine à partir d'extraits de cerveau de rat et de boeuf montre qu'il s'agit d'un dimère des sous unites >ou= 14 500 daltons pour le boeuf et de >ou= 16 000 daltons pour le rat. L'analyse de la composition en acides aminés fait ressortir un taux élevé de glycine et la richesse en acides aminés acides. L'isolement de la galaptine a permis de confirmer que certains effets des extraits bruts étaient bien liés à la présence de cette lectine dans les extraits

Apesar de sua importância clínica e epidemiológica, a fisiopatologia da préeclâmpsia ainda não foi completamente compreendida. Sabe-se que a doença constitui-se de uma fase pré-clínica e um estágio clínico. Durante a última década muito esforço tem se concentrado na identificação precoce da doença, ainda em sua fase pré-clínica. A literatura científica tem demonstrado claramente um desequilíbrio na regulação da angiogênese das gestantes com pré-eclâmpsia, marcado por níveis elevados do fator antiangiogênico soluble fms-like tyrosine kinase-1 (sFlt-1) e níveis diminuídos do fator pró-angiogênico placental growth fator (PlGF). Embora um número crescente de estudos em populações de alto risco tenha avaliado o papel desses biomarcadores no diagnóstico de pré-eclâmpsia, dados sobre sua utilização para a predição de pré-eclâmpsia superajuntada, cujo diagnóstico pode ser particularmente difícil, permanecem relativamente escassos e controversos. Com o presente estudo pretendemos avaliar o desempenho de medidas seriadas dos níveis maternos circulantes dos fatores sFlt-1 e PlGF, bem como da razão sFlt-1/PlGF, para predição de pré-eclâmpsia superajuntada e compará-lo ao seu desempenho na predição de pré-eclâmpsia em sua forma \"pura\", não superajuntada. Para este propósito, estudamos uma coorte prospectiva composta de dois braços, um de gestantes com hipertensão arterial crônica e outro de gestantes normotensas, e avaliamos os níveis séricos de sFlt-1 e de PlGF e a razão sFlt-1/PlGF nas idades gestacionais de 20, 26, 32 e 36 semanas, tendo como desfecho principal o diagnóstico de pré-eclâmpsia. Um total de 97 gestantes foram acompanhadas, 37 normotensas e 60 com hipertensão arterial crônica. Entre elas, 4 (10,8%) desenvolveram pré-eclâmpsia e 14 (23,3%) desenvolveram pré-eclâmpsia superajuntada. Para predição de pré-eclâmpsia, a análise ROC (Receiver Operating Characteristics) apresentou área sob a curva (AUC - area under curve) de 0,83 (IC 95% = 0,68-0,99, P = 0,035) para dosagem de PlGF com 20 semanas e AUC = 0,92 (IC 95% = 0,81 - 1,00, P = 0,007) para a razão sFlt-1/PlGF com 26 semanas de gestação. A variação percentual dos níveis de PlGF entre 26 e 32 semanas de gestação apresentou AUC = 0,96 (IC de 95% = 0,89-1,00, P = 0,003). Para a predição de pré-eclâmpsia superajuntada, a razão sFlt-1/PIGF na idade gestacional de 32 semanas apresentou AUC = 0,69 (IC de 95% = 0,53-0,85, P = 0,039). Entre 20 e 26 semanas de gestação, a variação percentual do PIGF e da razão sFlt-1/PlGF apresentaram, respectivamente, AUC = 0,74 (IC de 95% = 0,58-0,90, P = 0,018) e AUC = 0,71 (IC 95% = 0,52-0,91, P = 0,034). Por nossos resultados podemos concluir que, embora os níveis de PlGF e a razão sFlt-1/ PlGF tenham apresentado bons desempenhos na predição de pré-eclâmpsia, é preciso ter cuidado ao usá-los para a predição de pré-eclâmpsia superajuntada. Nessas gestantes, a dosagem dos fatores angiogênicos apresenta capacidade de predição menor e mais tardia. Avaliações seriadas dos fatores podem melhorar o desempenho dos testes para predição de pré-eclâmpsia superajuntada em idades gestacionais mais precoces
Despite being a major public health problem, the pathophysiology of preeclampsia is incompletely understood. Preeclampsia progression comprises a pre-clinical stage and a clinical stage. During the last decade much work has focused on identifying the pre-clinical stage of preeclampsia. Many researchers have clearly demonstrated an anti-angiogenic imbalance that is marked by higher levels of soluble fms-like tyrosine kinase-1 (sFlt-1) and lower levels of placental growth factor (PlGF) in the subjects who develop preeclampsia compared with those who do not. Although a growing number of studies in the high-risk population have shown the role of these biomarkers in diagnosing preeclampsia, superimposed preeclampsia, which can be a challenging diagnosis, remains partially understudied and the literature regarding this subject continues to be relatively scarce as well as controversial. By this study, we aimed to evaluate the performance of serial measurements of maternal circulating sFlt-1 and PlGF levels for the prediction of superimposed preeclampsia in chronic hypertensive subjects and to compare it to the prediction of preeclampsia in normotensive control subjects. For this purpose, we evaluated a two-armed prospective cohort of women with normotensive and chronic hypertensive pregnancies and assessed the serum levels of sFlt-1 and PlGF and the sFlt-1/PlGF ratio at gestational ages of 20, 26, 32 and 36 weeks, having preeclampsia as the primary outcome to be predicted. A total of 97 women were followed-up, 37 in the normotensive group and 60 in the chronic hypertensive group. Among them, 4 (10.8%) women developed preeclampsia and 14 (23.3%) developed superimposed preeclampsia. For predicting preeclampsia, PlGF at 20 gestational weeks presented an AUC=0.83 (CI 95% = 0.68 - 0.99, P=0.035) and the sFlt-1/PlGF ratio at 26 gestational weeks presented an AUC=0.92 (CI95% = 0.81 - 1.00, P=0.007). The percent change of the PlGF levels between 26 and 32 gestational weeks presented an AUC=0.96 (CI 95% = 0.89 - 1.00, P=0.003). For predicting superimposed preeclampsia, the sFlt-1/PlGF ratio at 32 gestational weeks presented an AUC=0.69 (CI 95% = 0.53 - 0.85, P=0.039). Between 20 and 26 gestational weeks, the percent change of PlGF and the sFlt-1/PlGF ratio presented, respectively, an AUC=0.74 (CI 95% = 0.58 - 0.90, P=0.018) and an AUC=0.71 (CI 95% = 0.52 - 0.91, P=0.034). By our results, we concluded that, although the PlGF level and the sFlt-1/PlGF ratio present good performances in the prediction of preeclampsia, caution is required when using them for the prediction of superimposed preeclampsia. Sequential assessments slightly improve the test performances for predicting superimposed preeclampsia at earlier gestational ages

La maladie d’Alzheimer est caractérisée par un déclin progressif des capacités cognitives. Les Aßo induisent des dysfonctionnements de la transmission via une altération des récepteurs au glutamate et une perte de synapses.Nos récents résultats démontrent que le VEGF facilite la plasticité synaptique et la mémoire chez des souris via son action sur son récepteur VEGFR2. Nous avons montré que le VEGF stimule l’insertion synaptique des récepteurs glutamatergiques et la formation de synapses, suggérant ainsi un rôle dans la modulation des altérations synaptiques observées dans la maladie d’Alzheimer.Notre objectif est d’étudier le rôle du VEGF, spécifiquement dans la maladie d’Alzheimer. Tout d’abord, nous avons examiné son expression en relation avec les plaques séniles chez des patients et dans un modèle de la maladie d’Alzheimer. Nos résultats ont démontré une colocalisation entre le VEGF et ces plaques.Afin d’examiner plus finement l’interaction Aß-VEGF, nous avons analysé la liaison entre les Aßo et le VEGF en test ELISA et puces à peptides. Nous avons ainsi démontré un potentiel blocage de l’interaction entre le VEGF et son récepteur, menant à des défauts de son activation.Enfin, nous avons examiné si le VEGF prévient les altérations synaptiques par des approches électrophysiologiques, biochimiques et immunocytochimiques. Nos résultats démontrent que lors d’un traitement aux Aßo, le VEGF restaure la LTP, l’expression des récepteurs au glutamate et limite la perte synaptique.Dans l’ensemble, nos résultats suggèrent que l’interaction Aß-VEGF altère la voie du VEGF chez les patients. De plus, le VEGF réduit la toxicité induite par les Aßo sur les synapses
Alzheimer disease (AD) is characterized by a progressive decline in cognitive abilities. Amyloid-ß oligomers (Aßo) trigger synapse dysfunction through defects in glutamate receptor function and subsequent dendritic spine loss. These synaptic impairments compromise memory and contribute to cognitive deficits.Our recent findings revealed that VEGF facilitates synaptic plasticity and memory in mice through its VEGFR2 receptor in neurons. We showed that VEGF promotes glutamate receptor synaptic insertion and stimulates dendritic spine formation, suggesting it may be a key candidate for alleviating synapse damage in AD.Our objective is to study the role of VEGF in synapse protection in AD models and unravel the underlying mechanisms.First, we examined the VEGF expression pattern in postmortem brain tissue from AD patients and APPPS1 model of AD. Our results showed a partial colocalization between VEGF and Aß plaques in AD patients and APPPS1 brains.To further investigate the Aß-VEGF interaction, we used Elisa assay and peptide arrays and demonstrated that Aßo binds several domains of VEGF, impedding VEGFR2 activation.Finally, we examined whether VEGF can prevent synapse damage induced by Aßo using electrophysiological, biochemical and 3D modelling approaches. Our results demonstrated that VEGF treatments can restore LTP in Aßo-treated hippocampal slices, glutamate receptor content at synapses and increase dendritic spine density.All together, our results suggest that Aß-VEGF interaction may alter VEGF pathway in AD and that VEGF reduces Aßo-induced toxicity at synapses by modulating glutamate receptor expression and promoting spine formation and/or stabilization

Lors du développement du système nerveux, l’élongation neuritique, à la base de la morphogenèse neuronale, requiert l’apport de matériel membranaire et protéique, tels les récepteurs aux facteurs de croissance ou les molécules d’adhérence, au niveau du cône de croissance. Nous avons montré précédemment l’importance de la voie d’exocytose impliquant la SNARE vésiculaire TI-VAMP dans la croissance neuritique, mais ses régulations sont mal connues. L’identification de nouveaux partenaires de TI-VAMP permet de lier TI-VAMP avec la machinerie de fusion ainsi qu’avec les cytosquelettes d’actine et de microtubule et les facteurs de polarisation neuronale. En particulier, Varp, partenaire de TI-VAMP, est un facteur d’échange pour Rab21 et est le premier lien moléculaire entre une v-SNARE et une Rab. Je montre que Rab21 régule la capacité des cellules PC12 à étendre des neurites et favorise la dynamique antérograde des vésicules TI-VAMP. Rab21 régule donc le transport de TI-VAMP vers le cône de croissance, mécanisme indispensable à la maturation neuronale. Ce travail jette les bases de la régulation du transport des vésicules TI-VAMP au cours de la neuritogenèse

Las hormonas recombinantes eritropoyetina (EPO) y hormona de crecimiento (GH), prácticamente iguales a las endógenas y de corta vida media en circulación, son de difícil detección directa en el control antidopaje.
Se determinaron los valores poblacionales de los biomarcadores indirectos EPO, receptor soluble de la transferrina, insulin-like growth factor-I (IGF-I) y procolágeno tipo III péptido (P-III-P), en poblaciones seleccionadas de deportistas, y el efecto del ejercicio y los distintos tipos de entrenamiento sobre su concentración sérica.
La comparación de resultados obtenidos mediante distintos ensayos demostró la necesidad de una validación exhaustiva previa a su utilización. A excepción del P-III-P, los biomarcadores séricos propuestos para la detección de rhEPO y rhGH no se encuentran directamente afectados por el nivel atlético, el ejercicio o la distinta carga de entrenamiento realizada a lo largo de la temporada deportiva. La edad es la principal influencia sobre las concentraciones séricas de IGF-I y P-III-P.
Direct detection of recombinant peptidic hormones erythropoietin (EPO) and growth hormone (GH), very similar to endogen molecules and with a short half life in blood, is difficult in antidoping control.
The main objective of this work is to determine indirect biomarkers' values of EPO, soluble transferrin receptor, insulin-like growth factor-I (IGF-I) and procollagen type III peptide (P-III-P), in selected populations of athletes, and the effect of exercise and different types of training on their concentration in serum.
The comparison of results obtained by the different assays showed the need of extensive validation of the analytical techniques before their use in the antidoping field. Excepting P-III-P, proposed biomarkers for the detection of rhEPO and rhGH abuse are not directly influenced by the athletic level, exercise or different training workload along the sport season. Age is the main factor affecting IGF-I and P-III-P concentrations in serum.

Les plaquettes sanguines sont des cellules qui ont un rôle majeur au cours des processus de l’hémostase primaire et jouent un rôle primordial dans l’immunité innée mais aussi adaptative. Ces cellules anucléées ont une capacité sécrétoire très importante de facteurs solubles notamment de cytokines, de chimiokines (CK/CH) et de facteurs immunomodulateurs. L’émergence du rôle inflammatoire des plaquettes sanguines dans la communauté scientifique a soulevé de nombreuses questions auxquelles nous essayons de répondre dans ce manuscrit. La majorité de ces questions repose sur la capacité de ces cellules anucléées à répondre de manière régulée à des stimuli complexes. Nos investigations pour répondre à ces questions ont été réalisées dans un contexte transfusion sanguine. Au cours de nos travaux, nous avons mis en évidence la corrélation des profils de sécrétion plaquettaire avec les récepteurs membranaires et les voies de signalisations intraplaquettaires engagées. Les plaquettes expriment plusieurs récepteurs immunitaires sur leur surface notamment les « Pattern recognition receptors » (PRR) et des récepteurs aux CK/CH. Nous avons démontré et caractérisé la fonction d’un nouveau récepteur plaquettaire, le Siglec-7. Ce récepteur est localisé dans les granules a ; son expression sur la membrane est corrélée avec l’état d’activation plaquettaire. Le Siglec-7 a une avidité élevée avec les molécules composées d’α2,8-disialyl (NeuAcα2,8NeuAcα2,3Gal) et de α2,6-sialyl (Gal-b1,3[NeuAcα2,6]HexNAc) (comme les gangliosides GD2, GD3 et GT1b). L’engagement de ce récepteur peut induire l’apoptose plaquettaire par la voie intrinsèque et extramitochondriale. Ce processus nécessite l’engagement du récepteur GPIIbIIIa et P2Y1 et la signalisation de la voie de PI3k. Nous avons également étudié et mis en évidence une composante inflammatoire multifactorielle dans les effets indésirables des receveurs (EIR) et trouvé dans les concentrés plaquettaires (CP), plusieurs facteurs solubles ayant une valeur prédictive élevée pour la survenue des EIR, notamment le sCD40L et l’IL-13. Nous avons confirmé que la concentration de ces facteurs augmente au cours de temps de stockage des CP, étant, en partie, responsable du taux élevé de l’EIR des CP âgés. Enfin, en plus de la conservation, les processus de préparation des CP peuvent aussi avoir des impacts sur les propriétés inflammatoires des plaquettes. Ces travaux montrent que la réponse inflammatoire plaquettaire est régulée en fonction du stimulus, permettant d’argumenter sur le rôle présumé de sentinelle des plaquettes sanguines humaines. Ainsi, mes travaux s’inscrivent dans la ré-exploration de la fonction inflammatoire des plaquettes sanguines et l’étude du rôle des plaquettes comme cellules de l’immunité à composante inflammatoire
Blood platelets are non-nucleated cells and play a major role in primary hemostasis and a key role in inflammation, innate and adaptive immunity. They secrete a large variety of soluble factors including cytokines/chemokines (CK/CH) and immunomodulator factors. The emergence of their inflammatory role has raised numerous questions based on the ability of platelets to respond to complex stimuli. Our investigations to answer these questions were realized in the context of platelet component transfusion. In our study, we demonstrated the correlation between the platelet secretion of soluble factors with their membrane receptors and the signaling pathways involved. Platelets express many immune receptors on their surface, including "Pattern recognition receptors" (PRRs) and receptor for CK/CH. We discovered and characterized the function of a new platelet receptor, the Siglec-7. This receptor is located in the granules a and its expression is correlated to the platelet activation level. The Siglec -7 has a high avidity with the molecules composed of α2,8-disialyl (NeuAcα2,8NeuAcα2,3Gal) and of α2,6-sialyl (Gal-b1,3[NeuAcα2,6]HexNAc) (ganglioside GD2 , GD3 and GT1b). Stimulation of this platelet receptor may induce platelet apoptosis by the intrinsic and extramitochondrial pathway. This process requires the engagement of GPIIbIIIa and P2Y1 receptor and the PI3K pathway. We also demonstrated a multifactorial inflammatory component in adverse effects issuing from platelets transfusion, and identified many soluble factors which have a high predictive value of Acute Transfusion Reactions (ATR) occurrence, such as sCD40L and IL- 13. We confirmed that the concentration of these factors increases during storage time of platelet component (PC), being partly responsible for the high rate of ATR by old PC. Finally, in addition to the PC conservation, the process of PC preparation may also have impacts on the inflammatory properties of platelets. These studies showed that the platelet inflammatory response is regulated by the stimulus, explaining the sentinel role of human blood platelets. Therefore, my work contributes to the re-exploration of inflammatory function of these cells and studies their role as an immune cell with an inflammatory component

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Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (Capes)
Funda??o de Amparo ? Pesquisa do estado de Minas Gerais (FAPEMIG)
Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq)
Introdu??o: Recentemente, a vibra??o de todo o corpo (VTC) tem sido um m?todo de exerc?cio f?sico indicado para aumentar o desempenho e a capacidade f?sico-funcional de idosos com osteoartrite (OA) de joelho. No entanto, os mecanismos relacionados aos efeitos produzidos por essa modalidade ainda n?o foram completamente investigados. Objetivos: O objetivo deste estudo foi investigar os efeitos da adi??o de VTC aos exerc?cios de agachamento na concentra??o plasm?tica de marcadores inflamat?rios e no desempenho e capacidade funcionais de idosos com OA de joelho na fase remissiva da doen?a (Estudo 1) e investigar os efeitos da adi??o do treinamento de VTC ao exerc?cio de agachamento em mulheres idosas com OA de joelho na fase remissiva da doen?a nos seguintes par?metros: 1) for?a isom?trica do m?sculo quadr?ceps; 2) concentra??o plasm?tica de BDNF; e 3) na concentra??o salivar da raz?o testosterona/cortisol (Estudo 2). Investigar a rela??o entre os n?veis plasm?ticos e no l?quido sinovial do TNF-? e seus receptores sol?veis (sTNFR1 e sTNFR2) assim como de BDNF em idosos com OA de joelho e ainda verificar a rela??o destes com a gravidade da OA e o autorrelato de dor, rigidez e fun??o f?sica com o WOMAC (Western Ontario and McMaster University Osteoarthritis Index) em idosos com OA de joelho na fase inflamat?ria aguda (Estudo 3). Metodologia: No estudo 1, trinta e dois idosos com OA de joelho foram divididos em tr?s grupos: grupo que realizou exerc?cio de agachamento associado a plataforma vibrat?ria (grupo plataforma N=11), grupo que realizou exerc?cio de agachamento sem vibra??o (grupo agachamento N=10) e o grupo controle (N=11). Um programa estruturado de exerc?cios de agachamento foi executado tr?s vezes por semana em dias alternados por doze semanas nos grupos plataforma e agachamento. A concentra??o plasm?tica de receptores sol?veis de TNF-? (sTNFR1 e sTNFR2) foi analisada usando a t?cnica de ELISA. O WOMAC foi usado para avaliar o autorrelato da fun??o f?sica, dor e rigidez. O teste de caminhada de 6 minutos, a escala de Berg e o teste de velocidade da marcha foram utilizados para avaliar a fun??o f?sica. No estudo 2, foram selecionadas quinze mulheres idosas com idade maior ou igual a 60 anos que tinham sido diagnosticadas com OA em pelo menos um joelho. A interven??o consistiu de doze semanas seguidas de exerc?cios de agachamento, 3 vezes por semana. O protocolo de exerc?cio foi similar em ambos os grupos diferindo apenas da presen?a de vibra??o. J? no estudo 3 participaram vinte e sete idosos diagnosticados com osteoartrite de joelho e dezenove idosos saud?veis. Radiografias ?ntero-posteriores do joelho foram realizadas para determinar a gravidade da doen?a no joelho afetado. A classifica??o radiogr?fica da OA do joelho foi realizada utilizando os crit?rios Kellgren-Lawrence. Os n?veis de TNF-?, sTNFR1, sTNFR2 e BDNF no plasma e no l?quido sinovial foram determinados por ELISA. Resultados: No estudo 1, o grupo que realizou exerc?cios de agachamento na plataforma vibrat?ria mostrou diminui??o nas concentra??es plasm?ticas dos marcadores inflamat?rios sTNFR1 e sTNFR2 (p<0,001 e p<0,05, respectivamente), no autorrelato da dor (p<0,05), melhora no equil?brio (p<0,05) e na velocidade e dist?ncia caminhada (p<0,05 e p<0,001, respectivamente) comparado com o grupo controle. O teste de velocidade da marcha tamb?m apresentou aumento no grupo plataforma quando comparado ao grupo agachamento (p<0,01). Os resultados do estudo 2 demonstraram uma varia??o (?) positiva dos valores da for?a isom?trica muscular do quadr?ceps (p=0,02) e da concentra??o plasm?tica de BDNF (p=0,03) no grupo vibra??o ap?s o per?odo de interven??o. A varia??o (?) na raz?o testosterona/cortisol (T/C) n?o diferiu significativamente, entre os grupos (p=0,61). No estudo 3, os n?veis de BDNF no l?quido sinovial correlacionou-se significativamente com dor autorrelatada [WOMAC] (rs = 0,39, p=0,04). Com rela??o aos receptores sol?veis para TNF-?, observou-se uma diferen?a entre os n?veis de sTNFR1 e de sTNFR2, tanto no plasma quanto no l?quido sinovial em pacientes com OA do joelho (1091 ? 99,48 pg / mL versus 2249 ? 126,3 pg / mL e 2587 ? 66,12 pg / mL versus 2021 ? 107,0 pg / mL, respectivamente). Al?m disso, os n?veis de sTNFR1 no l?quido sinovial foram, negativamente, correlacionadas com a dor e a fun??o f?sica autorrelatada (rs -0,6785, p<0,0001 e rs -0,4194; p=0,03, respectivamente). Ao passo que, os n?veis de sTNFR2 no l?quido sinovial foram negativamente correlacionadas com dor e rigidez articular (rs -0,5433, p=0,01 e rs -0,4249; p=0,02, respectivamente). Conclus?es: Os resultados dos estudos supracitados indicam que a adi??o da vibra??o de todo o corpo ao treinamento com exerc?cio de agachamento, nas condi??es experimentais propostas, melhora o equil?brio est?tico e din?mico e o desempenho da marcha e ao mesmo tempo reduz a autopercep??o de dor e a concentra??o de marcadores inflamat?rios (sTNFR1 e sTNFR2) em idosos com OA de joelho na fase de remiss?o da doen?a. Al?m disso, a associa??o da vibra??o de todo o corpo ao exerc?cio de agachamento promove uma melhora na for?a muscular de membros inferiores em mulheres idosas com OA de joelho na fase de remiss?o da doen?a e proporciona uma resposta adaptativa na concentra??o de BDNF sem altera??o na rela??o muscular de anabolismo/catabolismo. J? os resultados da rela??o entre sist?mico e local, indicam que os n?veis de BDNF sist?micos est?o associados com o mecanismo da dor articular na OA de joelho. Com rela??o aos receptores sol?veis de TNF-?, evidenciou-se a presen?a de receptores sol?veis para TNF-? no l?quido sinovial de pacientes com OA prim?ria de joelho e a rela??o destes receptores com par?metros cl?nicos.
Tese (Doutorado) ? Programa Multic?ntrico de P?s-Gradua??o em Ci?ncias Fisiol?gicas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2013.
ABSTRACT Introduction: Recently, whole body vibration (WBV) has been an alternative method of exercise that has been indicated to improve the physical performance of the elderly with osteoarthritis (OA) knee. However, the mechanisms related to the effects produced by this training mode have not been fully elucidated in the literature. Objectives: 1) To investigate the effects of the adittion of whole-body vibration to squat exercises on the plasma concentration of inflammatory markers and the functional performance of elderly individuals with knee OA remission phase (Study 1) and investigate the effects of WBV in addition to squat exercise training in elderly women with knee OA remission phase on the following parameters: 1) isometric strength of the quadriceps muscle; 2) BDNF plasma concentration; and 3) the testosterone/cortisol salivary concentration ratio (Study 2). 3). To analyze the concentrations of TNF-?, soluble receptors (sTNFR1 and sTNFR2) and BDNF in both plasma and synovial fluid of patients with inflammatory acute phase primary knee osteoarthritis during inflammatory acute phase, and to determine the possible correlations of plasma and synovial fluid TNF-?, soluble receptors (sTNFR1 and sTNFR2) and BDNF with the radiographic grading of knee OA and with self-reported pain, stiffness and physical function (Study 3). Methods: In study 1 thirty-two elderly subjects with knee osteoarthritis were divided into three groups [i.e., squat exercises on a vibratory platform (platform group N= 11), squat exercises without vibration (squat group N= 10) and the control group (N=11)]. The structured program of squat exercises in the platform and squat groups was conducted three times per week, on alternate days, for twelve weeks. The plasma concentration of TNF-? soluble receptors (sTNFR1 and sTNFR2) were analyzed using ELISA. The Western Ontario and McMaster University Osteoarthritis Index (WOMAC) questionnaire were used to evaluate self-reported physical function, pain and stiffness. The 6-minute walk test, the Berg balance scale, and gait speed were used to evaluate physical function. In study 2 the eligible patients were fifteen (15) elderly women ? 60 years of age who had been diagnosed with OA in at least one knee. The intervention consisted of uninterrupted squatting exercises for twelve weeks, 3x/week. The exercise protocol was similar in both groups differing only in the presence of vibration. In study 3 samples of plasma taken from the peripheral blood and of synovial fluid taken from the knee of patients with osteoarthritis (OA) were collected (n=27). Anteroposterior knee radiographs were taken to determine disease severity in the affected knee. Radiographic grading of OA in the knee was performed using the Kellgren-Lawrence criteria. Furthermore, plasma was collected from the peripheral blood of 19 healthy individuals, with no radiographic change in the hips and knees. The Western Ontario and McMaster University Osteoarthritis Index (WOMAC) questionnaire was used to evaluate self-reported physical function, pain and stiffness. ELISA measured the TNF-?, sTNFR1, sTNFR2 and BDNF levels in the plasma and synovial fluid. Results: In the study 1 the group that performed squat exercises on a vibratory platform, there were significant differences in plasma concentrations of the inflammatory markers sTNFR1 and sTNFR2 (p<0.001 and p<0.05, respectively), self-reported pain (p<0.05), balance (p<0.05) and speed and distance walked (p<0.05 and p<0.001, respectively) compared with the control group. The gait speed test also showed significant differences between the squat and platform groups (p<0.01). In the results of the study 2, the VG group demonstrated a significantly greater change (?) in IQMS values (p = 0.02) and in BDNF plasma concentrations (p = 0.03) after the intervention period compared with the EG group. The change (?) in T/C ratio showed no difference between the groups (p = 0.61). In the results of the study 3, the plasma BDNF levels significantly correlated with self-reported pain [WOMAC] (rs=0.39, p=0.04). According to soluble receptors to TNF-?, there was a difference between sTNFR1 and sTNFR2 levels in plasma as well as in synovial fluid in patients with knee (1091 ? 99,48 pg / mL versus 2249 ? 126,3 pg / mL e 2587 ? 66,12 pg / mL versus 2021 ? 107,0 pg / mL, respectively). Synovial fluid sTNFR1 levels were negatively correlated with pain and physical function self-reported (rs-0.6785, p<0.0001 and rs-0.4194, p=0.03, respectively). Synovial fluid sTNFR2 levels were negatively correlated with pain and joint stiffness (rs-0.5433, p=0.01 and rs-0.4249, p=0.02, respectively). Conclusions: The results of the above studies indicate that the addition of vibration training the whole body to squat exercise in the experimental conditions resulted in improvement in static and dynamic balance and gait performance and reduced the self-perception of pain and the concentration of inflammatory markers (sTNFR1 and sTNFR2) in elderly patients with knee OA. We also demonstrate that the combination of vibration training the whole body to squat exercise can promote an improvement in lower limb muscle strength in elderly women with knee OA and still provide an adaptive response to the concentration of BDNF compared with no change in muscle anabolism/catabolism. The results of the relationship between systemic and local concentration indicate that the systemic BDNF levels are associated with the mechanism of joint pain in knee OA. With respect to TNF-? soluble receptors, the findings demonstrated the presence of soluble receptors for TNF-alpha, particularly sTNFR1, in the synovial fluid of patients with primary knee OA and the relationship between these receptors and clinical parameters.

The presence of membrane microdomains or rafts within cell membranes is an area of intensive research and debate, with the notion of intracellular membrane rafts proving particularly contentious. The full extent of their biological importance is unknown but, they do appear to have vital roles in key processes such as membrane transport and cell signalling. They have been shown to have significant involvement in the formation of the immunological synapse and subsequent immune cell activation. Their cellular functions are also hijacked by various pathogens such as the human immunodeficiency virus and prion diseases, establishing them as important pharmaceutical targets.

As síndromes talassêmicas (?- e ?-talassemia) são as desordens mais comuns e frequentes associadas com eritropoese ineficaz. O desbalanço na produção das cadeias ?- e ?-globinas resulta no comprometimento da produção de eritrócitos, em anemia e aumento de progenitores eritroides no sangue periférico. Enquanto os pacientes homozigóticos afetados por essas desordens demonstram alterações características dos parâmetros relacionados a eritropoese, a relação entre grau de anemia, eritropoese alterada e disfunção do metabolismo de ferro ainda não foram investigados nos indivíduos com ?+-talassemia heterozigótica ou ?+-talassêmia. Duzentos e vinte seis indivíduos (75 do gênero feminino e 151 do gênero masculino) foram recrutados e divididos em 5 grupos: Controle (n=28), doadores de sangue regulares (DSR, n=23), ?+-talassemia heterozigótica (TAT, n=14), ?+-thalassemia (traço ?-talassêmico, TBT, n=20) e ?0-talassemia, (?-talassemia maior, BTM, n=27). As amostras foram analisadas para parâmetros hematológicos (Micros ABX 60); ferro sérico, capacidade total de ligação ao ferro e saturação de transferrina por método colorimétrico (Pointe Scientific, Inc., Canton, MI, USA), ferritina e proteína C-reativa ultra sensível por imunoensaio (Immulite 1000); receptor solúvel de transferrina, eritropoetina, fator de diferenciação do crescimento 15 (R&D Systems) e hepcidina (Intrinsic LifeSciences, La Jolla, CA) por ELISA. As razões sTfR/log ferritina e (hepcidina/ferritina)/sTfR foram calculadas para avaliar o metabolismo do ferro. sTfR/log ferritina pode distinguir depleção dos estoques de ferro de eritropoese deficiente de ferro, enquanto (hepcidina/ferritina)/sTfR pode avaliar os estímulos contrários (disponibilidade de ferro e atividade eritropoética) que controlam a síntese de hepcidina e a absorção de ferro, na ausência de estímulos inflamatórios. Foi demonstrado que TAT teve significativa redução da hepcidina e aumento do receptor solúvel de transferrina, com parâmetros hematológicos relativamente normais. Em contraste, todos os parâmetros hematológicos de TBT foram significativamente diferentes do Controle, incluindo aumento dos níveis do receptor solúvel de transferrina, ferritina, eritropoetina e fator de diferenciação do crescimento 15. Essas alterações em ambos os grupos sugerem um balanço alterado entre eritropoese e metabolismo de ferro. Os índices sTfR/log ferritina e (hepcidina/ferritina)/sTfR estão, respectivamente, aumentado e reduzido comparados ao Controle, proporcional a severidade de cada grupo talassêmico. Em conclusão, destacamos que, pela primeira vez, foram descritas alterações no metabolismo de ferro em indivíduos com ?+-talassemia heterozigótica. Esses dados demonstram que, no contexto da saúde pública, são necessários identificação e acompanhamento dos portadores de ?+-talassemia.
The thalassemia syndromes (?- and ?-thalassemia) are the most common and frequent disorders associated with ineffective erythropoiesis. Imbalance of ?- or ?-globin chain production results in impaired red blood cell synthesis, anemia and more erythroid progenitors in the blood stream. While patients affected by these disorders show definitive altered parameters related to erythropoiesis, the relationship between the degree of anemia, altered erythropoiesis and dysfunctional iron metabolism have not been investigated in both carriers of ?-thalassemia and ?-thalassemia. 226 subjects (75 females and 151 males) were recruited to this study and divided in 5 groups: Control (n=28), repeat blood donors (DSR, n=23), ?+-thalassemia heterozygous carriers (TAT, n=14), ?+-thalassemia (?-thalassemia trait, TBT, n=20) and ?0-thalassemia, (?-thalassemia major, BTM, n=27). Samples were tested for hematological parameters (Micros ABX 60); serum iron, total iron binding capacity, and transferrin saturation by the colorimetric method (Pointe Scientific, Inc., Canton, MI, USA), ferritin and high sensitive C-reactive protein by immunoassay (Immulite 1000); soluble transferrin receptor, erythropoietin and growth differentiation factor 15 (R&D Systems) and hepcidin (Intrinsic LifeSciences, La Jolla, CA) by ELISA. Were calculated the ratios sTfR/log ferritin and (hepcidin/ferritin)/sTfR to evaluate iron metabolism. sTfR/log ferritin can distinguish storage iron depletion from iron-deficient erythropoiesis, while (hepcidin/ferritin)/sTfR can be utilized to explore and quantify the opposing forces (i.e. iron availability and erythropoietic activity) regulating hepcidin synthesis and iron absorption in absence of inflammatory stimuli. We demonstrate that TAT have a significantly reduced hepcidin and increased soluble transferrin receptor levels but relatively normal hematological findings. In contrast, TBT have all hematological parameters significantly different from controls, including increased soluble transferrin receptor, ferritin, erythropoietin and growth differentiation factor 15 levels. These changings in both groups suggest an altered balance between erythropoiesis and iron metabolism. The indexes sTfR/log ferritin and (hepcidin/ferritin)/sTfR are respectively increased and reduced relative to controls, proportional to the severity of each thalassemia group. In conclusion, we emphasize that, for the first time in the literature, subjects with heterozygous ?+-thalassemia have altered iron metabolism. Our data demonstrate that within the context of public health, identification and monitoring of patients with ?+-thalassemia are needed.

Objective: Bone invasion represent significant problem in managing head and neck cancers, however the mechanisms of interactions between oral squamous cell carcinoma (OSCC) and bone cells are poorly understood. We hypothesized that tumor cells can directly stimulate osteoclastogenesis. Methods: OSCC cell lines, bone-invasive BHY and metastatic but not bone-invasive HN were cultured and conditioned medium (CM) was collected. Osteoclast formation from RAW 264.7 mouse monocytic cell-line was assessed. Results: When RAW 264.7 were primed with receptor activator of nuclear factor κB ligand (RANKL) and then treated with BHY-CM, marked 2-6 fold induction of osteoclastogenesis was observed. In contrast, HN-CM did not significantly affect osteoclastogenesis. In addition, BHY-CM, but not HN-CM promoted survival of mature osteoclasts. Using pharmacological inhibitors, we found that Protein kinase C (PKC)/Extracellular signal-regulated kinase (ERK)1/2/Mitogen activated protein kinase (MAPK) p38 as well as Phosphatidyl-inositol 3-kinases (PI3K)/Serine/threonine protein kinase Akt/Mammalian target of rapamycin (mTOR) pathways mediate BHY-CM induced osteoclastogenesis. Conclusion: OSCC-cells produce soluble factors that stimulate osteoclastogenesis from RANKL-primed precursors. Tumor-derived factors act by stimulating ERK1/2 and p38 MAPK pathways in osteoclast precursors.
Objectif: L'invasion du tissu osseux est un problème majeur dans le traitement des cancers de la tête et du cou, cependant les mécanismes d'interactions entre le carcinome de cellules de squamous oral (OSCC) et les cellules du tissu osseux sont mal compris. Nous avons posé comme hypothèse que les cellules tumorales peuvent stimuler directement le phénomène d'ostéoclastogenèse. Méthodes: Deux différentes populations cellulaires de la lignée OSCC furent utilisées: les cellules BHY ayant un potentiel de colonisation du tissu osseux et les cellules HN ayant un potentiel métastatique mais non colonisant. Ces deux lignées cellulaires ont été cultivées et le milieu de culture conditionné (CM) a été collecté. La formation de cellules ostéoclastiques à partir de cellules de la lignée monocytaire de souris RAW 264.7 a été évaluée. Résultats: Une augmentation significative du phénomène d'ostéoclastogenèse d'un facteur 2 à 6 fut observée lors d'une activation des cellules RAW 264.7 avec RANKL suivit d'un traitement avec BHY-CM. De plus, la survie des cellules ostéoclastiques matures était favorisée en présence de BHY-CM uniquement. L'utilisation d'inhibiteurs pharmacologiques nous a permis de mettre en évidence que la stimulation du phénomène d'ostéoclastogenèse induite par BHY-CM est médiée par les voies de signalisations PKC/ERK/p38 et PI3K/AKT/mTOR. Conclusion : Les cellules OSCC produisent des facteurs solubles stimulant la formation d'ostéoclastes à partir de précurseurs activées par RANKL. Les facteurs dérivés de tumeurs agissent en stimulant les voies de signalisation ERK1/2 et p38 dans les précurseurs ostéoclastiques.

Lymph nodes contain complex vascular networks composed of lymphatic and blood vessels. The blood vasculature contains specialised high endothelial venules functioning to permit the efficient entry of blood-borne lymphocytes into the node, while the lymphatics contain antigen-presenting cells draining from tissues. In contrast to the well understood cellular interactions and signalling mechanisms driving development of the stromal networks upon which immune cell interactions occur, the processes by which the complex vascular networks develop are poorly characterised. This study aimed to determine the mechanisms by which vascularisation of lymph node anlagen occurs during development. The structure of developing lymph nodes was studied using confocal microscopy to observe the organisation of basement membranes and the different cell types involved in the process. Expression of angiogenesis-related genes was studied using quantitative real-time PCR and microarrays. No evidence for vascularisation of the anlagen was found, though the markers used may not have stained nascent vessels. The lymph sac surrounding the anlagen was shown to exist as a two-layered structure composed of anastomosing blood and lymphatic endothelium. In vitro models of vasculogenesis and angiogenesis were developed utilising human umbilical vein endothelial cells in three dimensional collagen gels, with and without smooth muscle coverage. A spheroid-based model of angiogenesis was used to study the net angiogenic environment in developing E14.5–E17.5 anlagen, which determined that a net anti-angiogenic environment existed at all timepoints in the lymph nodes and thymus, but not skin. Additionally, tensional forces were observed to affect angiogenic sprouting in addition to soluble growth factors. As a consequence of the double-layered lymph sac observed in vitro, and the influence of anti-angiogenic factors and tensional forces observed ex vivo, a model of lymph node development involving anlagen patterning and vascularisation as a result of condensation-induced tensional forces was proposed, complementary to soluble growth factor-driven angiogenesis.

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina
Convergence of pathways in motoneuron (MN) injury include microglia in the initiation and progression of Amyotrophic Lateral Sclerosis (ALS). Neuroinflammation is a pathological hallmark of ALS and microglia may acquire neurotoxic or neuroprotective properties in response to misfolded superoxide dismutase-1 (SOD1) or other molecules produced by the injured MN. We assessed: (i) the role of microglia in preventing/restoring MN dysfunction using a mixed culture of NSC-34 MN-like cells (mutated in G93A) and of N9 microglia cells, added at 0 or 2 days-invitro(M0, M2) and cultured till 4 and 7 days-in-vitro; (ii) neurodegenerative network in organotypic cultures from lumbar segments of spinal cord (SC) obtained from the ALS mice model TgSOD1-G93A at 7 day-old and aged for 10 days-in-vitro, as well as the response to lipopolysaccharide (LPS, 1μg/mL) immunostimulation. Western blot assays for SOD1, high-mobility-group-box-protein-1 (HMGB1) and toll-like receptor-4 (TLR-4), and fluorimetric/colorimetric assays for ATP, glutamate and nitric oxide (NO), were used. Microglia (M0/M2) decreased the accumulation of human/mouse mutated SOD1 (P<0.01). In addition, elevation of glutamate efflux (P<0.01), and reduction of extracellular ATP (P<0.01), MMP-2(P<0.05) and MMP-9 (P<0.01) was observed by M2 at 7 days-in-vitro. Reduction of NO (P<0.05) and MMP-2 (P<0.01) was obtained with M0. HMGB1 increased by M0 and decreased by M2, suggesting HMGB1 release from the cell. Accumulation of SOD1 was verified in SC organotypic cultures, but no changes in ATP or NO were obtained, although a slight decrease in ATP by LPS was verified. Downregulation of TLR-4 by LPS may indicate the exhaustion of the inflammatory response mechanisms in the aged SC culture. Together, these results suggest that microglia by inhibiting MMP activation and HMGB1 cytoplasmic translocation in the ALS model are key in modulating MN degeneration and should be considered as therapeutic targets in ALS.
supported by FEDER (COMPETE Programme) and by National funds (Fundação para a Ciência e a Tecnologia – FCT, Portugal) with the projects PTDC/SAU-FAR/118787/2010 to D.B. and PEst-OE/SAU/UI4013/2011 and 2012 to iMed.UL

Legionella pneumophila (Lp) est une bactérie ubiquitaire dans les environnements aqueux et responsable d’une pneumopathie potentiellement sévère : la légionellose. La majorité des souches impliquées appartiennent au sérogroupe 1 (Lp1) et à un sous- groupe spécifique de souches portant un épitope particulier dites mAb3/1+. Cependant, la différence de distribution entre les souches retrouvées dans l’environnement et celles impliquées en clinique n’est pas clairement élucidée. Notre travail a porté sur la détection de deux facteurs de virulence de Lp. Nous avons voulu mettre en évidence l’existence de Phenols Soluble Modulines (PSMs), peptides uniquement décrit chez Staphylocoques et avons ainsi pu démontrer l’activité de peptides prédits par analyse in silico chez Lp capables d’activer la réponse inflammatoire par la voie du NF-?B et sont dotés d’une action cytotoxique. Notre deuxième axe d’étude a porté sur le lipopolysaccharide (LPS) de Lp. Afin de vérifier si la prédominance de certaines souches était liée à un biais diagnostique, nous avons voulu tout d’abord vérifier la sensibilité de 3 tests urinaires diagnostiques envers le LPS extrait de souches de différents sous- groupes de Lp1 et sérogroupes de Lp et avons ainsi pu montrer que ces tests sont capables de détecter tous les LPS de Lp1. La sensibilité envers le LPS des autres sérogroupes est très variable mais reste insuffisante pour permettre leur détection. Nous avons ensuite utilisé ces LPS extraits pour vérifier la réponse immunitaire innée en fonction des souches de Lp1. Ainsi les souches mAb3/1+ activent moins le système immunitaire que les souches mAb3/1-, ce qui pourrait expliquer alors une moins bonne clairance de ces souches permettant leur multiplication à l’origine d’une infection. Au final, notre travail a permis d’étudier deux facteurs de virulence potentiels au sein de Lp, pouvant expliquer partiellement la prédominance de certaines souches en pathologie humaine
Legionella pneumophila (Lp) is a ubiquitous intracellular bacterium found widely in the environment and is the cause of an opportunistic infection named legionellosis. The majority of the strains involved belong to serogroup 1 (Lp1) and to a specific subgroup named mAb3/1+, linked to a specific epitope expressed at the cell membrane. However the distribution difference between the strains found in the environment and the ones involved in pathology is not fully understood. We here studied two virulence factors of Lp. We first demonstrated the existence of Phenols Soluble Modulines (PSMs), smalls peptides that only have been described for Staphylococcus and found that the peptides that were predicted for Lp by in silico analysis were able to activate the innate immune response by NF-?B pathway and were able to have a cytotoxic activity. We also studied the lipopolysaccharide (LPS) of Lp. To found out if the predominance of some strains was linked to a diagnosis biais, we first evaluated the sensitivity of 3 urinary antigens tests against extracted LPS of strains belonging to all the sous-groups of Lp1 and serogroups of Lp. We then demonstrated that those tests are able to detect all LPS of Lp1, independently of mAb3/1 character. The sensitivities of the 3 tests were very variable for the other serogroups of Lp, but were too low to be able to detect those LPS in practice. We then used these extracted LPS to evaluate the innate immune response for different strains of Lp1. We demonstrated that mAb3/1- strains needed lower dose of LPS to activate the innate immune response than mAb3/1+ strains, which could be linked to a better clearance of the bacteria from the host, which doesn’t develop an infection. This work has studied two potentially virulent factors of Lp, which could partially explain the predominance of some strains of Lp in human pathology

Malignant epithelium and associated stromal cells secrete soluble factors which may influence tumour evasion of host immunity. The effect of these factors on the proliferation and function of individual immune cell populations has been investigated along with the role of hypoxia. The conditioned medium (CM) from four HNSCC cell lines, primary-derived fibroblasts, cultured in both normoxic and hypoxic conditions, and overnight-dispersed tumour CM was collected. Cytokine profiles were determined using a Quantibody cytokine array™ and ELISA. The CM was added to Tregulatory cells (CD4⁺CD25⁺CD127ˡᵒ), Teffector cells (CD4⁺CD25⁻) and cytotoxic T cells (CD8⁺) isolated from healthy donors and HNSCC patients. MTS assays and flow cytometry were used to assess changes in proliferation and percentage of immune cells. CFSE suppression assays, ELISA and flow cytometry were undertaken to measure changes in function of Tregulatory cells, Teffectors cells and CD8⁺ T cells obtained from healthy PBMC. A significant increase in the proliferation of whole PBMC from patient and healthy donors was observed upon the addition all HNSCC-derived CM, with healthy PBMC proliferating to an even greater extent compared with the patient samples. Tregulatory and Teffector cell percentages within healthy PBMC increased while CD8⁺ T cell percentage decreased in many cases. Also, HNSCC-derived CM was able to increase the suppressive activity of Tregs in 40% of samples. The CM caused an increase in Th1 type cytokines IFN-γ and IL-2 in at least 50% of samples with little change to Th2 cytokine levels and was also able to significantly reduce the function of CD8⁺ T cells in at least 50% of samples. In conclusion, the secretome of HNSCC epithelial cells, primary-derived fibroblasts and overnight dispersed tumour has the ability to alter the proliferation and function of individual sets of immune cells, potentially to a greater extent in the presence of other cell types.

Le but de ce travail a ete de caracteriser les fonctions d'un variant tronque, soluble, du facteur tissulaire. Un systeme bacterien a ete utilise pour l'expression a grande echelle de la proteine. Nous avons ensuite etudie le mecanisme d'inhibition du complexe enzymatique facteur tissulaire-facteur viia par des anticorps monoclonaux unusuels, dont la propriete est d'inhiber fortement, soit le facteur tissulaire, soit le facteur viia. Dans la seconde partie du travail, il s'agissait de comprendre le mecanisme cinetique de la reaction d'activation du fvii (ou autoactivation) mediee par le facteur tissulaire. De plus, nous avons recherche les causes de la deficience biochimique du facteur tissulaire soluble. Les resultats montrent que la forme soluble du facteur tissulaire est capable de promouvoir les reactions d'activation des facteurs vii, ix et x, a condition que le complexe soluble soit associe a la membrane

Tumour infiltrating lymphocytes (TIL) in head neck squamous cell carcinoma (HNSCC) are enriched in Treg, a finding confirmed in the current study, which is thought to be a contributing factor to immunosuppression and tumour evasion. Soluble factors released by malignant HNSCC cells may contribute to the enrichment of Treg by inducing differentiation from naïve T cells and their migration to the tumour. Using multi-colour flow cytometry, increases in CD4+CD25hi, CD4+CD25+CD39+, CD4+CD25+CD26− and CD4+CD25+FoxP3+ Treg phenotypes were observed in TIL from HNSCC compared to PBMC. Following culture of CD4+CD25− T cells with conditioned medium (CM) collected from dispersed tumour samples, there was an increase in CD39 expression but not FoxP3 compared to control cultures that were cultured in complete growth medium. Furthermore these cells were unable to suppress the proliferation of CD4+CD25− T cells in CFSE assays. Soluble factors in CM from UMSCC cell lines and tumour-derived fibroblasts were unable to induce the expression of any Treg markers following culture with CD4+CD25− T cells. Culture with CM also had no effect on T cell apoptosis, with no significant increase in PI-AnexinV+ of CaspACE-binding in T cells cultured with CM compared to controls. The expression of the chemokine receptors, CXCR3, CCR4, CCR5 and CCR6 was analysed on T cell populations from HNSCC PBMC and TIL and healthy control PBMC using five-colour flow cytometry. Increased proportions of CXCR3+ and CCR5+ T cells were observed in HNSCC TIL compared to HNSCC PBMC but were no different between the patient and healthy control PBMC. CCR4 and CCR6 were expressed on a higher proportion of Treg from HNSCC PBMC compared to healthy controls but no difference was observed on CTL. In TIL the percentage expression of CCR4 and CCR6 were no different in that of HNSCC patient PBMC. Despite these observed differences in receptor expression, soluble factors in tumour dispersed CM was unable to induce T cell chemotaxis. Overall, although limited effects were observed from soluble factors in tumour CM, the different expression of chemokine receptors suggests there may be a role for soluble factors in Treg recruitment. However whether this is responsible for Treg accumulation or general to all T cells is unclear.

El sistema de complemento es parte fundamental de la inmunidad innata, actúa reconociendo patrones moleculares asociados a microorganismos y patógenos, provenientes de ambiente extracelular. Su función principal es la eliminación de dichos patógenos, pero además contribuye al mantenimiento de la homeostasis y a la prevención de la autoinmunidad. Los inhibidores solubles C4BP y Factor H son componentes importantes del sistema del complemento, coordinando su actividad de tal manera que limitan su acción impidiendo respuestas exacerbadas e inespecíficas que pueden resultar nocivas para el organismo. Por su parte C4BP, actúa en la vía clásica y de las lectinas, presente en el plasma sanguíneo, con una conformación polimérica, y dos subunidades diferentes, la isoforma mayoritaria α7β1, formada por siete cadenas idénticas α y una cadena β, y otra isoforma minoritaria α7β0, incrementada en estados de elevada inflamación (fase aguda). Por otro lado, FH es un regulador esencial de la vía alternativa del sistema complemento, también presente en el plasma sanguíneo y esencial para mantener el equilibrio de esta vía. FH es una glicoproteína compuesta por 20 dominios homólogos, llamados dominios CCP, y participa como cofactor de la proteína C3. Otro componente de la inmunidad innata son las células dendríticas, las cuales juegan un papel fundamental en la respuesta inmune y son nexo de unión entre la inmunidad innata y la adaptativa. En el presente estudio se abordó la caracterización funcional y molecular de C4BP β- y FH sobre células dendríticas derivadas de monocitos (MoDCs). Dichas MoDCs fueron obtenidas a partir de células mononucleares humanas de sangre periférica (PBMCs) bajo estimulación con interleuquina 4 (IL-4) y Factor estimulante de colonias de granulocitos y macrófagos (GM-CSF), y maduradas con LPS. Mediante ensayos de citometría de flujo, para el análisis fenotípico de estadíos de diferenciación y maduración de MoDCs, se pudo estudiar en profundidad la actividad tolerogénica de C4BP β- y/o FH sobre MoDCs, la viabilidad y la capacidad endocítica de las mismas. Dicho estado tolerogénico de MoDCs se caracterizó por un claro perfil anti- inflamatorio y una alteración en la expresión de las moléculas co-estimuladoras, indispensables para la activación de células T. De igual modo se estableció un modelo murino de enfermedad del xeno-injerto contra el huésped (xeno-GvHD), utilizando ratones inmunodeficientes NSG (NOD scid gamma), con el objetivo de evaluar si las MoDCs convertidas en tolerogénicas con las potenciales proteínas inmunomoduladoras caracterizadas en este trabajo C4BP β- y/o FH tenían la capacidad de retrasar o mitigar la enfermedad xeno-GvHD. Así, se observó si se prolongaba la supervivencia al realizar un trasplante de éstas a animales previamente xenoinjertados con PBMCs humanas. Además, se realizaron otros ensayos funcionales que indican un claro papel inmunomodulador C4BP β- y FH. Posteriormente, se inició un análisis de expresión a nivel de genoma humano completo con el objetivo de establecer las bases moleculares de dicha inmunomodulación. Como consecuencia, los resultados obtenidos a partir de la técnica de microarrays han permitido realizar predicciones sobre las probables vías de señalización que pueden estar involucradas en la respuesta anti-inflamatoria e inmunomoduladora dependiente de estas proteínas. Aunque no se ha podido completar aún mediante análisis funcionales la implicación de las vías de señalización predichas, la información obtenida será de gran utilidad para futuros diseños experimentales que permitan dilucidar los receptores celulares y vías de señalización sobre los cuales actúan C4BP β- y FH, y para la caracterización de nuevos fármacos anti-inflamatorios e inmunomoduladores que actúen como agonistas de los receptores moleculares de C4BP β- y/o FH.
The C4BP and Factor H (FH) complement soluble inhibitors are pivotal when this immune response is activated given their role to control and limiting the complement activity, thus protecting from unspecific recognizing potentially harmful for human health. In addition to the complement system, the dendritic cells also play a relevant role in the response of the innate immunity. They are considered as the major joint between the adaptive and innate immunities. This Ph.D. thesis aimed the functional and molecular characterization of C4BP and FH regarding their role on differentiation and maturation of monocyte-derived dendritic cells (MoDCs). The MoDCs were obtained from human peripheral blood mononuclear cells (PBMCs) maturated with bacterial lipopolysaccharide (LPS) prior differentiation with interleukin-4 (IL4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimuli. Monitoring of MoDCs differentiation and maturation was done by flow cytometer, an approach that was essential to assess the impact of FH and C4BP on dendritic cells and the phenotype resulting. The tolerogenic status triggered by FH and C4BP on dendritic cells was characterized by an attenuated endocytic capacity, the reverting towards an anti-inflammatory cytokine profile, and the down-regulation of co-stimulating molecules indispensable for T cell activation. Furthermore, the profound effects of FH and C4BP on dendritic cells were tested in animal models using an animal model of the graft-versus-host disease (GvHD) based on NSG immunodeficient mice. It was possible to reproduce the GvHD in NSG animals using the xenograft based on estimulated and activated human dendritic cells, and simultaneously it was observed that tolerogenic dendritic cells treated with FH and C4BP, and injected in those mice as therapy, delayed the disease onset. Finally, a genome-wide expression analysis was performed in order to shed light on the molecular pathways activated as a consequence of the FH and C4BP immunomodulation. As a consequence, the results retrieved from the microarray approach have permitted to predict certain molecular pathways probable involved in the anti-inflammatory response of dendritic cells dependent of FH and C4BP. The information presented in this thesis will be useful to design future studies addressed to the characterization of the surface receptors, and signaling pathways, by which FH and C4BP induce tolerogenicity on dendritic cells as well as characterize potential agonist of such molecules for therapeutic aims.

This thesis proposes a mechanism for the induction of peripheral tolerance to protein antigens. I have investigated the mechanism of tolerance induction to soluble protein antigens by targeting an antigen to small, resting B cells. For this purpose I have used a rabbit antibody directed at the IgD molecule found on the surface of most small, resting B cells but missing or lowered on activated B cells. Intravenous injection of normal mice with 100 μg of an ultracentrifuged Fab fragment of rabbit anti-mouse IgD (Fab anti-δ) makes these mice profoundly tolerant to challenge with nonimmune rabbit Fab (Fab NRG) fragments. This tolerance is antigen specific since treated mice make normal responses to an irrelevant antigen, chicken immunoglobulin (Ig). Fab fragments of rabbit Ig (rabbit Fab) not targeted to B cells do not induce tolerance as well as Fab anti-δ. Evidence suggests that the B cells must remain in a resting state for tolerance to be induced, since injection of F(ab)'2 anti-δ does not induce tolerance. Investigation of the mechanisms of the tolerance, by adoptive transfer, have shown that rabbit Fab specific B cell function has been impaired. The major effect however is in helper T cell function, as shown by adoptive transfer and lack of help for a hapten response. In vitro proliferation experiments show that the T cell response has not been shifted toward activation of different T cell subsets which do not help Ig production, nor is there any change in the Ig isotypes produced. Suppression does not appear to be the major cause of the helper T cell defect as shown by cell mixing experiments. This work shows that an antigen targeted to small B cells can induce tolerance to a soluble protein antigen, and suggests a role for small B cells in tolerance to self-proteins not presented in the thymus.

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One third of all childhood cancer diagnoses are leukemias, making it is the most common cancer found in children. While overall survival for acute lymphoid leukemia (ALL) has improved greatly over the past 20 years, the survival rate for acute myeloid leukemia (AML) is still lagging behind. There are many biological classes of AML, several of which are largely resistant to intensive chemotherapy, necessitating the investigation of alternative treatment approaches. In particular, aberrant activation of the ERK signaling pathway has been associated with resistance to chemotherapy and decreased survival. The RAF kinases are central components of the ERK signaling pathway that are rarely mutated but often activated in AML. To understand the therapeutic potential of RAF kinase inhibition in AML, we investigated the susceptibility of AML cell lines to the novel pan-RAF kinase inhibitor AZ628. This work identified classes of RAF kinase inhibition sensitive and resistant cell lines, with the resistance associated with secretion of soluble factors that enhance AML cell growth and survival. This therapeutically relevant finding led to the development of a new approach for the analysis of secreted proteomes elaborated by AML cells, with the identification of candidate autocrine survival factors. This knowledge promises to lead to the identification of improved targets of therapy in order to improve the treatment of patients with AML.

Human main histocompatibility complex is encoded in the short arm of chromosome 6 and can be divided into three main regions based on the proteins that it encodes. We have class I, class III and class II. Class I encodes for proteins that are expressed on the cell surface of almost all somatic cells and is related to the presentation of self-antigens. Class I can be sub-divided into two groups known as classical class I (Ia) and non-classical class I (Ib), and expressed on the cell surface as human leukocyte antigen (HLA). HLA-G is one of the members of HLA-Ib and, together with HLA-E and HLA-F, is thought to play a key role in maternal tolerance to the semi-allogenic embryo, one haplotype comes from the mother, and is shared with her, while the other comes from the father. At the trophoblast stage, embryos do not express HLA-II and only express HLA-C (Ia member). Based on these facts, important questions about the role of these antigens during pregnancy have arisen. Our premise is that PreImplantation Factor (PIF), a 15 amino acid peptide secreted only by viable embryos, seems to plays a key role in this regulation. In this study we have used the JEG-3 cell line as a human trophoblastic model to study the effect of PIF on HLA-I expression. JEG-3 cells were incubated at different concentrations and time points of PIF. Using a wide variety of techniques, we could detect that PIF significantly induced HLA-I expression, mainly HLA-G and -E, increased their invasion, proliferation in vitro. Also, PIF modified protein profile, detected by 2D electrophoresis. Compared with the untreated cells, 14 proteins were over-expressed and 8 were under-expressed. Our study suggests that PIF, has a regulatory effect on HLA-I in this cellular model and the fact that not only HLA-G was over-expressed can suggest new regulations pathways under the control of HLA-E.