Cryprosporidium parvum is a coccidian protozoan that colonizes epithelial cells lining respiratory and digestive tracts of animals and humans. Cryptosporidiosis is a well-recognized zoonotic disease infecting primarily neonates and immunocompromised hosts, including human immunodeficiency virus-infected patients. Clinical disease is manifested as a chronic diarrheal illness that is self-limiting in immunocompetent hosts and prolonged and often life-threatening in hosts with compromised immune systems.The lack of a suitable small animal model for screening anti-cryptosporidial drugs and for examining the pathogenicity and immunobiology of chronic cryptosporidiosis was the impetus for this research effort. The objectives of the present study were three-fold: to characterize chronic Cryptosporidium parvum infections in dexamethasone-immunosuppressed mice; evaluate the effects of Cryprosporidium parvum and dexamethasone on B and T lymphocyte proliferation; and determine the effects of the immunomodulator dehydroepiandrosterone on oocyst shedding intensities of mice infected with Cryptosporidium parvum. Adult C57BL/6N mice were immunosuppressed with the synthetic glucocorticoid dexamethasone, then infected with Cryprosporidium parvum (106 oocysts/mouse) investigated for their ability to sustain a four-month chronic infection. Dexamethasone was administered intraperitoneally (125 Jlg/mouse/day) or orally (8 Jlg/ml) in the drinking water ad libitum. Infection chronicity was characterized by evaluating mouse monality, oocyst excretion in the feces, tissue distribution of the parasite, and parasite-induced pathology. A progressive infection with Cryptosporidium parvum occurred in mice immunosuppressed intraperitoneally and orally as long as dexameth sone was administered. Mice receiving dexamethasone given intraperitoneally had a shoner prepatent period and a more consistent, although cyclic, oocyst shedding pattern when compared with mice given dexamethasone orally. Mice given dexamethasone orally exhibited a delayed prepatent period, with a steady increase in oocyst shedding. All mice receiving dexamethasone orally died within three months following oocyst inoculation. Clinical signs included dehydration, icterus, and reduction in spleen and body weights. Clinical signs were more abrupt in mice receiving oral dexamethasone. Parasite colonization involved the entire intestinal tract, including the pyloric ring and Peyer's patches, but was the heaviest in the terminal ileum. Parasites were present in the lungs, gallbladder, and pancreatic ducts. Pathologic abnormalities were isolated to the terminal small intestine and included blunting and fusion of intestinal villi and crypt hyperplasia. Cryptosporidium parvum and dexamethasone administered in vivo reduced B and T lymphocyte responses to the mitogens lipopolysaccharide and concanavalin A. Dehydroepiandrosterone and dehydroepiandrosterone-sulfate resulted in no significant reductions in cryptosporidial activity as determined by oocyst shedding in the feces.

According to the cancer stem cell hypothesis a subpopulation of cells within a tumour has the capacity to sustain its growth. These cells are termed cancer stem cells, and are most simply defined as the cells within a primary tumour that can self-renew, differentiate and regenerate a phenocopy of that cancer when transplanted in vivo. Cancer stem cells have now been prospectively identified from numerous human tumours and are actively sought in many cancer types, both clinical and experimental. The cancer stem cell hypothesis remains controversial, with evidence both supporting and challenging its existence in human tumours and in animal models of disease. Here we prospectively identify and study brain cancer stem cells in clinically representative mouse models of the medulloblastoma and glioma. Cancer stem cells from both mouse brain tumour types are prospectively enriched by fluorescent activated cell sorting freshly dissociated cells for the surface antigen CD15, display a neural precursor phenotype, exhibit the hallmark stem cell characteristics of self-renewal and multilineage differentiation, and regenerate a phenocopy of the original tumour after orthotopic transplantation. Additionally, novel mouse medulloblastoma and glioma cancer stem cell lines were established and studied in vitro as adherent cultures in the same serum-free media conditions that support the growth of normal neural stem cells. When mouse and human glioma stem cell lines were compared, many novel molecular mediators of the tumour phenotype were identified, as were chemical compounds that selectively inhibit their growth. Our results have important implications regarding the cancer stem cell hypothesis, the mechanisms that drive brain tumour stem cell growth and the therapeutic strategies that may prove effective for the treatment of glioma and medulloblastoma.

Increasing evidence suggests that normal pulmonary vascular morphogenesis is critical for the formation and maintenance of the lung parenchyma, both pre- and postnatally. Indeed, the disruption of angiogenic pathways, whether through inherent genetic predisposition or as a consequence of life-saving interventions, may underlie many pulmonary diseases of infancy, including alveolar capillary dysplasia (ACD) and bronchopulmonary dysplasia (BPD). To understand the etiology of – and advance treatments for – such diseases, we must first identify the fundamental genetic regulators that orchestrate normal parenchymal development. Neuropilin-1 (NRP1) is a transmembrane receptor that plays essential roles in normal and pathological vascular development, and binds two distinct ligand families: Vascular endothelial growth factor (VEGF) and Class 3 Semaphorins (SEMA3). Although VEGF-NRP1 interactions in systemic vascular development have been described, the importance of SEMA3-NRP1 signalling in systemic or pulmonary vascular morphogenesis is uncertain. We hypothesized that SEMA3-NRP1 and VEGF-NRP1 interactions are fundamental pathways in the orchestration of pulmonary vascular development in both the embryo and neonate. Disruption of these pathways would therefore lead to significant interruption of normal angiogenic and vascular maturation processes that are relevant to the pathogenesis of pulmonary diseases. Using extensive histopathological analyses of NRP1 loss-of-function mice, we show evidence of a significant role for SEMA3-NRP1 signalling in fetal microvascular development: congenital loss of SEMA3-NRP1 signalling resulted in severely attenuated development of the distal vasculature and alveolar-capillary interface, leading to fatal respiratory distress at birth that is reminiscent of clinical ACD. By contrast, VEGF-NRP1 and SEMA3-NRP1 signalling appear unessential for normal alveolar and vascular development in the postnatal period, per se, despite increased mortality. Our results demonstrate the critical involvement of SEMA3-NRP1 signalling in endothelial development and substantiate the idea that NRP1 mediates opposing and cooperative functions between SEMA3 and VEGF ligands.

Hepatitis C Virus (HCV) is one of the most important causes of chronic liver disease, affecting more than 170 million people worldwide. The mechanisms of hepatitis C pathogenesis are unknown. Viral cytotoxicity and immune mediated mechanisms might play an important role in its pathogenesis. HCV infection and alcohol abuse frequently coexist and together lead to more rapid progression of liver disease, increasing the incidence and prevalence of cirrhosis and hepatocellular carcinoma. The cytopathic effect of HCV proteins, especially the core, E1 and E2 structural proteins, which induce liver steatosis, oxidative stress and cell transformation may be amplified by alcohol abuse. The purpose of this study was to characterize the liver damage mechanisms induced by HCV structural proteins and alcohol and to determine the potential molecular mechanism(s) that may promote chronic, progressive liver damage. A transgenic mouse model expressing HCV core, E1 and E2 was used to investigate whether alcohol increased HCV RNA expression. Real-time RT-PCR analysis of genes involved in lipid metabolism and transport confirmed their abnormal expression in the alcohol-fed transgenic mice. In addition, light and electron microscopy analysis were performed on liver tissues of transgenic mice on an alcoholic diet versus those on a normal diet, in order to identify histological changes. The severe hepatopathy in HCV transgenic mice was exacerbated by alcohol. Mitochondria and endoplasmic reticulum had severe abnormalities in the electron microscopy analysis. The second part of this study focused on adaptive immune responses, which may also play an important role in HCV pathogenesis. I focused my analysis on dendritic cells (DC), which have been the main suspects to explain immune impairment in HCV infection. Their powerful antigen-presenting function allows them to stimulate the antiviral response of CD4+ and CD8+ T cells, the effector cells of the immune system. This unique function of the DC makes them possible targets for immune evasion by the Hepatitis C virus. In this study, DCs were generated from mouse bone marrow cells. I investigated their maturation capacity in the presence of structural proteins of HCV. The impact of HCV core/E1/E2 polyprotein on DCs cytokine expression and ability to activate T-cell lymphocytes was also analyzed. A dysfunctional CD4 T cell response was observed after exposure of DCs to core/E1/E2 polyprotein, indicating inefficient CD4 priming, which might lead to chronic HCV infection in humans. The presence of the core/E1/E2 polyprotein reduced the DC maturation capacity and the expression of certain cytokines (IL-12, IFNg, IL-6, MCP-1) important for stimulation and chemotaxis of T cells and other immune cells. My studies contribute to the understanding of HCV pathogenesis and may have implications to the development of better therapies for HCV infection.

Made available in DSpace on 2014-06-11T19:32:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-12-20Bitstream added on 2014-06-13T20:24:19Z : No. of bitstreams: 1 marques_sbs_dr_jabo_prot.pdf: 1156404 bytes, checksum: 4759c4421e1acf181f52d3ac10abd71c (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A aderência de bactérias patogênicas a receptores na superfície de . células epiteliais tem sido reconhecida como um importante evento inicial da colonização bacteriana. O principal mecanismo de patogenicidade da EPEC é uma lesão provocada pela sua aderência ao epitélio através do attaching and effacing (AlE), que é caracterizado pela íntima adesão da bactéria à célula do hospedeiro, esta adesão às microvilosidades do enterócito ocorre devido a formação de estruturas semelhantes a pedestais. O objetivo deste estudo foi desenvolver um modelo de infecção com a Escherichía calí enteropatogenica (EPEC) em camundongo do tipo mus musculus. A estes animais foram administrados um inoculo de 108 CFU de linhagens EPEC, sendo que a linhagem 3111-90 foi proveniente de cepas que podem causar diarréia infantil e as linhagens 537-1, 263, 304-3 e 988-2 foram provenientes de leite mastítico bovino. As linhagens foram introduzidas em camundongos com vinte dias de vida por via intraperitonial, e estes foram eutanasiados cinco horas após a inoculação. Como resultado através de cortes histológicos seriados, verificou-se que algumas das linhagens inoculadas levaram ao aparecimento de lesões suaves na mucosa intestinal, com a quebra da camada celular superficial da mucosa, aparecimento de células epiteliais com contornos irregulares e infiltração de células inflamatórias. / The aim of this study was to develop a enteropathogenic Escherichia colí (EPEC) infection model in mice. An inoculum of 108 CFU of EPEC strains 3111-90 from infantile diarrhea; 537-1, 263,304-3 and 988-2 from bovine mastitic milk to newborn mice lead to a mildly damage in the intestinal mucosa with breaking in the superficial stratum, epithelial cells with irregular shape and a inflammatory cell infiltration.

The epigenetics of the aging brain is a growing field of study that holds great promise for the discovery of mechanisms and potential treatments for neurodegenerative diseases. In this current study, a novel, accelerated aging murine model, the I-PpoI/Cre, or ICE (Inducible Changes in the Epigenome) mouse, is studied to test its potential for demonstrating the theory of the rearrangement of chromatin (RCM) as the main cause of aging, and in particular, the mechanism through which the brain ages. Immunohistochemistry and behavioral assays are utilized to determine whether there are morphological changes, inflammatory response, and changes in learning and memory. Results showed a significant increase in microglia and astrocytes, markers of inflammation, in I-PpoI/Cre mice compared to their Cre controls. Long term memory performance was also significantly decreased in the I-PpoI/Cre mice, demonstrated through contextual fear conditioning (CFC) testing, and Morris Water Maze (MWM) testing. Results from this study are in support of the I-PpoI/Cre mouse as a model of accelerated aging of the brain, with deficits in learning and memory. Further studies are needed to further characterize this murine model of accelerated aging.

Unlike other regenerative organs such as the liver, the adult mammalian heart does not regenerate tissue lost following injury such as myocardial infarction (MI). Instead a non-contractile fibrous scar develops that in the longer term leads to the development of heart failure (HF). In contrast to the adult, neonatal mammals, including mice and man, retain potent cardiac regenerative capacities and can replace myocardium lost following injury. Understanding the mechanisms underlying scar free repair in the neonate may help in development of new approaches to reduce the impact of myocardial injury in adults. In this thesis MI was induced by coronary artery ligation in mice at post-natal day 1 (P1). Novel electrocardiogram gated high resolution cardiac ultrasound was developed to permit non-invasive confirmation of injury 1 day later and regeneration 21 days later by loss, then restoration, of contractile function. Macrophages (MФ) play important roles in organ growth and homeostasis, and are required for scar-free regeneration of the neonatal mouse heart following MI. WNTs are secreted lipophilic proteins with multiple roles in development. MФ-derived WNTs are essential for scar free tissue regeneration following injury in the kidney, liver, and gut, but their role in the heart is unknown. The primary aim of this thesis was to investigate the role of MФ, and in particular MФ-derived WNTs in determining normal growth of the myocardium from neonate to adult and also in regeneration of the neonatal heart following injury. In wild-type neonatal mouse hearts, Csf1r-expressing cells density (mostly macrophages) was consistent across all time points studied. Three populations of resident cardiac mononuclear phagocytes were identified by flow cytometry: F4/80hi, CD11blo, Ly6C-ve - F4/80lo, CD11bhi, Ly6C-ve - F4/80lo, CD11bhi, Ly6C+ve. F4/80hi, CD11blo, Ly6C-ve cells were hypothesised to correspond to yolk-sac derived mononuclear phagocytes and F4/80lo, CD11bhi, Ly6C-ve - F4/80lo, CD11bhi, Ly6C+ve to foetal liver/bone marrow derived mononuclear phagocytes. Three phases of myocardial growth were identified by ultrasound and histological techniques: hyperplastic (P2-P8, with increased Ki67 and cardiac troponin immunopositive cells), hypertrophic/reorganisation (P8-P21, with increasing cardiomyocyte size and no change in left ventricle wall thickness), and finally hypertrophic solely (P21-P42, with increasing cardiomyocyte size and left ventricle wall thickness). Average coronary vessel size was shown to decrease between P2 and P8 whilst vessel density was increased. The number of α-smooth muscle actin (αSMA) coated vessels greatly increased between P8 and P42, indicating vessel maturation. Throughout all phases cardiac systolic function was maintained at steady state. Diastolic function was however shown to mature from a foetal to an adult pattern between P2 and P8, with reversal of the E:A wave ratio on Doppler ultrasound. In mice globally deficient in MФ due to a germline knock-out of the Csf1r gene (Csf1rnull mice), both body and heart weights were decreased from P7 onwards. The number of proliferating (Ki67+ve) cardiomyocytes at P1 and P7 was unchanged in Csf1r-null mice but there was a trend towards decreased cardiomyocyte size at P7, suggesting an influence on hypertrophic rather than hyperplastic growth of the myocardium. There was also a trend for slowed vascular network maturation, with a delay in the shift from large to smaller vessels in hearts from Csf1r-null mice. In mice with MФ-directed (Csf1r-icre mediated) depletion of Porcupine (Porcn), a gene encoding an enzyme required for WNT acylation and secretion cardiac growth, vascularisation, fibrosis and function were all similar in Cre-ve and Cre+ve animals until P41, when cardiomyocyte size and cardiac systolic function were both significantly increased in Cre+ve animals. However, the underlying mechanism is unknown. In the neonatal mice, Csf1r expressing cells, mostly MФ, were identified in association with regenerating myocardium after induction of MI at P1. Flow cytometry data showed that by P7 the putative resident yolk-sac derived population had mostly disappeared from the heart and was replaced by F4/80lo cells, similar to the pattern reported in the adult. In the regenerating myocardium, Axin2 expression was increased consistent with activation of canonical Wnt signalling. Expression of Wnt5b and Fzd2 receptor, both associated with fibrosis, was significantly increased relative to age matched uninjured hearts. MФ-directed depletion of Porcn did not influence either the functional decrease at day 1 or recovery at day 21 following induction of MI at P1. Coronary re-vascularisation was also unaffected by the genotype. However, retention of intra-myocardial fibrosis (picrosirius red staining) was significantly increased in hearts at day 21 post-MI from mice with MФ-directed depletion of Porcn. MФ-derived WNTs are therefore required for scar-free wound healing in the heart, as they are in the liver and the kidney where they regulate matrix metalloproteinase activity. In summary, novel ECG-gated high-resolution in vivo ultrasound developed in this project has allowed characterisation of cardiac structure and function during early post-natal growth and following injury and regeneration in neonatal mice. The resident MФ population of the heart is established pre-natally, and may play a role in determining maturation of the developing vascular network, although this does not involve MФ-derived Wnt signalling. Following MI, the MФ population may expand from bone marrow cells and MФ accumulate around the regenerating tissue. MФ derived WNTs are not required for regeneration of the neonatal myocardium but do have a role in ensuring scar free wound healing and this merits further investigation.

Paediatric intestinal failure following bowel resection causes significant morbidity and mortality. There is a pressing need for improved treatment modalities. Following loss of bowel, the remaining intestine undergoes a period of adaptation, characterised by an increase in height of the intestinal villi. Better understanding the factors that govern the formation and growth of villi may lead to therapeutic interventions that amplify the intrinsic adaptation response. This thesis aims to explore the processes by which intestinal villi form during embryological development, the contribution of intestinal stem cells to this, and candidate signalling pathways that may yield insights into new therapeutic interventions for patients with intestinal failure. Abstract: Aim I will examine the morphogenesis and morphology of intestinal villi by investigating three themes: 1) Villus morphogenesis: When and where do villi form along the gut tube? Can this process be quantified, both in vivo and in vitro? Is this initiated by a dominolike signaling-cascade along the bowel, or location-specific intrinsic triggers? 2) Stem cells: What is the spatiotemporal appearance of the Lgr5-expressing intestinal stem cells during development? How does this relate to the process of villus morphogenesis? 3) Signalling pathways: Can a genetic mutation mouse model help elucidate pathways by which post bowel resection adaptation might occur? Can this be used to help identify potential intestinotrophic agents? Abstract: Materials and Methods Three mice models were used as the foundation for this work. Embryonic tissue was analysed from wild-type CD1 and Lgr5-eGFP-IRES-CreERT2 mice, and adult intestinal tissue examined from tamoxifen-activated Villin-Cre-ERT2 Pten-/- Brafv600E mice. Culture of wild-type embryonic mouse intestine with and without segments removed and / or reversed was performed to investigate the question of what triggers the proximal-to-distal wave of villus morphogenesis. Immunohistochemical interrogation using anti-GFP antibodies was used in the Lgr5- GFP mice to identify the location of Lgr5-expressing cells during the development of villi. Bright-field microscopy, time-lapse in-incubator microscopy, and histological sections assessed villus morphology. The Villin-Cre-ERT2 Pten-/- Brafv600E mouse mutant was explored regarding the intestinal epithelial morphometric changes that occur following tamoxifen-induction. Abstract: Results The proximal-to-distal wave of villus morphogenesis was observed both in vivo and in vitro. Villus morphogenesis commences at embryonic day 14.5 in vivo and after three days in culture from e11.5 in vitro. The villus structures formed in vitro are significantly attenuated compared to in vivo development. An attempt was made to overcome this by providing intestinal explants with a blood supply to aid growth. Evidence is presented that suggest the proximal-to-distal wave of villus morphogenesis is driven by location specific factors intrinsic to each part of the bowel, rather than a domino-like signalling cascade travelling along the intestine. Lgr5-expressing intestinal stem cells were present in early development. Prior to villus morphogenesis they were uniformly distributed along the luminal surface of the intestinal epithelia. During the intense proliferation associated with villus morphogenesis they progressively congregated to the inter-villus spaces. Once villi are fully formed they were absent from the villi but identified in the inter-villus spaces. The Pten/Braf mouse mutant demonstrates villus morphological changes similar to those found following post-bowel resection adaptation. This suggests that there may be a role for Pten/Braf in the epithelial proliferation following extensive bowel resection. Signalling factors in these pathways may be candidate intestinotrophic agents for the treatment of short bowel syndrome. Abstract: Conclusions Before any processes that manipulate intestinal epithelia can be safely translated into therapies to aid adaptation in patients with intestinal failure, it is important to have a full and detailed understanding of the basic science principles that underpin the behaviour of the epithelial cells, both during development and in adulthood. I have explored and quantified the process of villus morphogenesis in the embryonic mouse, investigated the timing of appearance of Lgr5 intestinal stem cells, and interrogated a genetic mouse model with morphometric changes similar to those seen following small bowel resection. I propose two candidate intestinotrophic agents that may hold regenerative potential to augment post small bowel resection adaptation. The next stage of investigation would be to use a mouse model of small bowel resection with manipulation of cell signalling factors to assess impact on post resection adaptation. The ultimate goal would be to investigate epithelial activity in human neonatal intestine and explore methods of modulating this to improve the outcomes from post bowel resection intestinal failure.

Synapses are an early pathological target in a wide range of neurodegenerative conditions including adult-onset Alzheimer’s and Parkinson’s, and diseases of childhood such as spinal muscular atrophy and neuronal ceroid lipofuscinoses (NCLs). However, our understanding of the mechanisms regulating the stability of synapses and their exceptional vulnerability to neurodegenerative stimuli remains in its infancy. To address this, we have used the NCLs to model the molecular alterations underpinning synaptic vulnerability. Our primary objective is to identify novel regulators of synaptic stability as well as highlight novel therapeutic targets which may prove effective across multiple neurodegenerative conditions where synapses are an early pathological target. The NCLs, are the most frequent autosomal-recessive disease of childhood. There are currently 14 individual genes whose mutations result in similar phenotypes including blindness, cognitive/motor deficits, seizures and premature death. This suggests that despite the difference in the initiating mutation and the degenerative processes across this collective group are likely to impact on overlapping pathways. Focusing on two murine models of NCL; one with an infantile onset - CLN1 disease (Ppt1-/-) and one with a juvenile onset - CLN3 disease (Cln3-/-) we made use of the temporo-spatial synaptic vulnerability pattern in these mice to plan proteomic and in silico analyses. This pipeline was utilised to identify perturbed protein candidates and pathways correlating with differential regional synaptic vulnerability. This ultimately allowed the generation of a list of candidate proteins, some of which were relevant to human NCL as they were altered in post mortem brain samples. Interestingly, many of the correlative candidates also appear to show conserved alterations in both NCL forms examined and other neurodegenerative diseases. Next, candidates were genetically and/or pharmacologically targeted to study their modulatory effects on neuronal stability in vivo. This was done using CLN3 Drosophila as a rapid screening assay and led to the successful characterisation of a subset of candidates as either enhancers or suppressors of the CLN3-induced phenotype in vivo. As well as identifying regulators of neuronal stability, following a similar pipeline, we identified a set of putative biomarkers of disease progression in muscle and blood in the Ppt1- /- mice, a subset of which appeared conserved in Cln3-/- mice. One of these conserved candidates presented the same directionality of change in human post mortem brain samples, indicating its relevance to the human NCL. Following this workflow from spatio-temporal profiling of murine synaptic populations, to in silico analyses and in vivo phenotypic assessment, we demonstrate that we can identify multiple protein candidates capable of modulating neuronal stability in vivo and identified putative biomarkers that tracked disease progression.

Hepatitis C Virus (HCV) is one of the most important causes of chronic liver disease, affecting more than 170 million people worldwide. The mechanisms of hepatitis C pathogenesis are unknown. Viral cytotoxicity and immune mediated mechanisms might play an important role in its pathogenesis. HCV infection and alcohol abuse frequently coexist and together lead to more rapid progression of liver disease, increasing the incidence and prevalence of cirrhosis and hepatocellular carcinoma. The cytopathic effect of HCV proteins, especially the core, E1 and E2 structural proteins, which induce liver steatosis, oxidative stress and cell transformation may be amplified by alcohol abuse. The purpose of this study was to characterize the liver damage mechanisms induced by HCV structural proteins and alcohol and to determine the potential molecular mechanism(s) that may promote chronic, progressive liver damage. A transgenic mouse model expressing HCV core, E1 and E2 was used to investigate whether alcohol increased HCV RNA expression. Real-time RT-PCR analysis of genes involved in lipid metabolism and transport confirmed their abnormal expression in the alcohol-fed transgenic mice. In addition, light and electron microscopy analysis were performed on liver tissues of transgenic mice on an alcoholic diet versus those on a normal diet, in order to identify histological changes. The severe hepatopathy in HCV transgenic mice was exacerbated by alcohol. Mitochondria and endoplasmic reticulum had severe abnormalities in the electron microscopy analysis. The second part of this study focused on adaptive immune responses, which may also play an important role in HCV pathogenesis. I focused my analysis on dendritic cells (DC), which have been the main suspects to explain immune impairment in HCV infection. Their powerful antigen-presenting function allows them to stimulate the antiviral response of CD4+ and CD8+ T cells, the effector cells of the immune system. This unique function of the DC makes them possible targets for immune evasion by the Hepatitis C virus. In this study, DCs were generated from mouse bone marrow cells. I investigated their maturation capacity in the presence of structural proteins of HCV. The impact of HCV core/E1/E2 polyprotein on DCs cytokine expression and ability to activate T-cell lymphocytes was also analyzed. A dysfunctional CD4 T cell response was observed after exposure of DCs to core/E1/E2 polyprotein, indicating inefficient CD4 priming, which might lead to chronic HCV infection in humans. The presence of the core/E1/E2 polyprotein reduced the DC maturation capacity and the expression of certain cytokines (IL-12, IFNg, IL-6, MCP-1) important for stimulation and chemotaxis of T cells and other immune cells. My studies contribute to the understanding of HCV pathogenesis and may have implications to the development of better therapies for HCV infection.