EXPERIMENTAL PATHOLOGY

Author: Jiménez-Prada de Miguel Marta.
Year: 2004.
University: VALLADOLID.
Place of defense: Facultad de Medicina.
Place of preparation: Facultad de Medicina.

Summary: In transplants, the body is subjected to graft inevitably to a period of ischemia. In the case of lung cancer, while lung cells are slow to die after the stop circulatory, is not determined accurately. Being so it is of interest to deepen the knowledge of the processes that occur during both ischemia and during reperfusion secuente to restoring circulation. To investigate these processes, it requires the standardization of experimental models and better coordination between research teams. The understanding of the phenomena that determine the order of viability will improve lung preservation techniques and increase the possibility of transplant, making it possible to use organs from donors and other distant until recently considered inappropriate, as are those of cadaver "No beating." This pilot study, conducted in rabbits, analyzes the changes taking place in the lung during ischemia, trying to assess their injuries morphological and functional consequences. It also seeks to assess the potential viability post-mortem lung and experimental validation of the method used. The model, adapted from those of other authors developed two types of experiments: EXPERIMENT A) Consisting of lung submit to a period of ischemia normotérmica "on the spot" 60-minute periods followed by reperfusion of 60120 or 180 minutes in sub 1.2 and 3 respectively. The ischemia is achieved by ligation block of lung hilio left for one hour. Once released ligation, it is likewise hilio contralateral, upon a period of 5-10 minutes hemodynamic stabilization, prior to considering the time of reperfusion. The subgroup 4 (control) was submitted to the same surgical protocol but not to "clampajes" in order to rule out the possible influence of the technical results. Every 30 minutes were conducted measurements of heart rate, systolic blood pressure, central venous pressure, the air pressure and temperature. It also took blood samples for determination of arterial pH, PaO2, PaCO2 and saturation O2. The lung samples obtained, studied under optical microscopy revealed alterations histógicas unspecified only significant in comparison with the control, especially in the subgroups that had longer periods of reperfusion. The infiltration was the note macrophagic more prominent. Among the determinations functional PaO2 proved most useful to assess changes induced by ischemia and reperfusion by. EXPERIMENT B) Consisting of a post-mortem carried out to the 60 minutes of slaughter, previously submitted, or not, to a severe hypovolemia (subgroups 3 and 1, respectively). That hypovolemia extraction was performed using a 30% of total circulatory volume through the jugular cannula. The sampling for the study was immediately after the death of the animal in sub 2 (subject to hypovolaemia pre-mortem) and 4 (control). Samples pulmonary allowed valuing the water content of the tissue (calculated as the difference between the dry and wet weight), the reserve energy (expressed as a level of ATP) and viabilildad cell (determined by Trypan Blue staining vital). Regarding the tissue water content, no differences were found between the various subgroups nor with the control subgroup. The levels of ATP revealed no significant differences between subgroups, but we see a slightly higher rate in the subgroup 3, which could lead to speculation about the phenomenon of "ischemic preconditioning." Trypan Blue staining with significant differences entr 8 and the its 573 bgrupos and between them and control, revealing a decrease in the number of viable cells that could be related to functional disturbances occurred. Ultimately, the mock design is simple and useful for studying the processes of ischemia. The feasibility lung was gradually narrowing in the period post-mortem, but 1 hour after the cessation circulatory remains still a considerable percentage of viable cells. The phenomenon of "ischemic preconditioning," whose ultimate mechanism is elucidated appears to improve the resistance of tissue ischemia, a fact to keep in mind with a view to the use of organs from cadaver "is not beating," and in certain clinical situations.

Author: DIEZ ORTEGO IRENE.
Year: 2004.
University: AUTÓNOMA DE MADRID.
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: FACULTAD DE MEDICINA.

Summary: In this thesis we studied the effect modulator exercising PGE2 on the recruitment of leukocytes in the synovial inflammation. The objectives were 1) to study the effect of inhibition of PGE2 on the recruitment cell in an experimental model of acute antigen-induced arthritis (AIA) in rabbits, 2) to study the effect of PGE2 on the synthesis and expression of gene MCP-1 and the effect of its inhibition in cells in culture sinoviales stimulated with IL-1, and 3) studying the role of different types of receivers PGE2 on gene expression of MCP-1 in cultured cells stimulated sinoviales with IL-1, and the involvement of the transcription factor NF- B. In the experimental model of IAA in rabbits, NSAIDs meloxicam (MXC) and diclofenac (DCF) produced a significant decrease in the level of PGE2 in the synovial fluid (LS) compared to untreated animals that measured by ELISA. We also note with both treatments, a decrease in the volume of LS and in the number of polymorphonuclear cells, both in the LS in the membrane, which were only significant in the case of MXC. However, none of these NSAIDs see a decrease in the number of monocytes, rather showed a tendency to rise. These results were correlated with the subsequent analysis by RT-PCR and immunohistochemistry for the presence and expression of IL-8 and MCP-1 in the inflamed synovium: we observe a decrease of IL-8 in the synovial tissue with both treatments, but not MCP-1 with respect to untreated animals. In studying the effect of PGE2 on MCP-1 in sinoviales cultured cells stimulated with IL-1, we note that PGE2 reduces time and dose form of the expression of MCP-1 in rabbit cells by RT-PCR and human cells by immunocytochemistry. The inhibition of the synthesis of PGE2-mediated MXC DCF and led to a significant increase in gene expression of MCP-1 in both rabbit cells by RT-PCR in human cells by Northern-blot stimulated with IL-1. Moreover, the increase in the expression of MCP-1 was reversed by the addition of PGE2 exogenous. The selective agonist-receptor EP2/EP4, 11-deoxi-PGE1 and EP2, butaprost, reproduced the inhibitory effect of PGE2 on the expression of MCP-1 in sinoviales rabbit cells in culture stimulated with IL-1. However, the selective agonist-receptor EP1/EP3, sulprostone, did not produce this effect by RT-PCR. In cultured human cells sinoviales the PGE2 produced inhibition of cellular activation of the transcription factor NF- B-mediated IL-1, like the selective agonist-receptor EP2/EP4, 11-deoxi-PGE1. In conclusion, our results show that the activation of receptor EP2/EP4 may regulate the recruitment of leukocytes to the inflamed synovial tissue, and therefore, drugs that block the synthesis of PGE2 might interfere with the anti-inflammatory effect of this mechanism autorregulatorio.

Author: MANSILLA APARICIO ALICIA.
Year: 2004.
University: AUTÓNOMA DE MADRID.
Place of defense: FACUILTAD DE CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS.

Summary: In previous studies we have shown expression of proinsulina Extra in the chicken embryo from gastrulation to early organogenesis, as well as in the retina during neurogenesis. At this stage the precursor of insulin, proinsulina, remains raw and acts as a factor for survival, regardless of the classical role of insulin in maintaining glucose homeostasis. In characterizing the mRNA for proinsulina in the embryo prepancreático we have found a transcribed having the same region coding that pancreatic but has an extension of 32 nucleotides in the region 5 'untranslated, which call Pro1B. In translation experiments on cells in culture and in a system of reticulocytes we found that the embryo has transcribed very small translating proinsulina regard to pancreatic transcribed by the presence of two upstream AUGs in the region 5 'widespread. When these AUG mutate to AAG, recovering levels of translation igualandose the pancreatic mRNA. In the mouse embryo and the chicken at early stage, there is also the messenger Pro1B another messenger of proinsulina he failed to processing the first intrón (as we call Pro1B1). At least in the case of the chicken embryo, the messenger acts as a messenger mature and is not retained in the nucleus, although according to the translation experiments on cells in culture and in rabbit reticulocytes, we found that its translation capabilities proinsulina is almost blocked. The expression of transcribed with this regulated intrón withheld temporarily, increasing the expression with the progress of development, and spatially; in the early organogenesis barely expressed in the optic vesicle is the majority but in the hearts and the region presomítica. The presence of two messengers suggests the existence of two levels of regulation, which demonstrates the need to maintain high levels of proinsulina low in the embryo, to test this hypothesis we have added proinsulina to embryos in ovo and as we have seen this proinsulina decreases death programmed cell and causes a number of abnormalities especially in the nervous system. The programmed cell death is a process essential for the proper development morphogenetic, and it is therefore necessary to allow a certain level of death, maintaining levels of survival factors such as proinsulina thinly regulated. Besides managing proinsulina in the area of field precardiogénico by implantation of acrylic balls in stadiums HH4-5, but not in subsequent stages, it is capable of inducing cardiac tissue so Ectopic. The ability inducing seems decline when transcribed which has blocked the translation (Pro1B1) begins to be a majority, establishing a parallel between temporary action of the proinsulina and inducing the expression of transcribed best results.

Author: Esteban Vázquez Vanesa.
Year: 2005.
University: AUTÓNOMA DE MADRID.
Place of defense: Facultad de Medicina de la UAM (La Pagoda).
Place of preparation: Facultad de Medicina (Fundación Jiménez Díaz) UAM.

Summary: AngII is the main peptide of the renin angiotensin system (RAS). AT1 receptor is involved in the pathogenesis of several cardiovascular and renal diseases. Several evidences suggest that AT2 receptor participates in the renal inflammatory response. Besides AngII other peptides of the RAS, such as Ang(1-7) and AngIV, also possess biological activities acting through their specific receptors (Mas and IRAP/AT4). The first aim of this thesis was to evaluate receptor subtype involved in the inflammatory response in the kidney. For this reason we have investigated the NF- B pathway and related genes, using different experimental models of renal damage, pharmacological tools and mice genetically modified. Our results show that the blockade of both AT1 and AT2 receptors is necessary to stop the inflammatory response. AT2 expression was increased in experimental models of renal damage and in patients with diabetic nephropathy, showing the important role of this receptor in renal diseases. Our next aim was to determine the role of the axis Ang(1-7)/Mas/ECA2 in renal damage, using diverse experimental models and Mas receptor knockout mice. In pathological settings, there is an increased of renal ECA2. Ang(1-7) via Mas receptor, increased the number of inflammatory cells in the kidney, through a mechanism mediated by NF- B activation and upregulation of proinflammatory genes (in vivo and in vitro). Finally, we evaluated whether AngIV could posses proinflammatory prope 8 rties. I 47b n culture cells (renal and vascular), AngIV via IRAP/AT4 activated NF- B pathway and increased proinflamatory factors. In summary, these results show that the angiotensin peptides (AngII, Ang1-7 and AngIV), acting through its specific receptors (AT1, AT2, Mas and IRAP/AT4), present proinflammatory properties and participate in the pathogenesis of several cardiovascular and renal diseases. The selective blockade of these receptors opens new avenues for therapeutic strategies in these pathologies.

Author: PEREDA CERVERA JAVIER.
Year: 2005.
University: VALENCIA.
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: DEPARTAMENTO DE FISIOLOGÍA,.

Summary: Introduction: The acute pancreatitis is a disease of great importance because of their relatively high frequency and an increasing incidence nowadays associated with gallstones and alcoholism. In addition, severe pancreatitis presents a high percentage of mortality. The molecular mechanisms causing pancreatitis as well as those that produce local and systemic complications that trigger an acute pancreatitis are not entirely clarified. The glutathione, which is a major intracellular antioxidant, it depleciona in the pancreas during acute pancreatitis. This depletion affects mortality cell and the prediction of experimentally-induced pancreatitis. Moreover, the replenishment by glutathione ester prevents much of the harmful effects of the disease. Objectives: Our goal has been to determine the mechanisms responsible for the depletion of glutathione in the pancreas in experimental acute pancreatitis. To do this, we studied the role of -glutamil cysteine synthetase (GCS), limiting enzyme for the synthesis of glutathione from cysteine, the relationship between the depletion of glutathione and TNF, as well as the role of proteases and proteín kinases activated by mitogenic (MAP kinase). In addition we studied the relationship between depletion of glutathione and cell death in cells AR42J, which is a cell line derived from rat exocrine pancreas. Materials and methods: To accomplish these objectives, we used an in vivo model of experimental acute necrotic pancreatitis using a retrograde infusion of taurocolato to 3.5% in the conduit biliopancreático, edematous and a model of acute pancreatitis induced pilot with ceruleína in rats. We have also employed a model in mice using ceruleína. We have measured the reduced glutathione through techniques espectofotometría and gene expression of GCS using real time RT-PCR. We have also studied the transcriptional regulation of these genes using techniques inmunoprecipitación of chromatin. By Western blot, we have studied the phosphorylation of MAP kinase and protein expression of GCS. These studies were supplemented by confocal microscopy techniques using fluorochromes specific to the study of glutathione and the type of cell death. Results and conclusions: We confirm an early and severe depletion of reduced glutathione in acute necrotizing pancreatitis experimental rats. This depletion is maintained even 9 hours post-inducción. It also produces a strong depletion of glutathione in the model of acute pancreatitis edematosa after treatment with ceruleína in rats. There is an ineffective induction of -glutamil cysteine synthetase in the pancreas during the severe acute pancreatitis that could contribute to the depletion of glutathione maintained. In acute necrotizing pancreatitis induced taurocolato does not increase the expression of the subunit of the -glutamil cysteine synthetase during the early hours, despite the binding of RNA polymerase II both promoters and the exons of the gene encoding the subunits of this enzyme. However, in oedematous induced acute pancreatitis in rats with ceruleína occurs at an early stage for a sharp increase in the expression of the catalytic subunit of the -glutamil cysteine synthetase. This marked induction is mainly due to the union of transcription factors NF- B, SP-1 and c-Myc the sponsor. In acute pancreatitis pilot induced taurocolato occurs so early an increase in the phosphorylation of the qu 8 inasas E 970 RK 1 / 2, JNK and p38 in the pancreas. Then gradually descends the phosphorylation of them to levels similar to the control six hours after induction. Treatment with pentoxifylline prevents phosphorylation of ERK 1 / 2 and JNK, while the oxipurinol prevents phosphorylation of p38. Combined treatment with pentoxifylline and oxipurinol prevents both the phosphorylation of the three main families of MAP kinases. The TNF-alpha has no significant role in the depletion of glutathione in acute pancreatitis since the depletion of glutathione associated with acute pancreatitis induced ceruleína occurs both in mice and in wild-type control mice knockout of TNF-alpha or TNF-alpha receptor. The inhibition of ERK prevents via glutathione depletion induced taurocolato in acinar cells both in vitro and in cells AR42J. However, JNK and p38 not influence significantly in this depletion of glutathione. The proteases AEBSF prevents inhibitor of glutathione depletion induced taurocolato cells AR42J. However, proteases lisosomales, the proteasome, calcium and -glutamil transpeptidase do not play a significant role in the depletion of glutathione. The AEBSF, inhibitor of glutathione depletion decreases necrosis induced taurocolato cells AR42J, increasing cell death by apoptosis. Based on these results, our results suggest that depletion of glutathione in the pancreas in acute pancreatitis is due to a lysosomal protease or peptidasa not dependent on MAP kinase, not dependent on calcium and inhibible by AEBSF.

Author: SANDOVAL MARTINEZ NIDIA RAQUEL.
Year: 2005.
University: SALAMANCA.
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: CENTROS DE INVESTIGACIONES TROPICALES.

Summary: The schistosomiasis represents a growing problem in non-endemic areas because of the increasing number of immigrants and tourists who contracted this disease. The acute schistosomiasis is not diagnosed early due to the lack of diagnostic tools that are sufficiently sensitive to detect the parasite during the first few weeks of infection. The methods currently available for the diagnosis of schistosomiasis human problems of specificity and sensitivity. Recently, several authors have developed diagnostic methods more specific and sensitive, mainly based on the technique of chain reaction (PCR) polymerase. However, these new approaches have been applied only for the diagnosis of one of the five species that affect humans (Schistosoma mansoni). Moreover, the application of this specific PCR alone has been demonstrated in samples of blood and feces. In this paper, we present the development of a highly sensitive PCR, which allows for the amplification gender and especie-específica of the four species of Schistosoma that mainly cause illness in humans. In addition, we apply this technique successfully for the detection of parasite DNA in urine samples easy to obtain and manage, in patients with schistosomiasis. With these samples, we found a 94.4% sensitivity and a 99.9% specificity of the primers to implement gender (Schistosoma spp.) -específicos, And a 100% sensitivity and a 98.9% specificity in PCR kind (S. mansoni) -específica. We have also developed a diagnostic approach based on the detection of parasite DNA by PCR in urine, comparing the usefulness of this new approach with that of the two most commonly used tools in the diagnosis of schistosomiasis (Kato - Katz and ELISA) and as with PCR in faeces. This comparison was made in an experimental model of murine Schistosoma mansoni, which allows tracking the parasite from the acute phase to chronic infection. Our results suggest that this new PCR could be useful for the detection of acute schistosomiasis in clinical samples easy to obtain and manipulate, as urine. Also extendimos these studies, using PCR samples both immigrants (n = 109) as passengers (n = 35) from endemic areas, comparing its usefulness with that of parasite and serological diagnostic methods (AWA Sb ELISA). Our results show that the PCR could be useful for (i) exclude false positive ELISA and (ii) to obtain greater sensitivity than with the serological methods in the diagnosis of patients with both chronic and acute schistosomiasis.

Author: ESTEBAN VÁZQUEZ VANESA.
Year: 2005.
University: AUTÓNOMA DE MADRID.
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: FACULTAD DE MEDICINA (FUNDACIÓN JIMÉNEZ DÍAZ) UAM.

Summary: The angiotensin II (AngII) is the principal peptide of the renin angiotensin system (MRS). By activating receptor AT1 contributes wing pathogenesis of cardiovascular disease and kidney. Some data suggest that the recipient AT2 involved in the inflammatory response kidney. In addition, the AngII, there are other biologically active peptides of the SRA, as Ang1-7 and AngIV, which act by binding to specific receptors (Mas and IRAP/AT4). The first objective of this thesis was to evaluate the type of receptor AngII involved in the inflammatory response kidney. We have investigated the route NF-By genes associated using experimental models of renal damage, therapeutic strategies and genetically modified mice. Our results show that for inhibiting the inflammatory response is necessary kidney blocking both receivers AT1 and AT2. In various experimental models and in patients with diabetic nephropathy, the expression of the receptor AT2 increases during renal damage, demonstrating the important role of this receptor in the kidney pathology. Our next goal has been to determine the role of the shaft Ang (1-7) / Mas/ECA2 in renal damage. In pathological situations there is a rise in ECA2 kidney. Using mice deficient in the gene receptor Mas (Mas- /-), we have observed that prevents kidney damage caused by unilateral ureter obstruction. The Ang (1-7), via receiver Mas, caused by the presence of inflammatory cells in the kidney by a mechanism mediated by the activation of NF-Be induction of genes proinflamatorios (in vivo and in vitro). Finally, we evaluated whether AngIV presents proinflammatory properties. In cultured cells (kidney and vascular), AngIV channel receiver AT4/IRAP active route NF-B increases proinflamatorios factors. Taken together, these results show that the peptides of Ang (AngII, Ang1-7 and AngIV), each acting through its binding to specific receptors (AT1, AT2, Mas and IRAP/AT4) have proinflammatory properties and participate in the pathogenesis of kidney disease and cardiovascular. The selective blocking of each of these receptors opens up new therapeutic strategies with significant implications in these conditions.

Author: DURÁN FRAGUAS MAXIMINA CRISTINA.
Year: 2006.
University: LEÓN.
Place of defense: FACULTAD DE CIENCIAS BIOLÓGICAS Y AMBIENTALES..
Place of preparation: FACULTAD DE VETERINARIA.

Summary: The hepatocarcinogénesis induced in rats has been shown by the existence of histopathological changes characteristics of hepatocarcinoma and increased the expression of glutatión S-transferasa placental tissue in the liver. Treatment with NPT-470 prevents damage to the liver tissue and decreases the expression of placental glutatión S-transferasa. All of this indicates the ability of the drug to inhibit tumor growth in our model for hepatocarcinoma. The hepatocarcinoma produces an increase in the rat liver weight and the relationship hepatosomática and reduced body weight gain. Treatment with NPT-470 decreases the liver weight and the relationship hepatosomática in the group of animals with hepatocarcinoma and produces weight loss in all treated animals. Noting that despite its beneficial effect of treatment with NPT-470 shows a slight toxicity. The hepatocarcinoma increases the plasma concentration angiogénico growth factor VEGF and the expression of hepatic growth factor angiogénico HGF. The administration of the NPT-470 to the group of animals with hepatocarcinoma prevents these increases, confirming this model in effect antiangiogénico. The hepatocarcinoma involves changes in antioxidant defenses to reduce the activity of liver enzymes catalase, glutathione peroxidase both cytosolic and mitochondrial and cytosolic superoxide dismutase (SOD-Cu/Zn), as well as increased activity of the liver enzyme superoxide mitochondrial dismutase (SOD-Mn). These changes are accompanied by a sharp increase in the hepatic expression of SOD-Mn and without changes in the expression of other liver enzymes. Treatment with NPT-470 maintains the activity of the enzyme and prevents the increase in the expression of SOD-Mn, indicating a strong antioxidant effect of the drug. The hepatocarcinoma is associated with increased expression of the hepatic ciclinas DyEy their kinases associated CDK4 and CDK2. The administration antiangiogénico NPT-470 decreases the expression of both the ciclinas as the kinases, which shows a reduction in cell cycle progression. The hepatocarcinoma induces a decrease in hepatic nuclear expression of the protein p53, and in the hepatic expression of the protein p21. The administration of the NPT-470 prevents the decline indicating that the inhibition of the cell cycle by antiangiogénico is mediated, at least in part, by activation of p21WAF1/CIP1 due to a mechanism dependent on p53.

Author: DE SOUSA RESENDES ANA ROSA.
Year: 2006.
University: AUTÓNOMA DE BARCELONA.
Place of defense: FACULTAT DE VETERINARIA.
Place of preparation: U.D. HISTOLOGIA I ANATOMIA PATOLOGICA.

Summary: This thesis is focused on the involvement of apoptosis in the development of lymphocyte depletion and hepatitis lesions associated with postweaning multisystemic wasting syndrome (PMWS), a pig disease in which porcine circovirus type 2 (PCV2) is the essential infectious cause. PMWS is believed to be a "multifactorial" disease, and is clinically characterized by wasting and poor response to therapeutic treatments. Main pathological features are lymphoid depletion, lymphopenia, anaemia, hepatitis, nephritis, enteritis and pneumonic lesions. It is believed that PMWS affected pigs suffer from immunosuppression. Mechanisms underlying the pathogenesis of the disease still are poorly understood. Viruses often modulate the cell apoptotic machinery to succeed in host infection; these viral mechanisms may induce lymphocyte and hepatocyte death among other cell types. Apoptosis involvement in PMWS lesions development was suggested in the first pathologic descriptions of the disease based on morphological criteria. Further, apoptosis has been investigated to a certain degree in hepatitis and lymphoid lesion from PMWS affected pigs by the TUNEL technique. In the present thesis we evaluated apoptosis from PMWS cases by in situ detection of anti-active caspase-3 in hepatic and lymphoid lesions. Our results indicated that liver cell damage in PCV2 hepatitis is mediated by induction of apoptosis, but this does not seem to be the main mechanism involved in lymphoid depletion lesions. These results bring a new insight into the possible role of apoptosis involvement in PMWS. The potential effect of PCV2 ORF3 protein inducing apoptosis in vivo in pigs suffering from PMWS under conventional field circumstances seems feasible according to our results, at least in the liver. Our results also suggest that other mechanisms may be involved in the pathogenesis of lymphoid depletion, such as inhibited proliferation of thymocytes, supporting the hypothesis proposed by other studies. The main conclusions of the present thesis investigations are that (1) anti-active caspase-3 immunohistochemistry is a reliable method and is equivalent, in general terms to the TUNEL assay in general terms to detect apoptosis in pig lymphoid and hepatic tissues, (2) that apoptosis can differ significantly in pig lymphoid tissues according to the animal age, (3) that apoptosis does not seem to be a relevant phenomenon in the development of lymphoid depletion lesions, and (4) that apoptosis is involved in the pathogenesis of hepatitis lesions associated with PCV2 infection.