Thesis (M.S.)--Uniformed Services University of the Health Sciences, 2005. / Typescript (photocopy).

Human schistosomiasis popularly known as bilharzias in many regions of Africa is a freshwater snail-transmitted disease caused by parasitic flatworms known as schistosomes. The growth and development of schistosomes typically requires developmental stages in multiple hosts and transmission stages in freshwater. These life cycle environments present a plethora of stressors. Certain gene families including heat shock proteins (HSPs/Hsps) and universal stress proteins (USPs) help schistosomes to respond to unfavourable conditions. The availability of genomes sequences information for Schistosoma japonicum, Schistosoma mansoni and Schistosoma haematobium provide unique research resources to apply bioinformatics analysis of its associated USPs to predict regulatory features from sequence analysis. The objectives of the research were to (i) Infer the biochemical and environmental regulation of universal stress proteins of Schistosoma species; (ii) Identify biological function relevant protein sequence and structure features for prioritized universal stress proteins from Schistosoma species; (iii) Determine the distinctive structural features of a predicted regulator of Schistosoma adenylate cyclase activity that has possible influence on the functioning of universal stress proteins. The findings revealed that (i) schistosomes USPs are hydrophilic and very reactive in the water environment or in aqueous phase, which seems adaptive with their immediate environment and developmental stages; (ii) The functions of Smp_076400 and Sjp_0058490 (Q86DW2) are regulated by conserved binding site residues and metallic ions ligands (Ca2+, Mg2+ and Zn2+), particularly Ca2+ predicted to bind to both USPs; (iii) The S. mansoni life cycle and stress resistance pathway protein (Smp_059340.1) is regulated by Ser53, Thr188, Gly210 and Asp207 residues. The overall scope has highlighted the role of bioinformatics in predicting exploitable regulatory features of schistosome universal stress proteins and biological pathways that might lead to identification of putative functional biomarkers of common environmental diseases. The findings of this research can be applicable to other areas of environmental health and environmental genomics. / Environmental Sciences / (D. Litt et Phil. (Environmental Sciences)

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: The tolerance of Enterococcus mundtii ST4SA to stressful gastro-intestinal conditions in humans and animals is vital to its success as a probiotic. The need for new effective probiotics with stronger inhibitory (bacteriocin) activity has arisen due to the increasing number of antibiotic resistant pathogens. Enterococci are used in the fermentation of sausages and olives, cheese making and as probiotics. Their role as opportunistic pathogens in humans makes them a controversial probiotic (Moreno et al., 2005). Enterococci occur naturally in the gastro-intestinal tract which renders them intrinsic acid and bile resistance characteristics. E. mundtii ST4SA produces a 3950 Da broad-spectrum antibacterial peptide active against Gram-positive and Gram-negative bacteria, and viruses. The bacteria include Enterococcus faecalis, Streptococcus spp., Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae and Staphylococcus aureus. E. mundtii ST4SA inactivates the herpes simplex viruses HSV-1 (strain F) and HSV-2 (strain G), a measles virus (strain MV/BRAZIL/001/91, an attenuated strain of MV), and a polio virus (PV3, strain Sabin). This study focuses on the genetic stability of E. mundtii ST4SA genes when exposed to stress factors in the human and animal gastrointestinal tract. Based on results obtained by real-time PCR, the expression of genes encoding bacST4SA, RecA, GroES and 23S rRNA by E. mundtii ST4SA were not affected when the cells were exposed to acid, bile and pancreatic juice. This suggests that these genes of E. mundtii ST4SA will remain stable in the intestine. This could indicate that other genes of E. mundtii ST4SA could remain stable in the host. Further studies on the stability of genes encoding antibiotic resistance and virulence factors should be conducted to determine their stability and expression in the host in stress conditions. Concluded from this study, E. mundtii ST4SA is an excellent probiotic strain. / AFRIKAANSE OPSOMMING: Enterococcus mundtii ST4SA se weerstandsvermoë teen stresvolle gastrointestinale kondisies is essensieel vir die sukses van hierdie organisme as ‘n probiotikum. Die aanvraag vir nuwe, meer effektiewe probiotika met sterker inhibitoriese (bakteriosien) aktiwiteit is as gevolg van die toename in antibiotikum weerstandbiedende patogene. Enterococci word algemeen gebruik as probiotika, sowel as in die fermentasie van worse, olywe en kaas. Hulle rol as oppertunistiese patogene in mense veroorsaak kontroversie as gevolg van hul toenemende gebruik as probiotika. Enterococci is deel van die natuurlike mikroflora in die gastrointestinale weg van mense en diere. Dit verleen aan hierdie spesies ‘n natuurlike weerstandsvermoë teen maagsure, galsoute en pankreatiese afskeidings. E. mundtii ST4SA produseer ‘n 3950 Da wye spektrum anti-bakteriese peptied, aktief teen Gram positiewe en Gram negatiewe bakterieë sowel as virusse. Hierdie bakterieë sluit Enterococcus faecalis, Streptococcus spp., Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae en Staphylococcus aureus in. E. mundtii ST4SA inaktiveer die herpes simpleks virus HSV-1 en HSV-2, ‘n masels virus (MV/BRAZIL/001/91), en ‘n polio virus (PV3, stam Sabin). Hierdie studie fokus op die genetiese stabiliteit van E. mundtii ST4SA gene, wanneer hulle blootgestel word aan stress faktore in die mens en dier gastrointestinale weg. “Intydse” PKR data gebasseer op die uitdrukking van die bacST4SA, RecA, GroES en 23S rRNA gene in stresvolle kondisies dui aan dat E. mundtii ST4SA nie geaffekteer word wanneer die sel blootgestel word aan suur, gal en pankreatiese vloeistowwe nie. Hierdie resultate dui aan dat hierdie gene van E. mundtii ST4SA stabiel sal bly in die intestinale weg van die mens en dier. Dit kan aandui dat ander gene van E. mundtii ST4SA soos die wat kodeer vir virulensie faktore en antibiotikum se weerstandsvermoë stabiel mag bly in die gasheer. Verdere studies wat fokus op die stabiliteit van gene wat kodeer vir antibiotikum weerstandbiedendheid en virulensie faktore moet uitgevoer word om hulle stabiliteit en uitdrukking in die gasheer te bepaal. Bevindings van hierdie studie dui aan dat E. mundtii ST4SA goeie potensiaal het as ‘n probiotikum.

Heat shock proteins increase their gene expression after exposure of cells or organisms to some forms of stress, which may be high temperature, infection, inflammation, hypoxia, lack of nutrients and water. Stressful situations for the body are also pregnancy-related complications associated with placental insufficiency - preeclampsia and IUGR, as well as other pregnancy-related complications such as fetal growth restriction and gestational hypertension. Therefore, I examined whether the occurrence of pregnancy-related complications (preeclampsia, fetal growth retardation, gestational hypertension) affect the gene expression of heat shock proteins. Five hsp systems was detected, namely Hsp27, Hsp60, Hsp70, Hsp90 and HspBP1 in placental tissue samples and whole maternal peripheral blood. Samples came from women with physiological pregnancy and from women with certain pregnancy-related complications (PE, FGR, GH). RNA was isolated from the samples. Detection of hsp expression was performed by using real-time RT-PCR and the comparative Ct method. Changes in gene expression between the test samples and reference sample were examined. To assess the difference between physiological pregnancies and pregnancies with selected pregnancy- related complications, an analysis of variance (ANOVA) was used....

La température est le paramètre abiotique qui influence de façon majoritaire les traits d’histoire de vie des espèces ectothermes. Pour appréhender les relations entre physiologie, environnement et histoire évolutive et leur influence respective sur la délimitation des aires de distribution des espèces, ce travail exploite les caractéristiques thermiques du milieu souterrain. Les résultats réfutent l’hypothèse de sténothermie des deux organismes hypogés étudiés Niphargus rhenorhodanensis et Proasellus valdensis et prouvent que l’histoire évolutive, la dispersion ou la compétition sont des paramètres importants dans l’établissement des aires de distribution des espèces souterraines. En outre, ce travail caractérise pour la première fois chez des organismes souterrains, un gène codant pour des protéines de choc thermique et montre l’importante sensibilité cellulaire de N. rhenorhodanensis face à une augmentation de température / Temperature is the abiotic factor that most influences the life-history traits of ectothermic organisms. In order to study the relationships between physiology, environment and evolutionary history and their respective role in the determination of species distribution areas, this work takes advantage of the thermal caracteristics of subterranean aquatic biotopes. Our results refuted stenothermy in both studied hypogean organisms Niphargus rhenorhodanensis and Proasellus valdensis and they showed that evolutionary history, dispersal and competition are important factors that determine the distribution of subterranean species. Moreover, this work characterized for the first time in subterranean organisms a gene encoding heat shock proteins and demonstrated the high cellular sensitivity of N. rhenorhodanensis to increased water temperature

Las proteínas pequeñas de choque térmico (sHSP) responden a una amplia variedad de estreses ambientales, estabilizando proteínas parcialmente desplegadas y evitando su agregación irreversible, en forma independiente de ATP. En plantas, las sHSPs son especialmente diversas siendo las sHSPs de organelas una característica única de las plantas. La estructura primaria de las sHSP incluye una secuencia N-terminal no conservada de longitud variable, un dominio α-cristalino conservado (ACD) y una secuencia C-terminal corta no conservada. El dominio ACD representa una característica conservada presente en todas las sHSPs, sin embargo, no todas las proteínas que contienen un dominio ACD son sHSP. Las sHSPs pertenecen a una gran superfamilia, siendo su importancia funcional y fisiológica en gran parte desconocida. El objetivo de este trabajo fue dilucidar el rol de sHSPs de localización mitocondrial (sHSPs-M) en Arabidopsis thaliana y Solanum lycopersicum en situaciones de estrés ambiental, y caracterizar probables promotores bidireccionales que regulan la expresión de genes codificantes de proteínas con dominio ACD con orientación cabeza a cabeza en el genoma de Arabidopsis. Este trabajo cubre aspectos desde la organización genómica y la función de sHSPs-M en Arabidopsis hasta el rol de las sHSPs-M en la respuesta al estrés por frío de frutos de tomate. Para ello, se generaron plantas mutantes de Arabidopsis y de tomate utilizando la tecnología de silenciamiento génico por micro ARNs artificiales. Las plantas mutantes fueron analizadas en su proteoma, metaboloma y lipidoma en distintas condiciones de estrés. A continuación, se presenta un resumen de los resultados obtenidos. En la primera parte de este trabajo, se realizó la caracterización funcional de genes con orientación cabeza a cabeza en el genoma de Arabidopsis, que codifican para proteínas con ACD y las regiones intergénicas correspondientes. Se lograron identificar y caracterizar cuatro distintos promotores bidireccionales, entre ellos el promotor del gen At5g51440 que codifica una sHSP de localización mitocondrial (sHSP23.5). Los resultados obtenidos sugieren que el promotor bidireccional contenido en el par At5g51430-At5g51440 es fuertemente inducido por altas temperaturas en una dirección, pero no así en la dirección opuesta. El promotor contenido en el par At1g06460-At1g06470 mostró una actividad alta en ambas direcciones, teniendo por ello, un alto potencial de aplicación en ingeniería genética. Los dos promotores restantes mostraron mayor actividad en una dirección y, por lo tanto, pueden ser considerados como promotores bidireccionales asimétricos. El estudio funcional de los promotores seleccionados reveló el potencial biotecnológico de los mismos ya que pueden ser inducidos específicamente en una determinada condición (como altas temperaturas) en una o en ambas direcciones cuando sea necesario. En la segunda parte, se presenta la caracterización funcional de sHSP mitocondriales en condiciones de estrés y durante el desarrollo de Arabidopsis. Se identificaron tres genes parálogos en el genoma de Arabidopsis (At5g51440, At4g25200, y At1g52560), y se diseñaron microARN artificiales específicos para la obtención de plantas mutantes por silenciamiento (amiR simple, doble y triple). Las plantas amiR simples y dobles (para sHSP23.5 y sHSP23.6) no mostraron un fenotipo evidentemente afectado, probablemente debido a la compensación o redundancia funcional de las sHSP mitocondriales. Por otro lado, las plantas triple mutante amiR23.5/23.6/26.5 muestran un fenotipo alterado en las etapas vegetativa y reproductiva. Estas plantas presentan hojas pequeñas, células epidérmicas con áreas reducidas, pero no reducción en el número de células epidérmicas por hoja. Además, exhiben hojas cloróticas, raíces cortas y menor producción de semillas en comparación con las plantas Col-0. Las plantas triple amiR son considerablemente pequeñas debido a una alteración en el proceso de expansión celular, pero no en la proliferación celular, lo que indica una profunda alteración en el programa de desarrollo de la planta. En el análisis proteómico de las mutantes amiR se observó un aumento significativo de proteínas implicadas en el metabolismo, y alteración en la abundancia de varias proteínas relacionadas con la traducción, y con el funcionamiento y la estructura de los ribosomas. La triple mutante exhibió un mayor número de proteínas con abundancia alterada involucradas en estos procesos en comparación con las mutantes simples y doble amiR23.5/23.6. Estos cambios tan amplios observados en proteínas relacionadas con el funcionamiento de los ribosomas sugieren una posible alteración en la función normal de los mismos. Los resultados presentados en este trabajo proporcionan evidencias sobre el importante rol de las sHSPs-M no sólo en la respuesta a las altas temperaturas, sino también durante el desarrollo de la planta de Arabidopsis. Los resultados indican que una compensación funcional podría ser responsable del fenotipo observado en las plantas mutantes con niveles reducidos de cada sHSPs-M individual. Sin embargo, la reducción simultanea de las tres sHSPs-M causó una profunda alteración en la función normal de mitocondrias y ribosomas, afectando gravemente el metabolismo energético y la homeostasis celular, lo que llevó a alteraciones en el desarrollo correcto de la planta. En la última parte de este trabajo, las consecuencias de la disminución de la proteína sHSP23.8 de localización mitocondrial en frutos de tomate fueron investigadas. Los frutos fueron analizados en su fenotipo y en la susceptibilidad al daño por frío. Los frutos de las plantas mutantes amiR23.8 que fueron conservados en frío mostraron mayor pérdida de agua y de electrolitos en el tejido pericárpico en comparación con frutos WT. El deterioro observado en los frutos amiR23.8 indica que estos frutos mutantes son mayormente susceptibles al estrés por frío desarrollando síntomas de daño por frío. El lipidoma de los frutos amiR23.8 frigoconservados mostró cantidades alteradas de glicerolípidos, y los niveles de lípidos saturados en amiR23.8 disminuyeron luego del tratamiento con frío, pero no por debajo de los niveles encontrados en frutos WT en condiciones normales. Lo opuesto se encontró en el porcentaje relativo de lípidos insaturados, con niveles significativamente más bajos de insaturaciones en frutos amiR23.8 en condiciones normales y después del enfriamiento. Los resultados presentados indican una degradación diferencial de lípidos extraplastídicos y plastídicos en frutos amiR23.8, y alteraciones en la remodelación del lipidoma luego del estrés por frío, lo que podría conducir a una mayor sensibilidad al daño por frío. Los resultados discutidos aquí indican que la proteína sHSP23.8 podría estar directamente involucrada en los mecanismos de protección contra el estrés por frío en frutos de tomate.

L’objectif de ce travail de thèse a été d’analyser les stratégies de réduction de la fatigue musculaire en course de trail et potentiellement d’identifier certains paramètres d’influence de cette fatigue. La course de trail est un nouveau sport en essor qui induit une combinaison spécifique de fatigue et dommages musculaires des principaux muscles locomoteurs. Afin de pouvoir conduire des études interventionnelles, une étude descriptive préliminaire a été conduite pour caractériser la fatigue spécifique et les dommages musculaires induits par ce type d’épreuve de trail. Ensuite, la reproductibilité du trail comme modèle de fatigue a été vérifiée afin de pouvoir l’utiliser dans un contexte d’intervention. Enfin, deux études visant à réduire la fatigue induite par le trail ont été conduites. D’une part l’utilisation des vêtements de compression - très à la mode en trail a été analysée comme stratégie d’optimisation de la performance. D’autre part, a aussi été étudié l’effet d’un réchauffement préalable du muscle sur les dommages musculaires : Dans cette optique, une étude contrôlée en laboratoire a été menée, examinant les effets d’un réchauffement passif sur les conséquences fonctionnelles de course en descente chez une population non-entraînée. En résumé, les travaux conduits au sein de cette thèse fournissent une description de la fatigue en trail, et valident l’utilisation du trail comme modèle reproductible de terrain pour investiguer les stratégies de réduction de la fatigue. De plus, ils relativisent l’effet positif des vêtements de compression sur la performance et montrent le lien fonctionnel entre le réchauffement musculaire et la réduction des dommages musculaires induits par un travail excentrique. / The aim of this thesis was to analyse strategies to reduce muscular fatigue in trail running and potentially draw conclusions on the underlying mechanisms. Trail running is a new and upcoming sport that induces a combination of fatigue and muscle damage in the main locomotor muscles. To obtain conclusive evidence on the effect of intervention studies a preliminary descriptive study was undertaken to characterise typical fatigue and damage. Subsequently a model was developed and validated that would allow the investigation of interventions in an applied field setting. A popular current strategy in trail running is the use of compression garments; therefore the effect of these on performance was studied as an intervention. Furthermore, prior heating is anecdotally considered beneficial and recent research has suggested a potential mechanism to link this with reduced muscle damage. Therefore a controlled laboratory study was conducted, examining the effects of passive heating on functional consequences of downhill running in an untrained population. In synopsis, the research conducted for this thesis provides descriptive evidence and a validated terrain model to further investigate fatigue reduction strategies in trail running. Additionally it adds to the current literature in disproving a positive effect of compression garments on performance and demonstrating the functional link between heating and eccentric-induced muscle damage reduction.

La formation et le dépôt de fibres d'α-Synucléine dans le cerveau humain sont à l‟origine de la maladie de Parkinson. Cette thèse documente le rôle de deux chaperons moléculaires dans l‟assemblage en fibres de l'α-Syn : Hsc70 (protéine de choc thermique constitutivement exprimée chez l‟Homme) et Ssa1p (son équivalent chez la levure). Le but était d'élargir le catalogue d'effets connus des chaperons moléculaires sur α-Syn, pour éventuellement ouvrir la voie à des applications thérapeutiques. Nous avons montré que Hsc70 inhibe l'assemblage de l'α-Syn en fibres, en se liant avec une forte affinité à la forme soluble de l'α-Syn. Hsc70 se lie préférentiellement aux fibres de l'α-Syn, et cette liaison a un effet cytoprotecteur puisqu'elle rend les fibres moins toxiques pour les cellules de mammifères en culture. Pareillement à Hsc70, Ssa1p inhibe l'assemblage de l'α-Syn en fibres, et a une plus forte affinité pour les fibres que pour la forme soluble de l'α-Syn. En revanche, la liaison de Ssa1p aux fibres de l'α-Syn n'a pas d'effet cytoprotecteur, sûrement due aux différences entre les séquences du site de liaison aux peptides des deux chaperons moléculaires, qui fait que Ssa1p a une affinité plus faible que Hsc70 pour les fibres d'α-Syn. Nous avons fixé le complexe entre Ssa1p et α-Syn avec des agents pontants, pour ensuite établir une carte du site d'interaction entre les deux protéines en utilisant la spectrométrie de masse. Ceci est indispensable si un « mini » Ssa1p, constitué des éléments nécessaires et suffisants sera utilisé comme agent thérapeutique pour réduire la toxicité des fibres d'α-Syn. / The formation and deposition of α-Synuclein fibrils in the human brain is at the origin of Parkinson’s disease. The objective of my thesis was to document the role of two molecular chaperones on the assembly of α-Syn into fibrils: Hsc70, a constitutively expressed human heat shock protein, and Ssa1p, its yeast equivalent. The aim was to expand the catalogue of known effects of molecular chaperones on the PD implicated protein, which could have therapeutic significance. We showed that Hsc70 inhibits the assembly of α-Syn into fibrils, by binding with high affinity to the soluble form of α-Syn. We documented that Hsc70 binds preferentially to α-Syn fibrils and that this binding has a cytoprotective effect, as it renders the fibrils less toxic to cultured mammalian cells. Similarly to Hsc70, Ssa1p inhibits the assembly of α-Syn into fibrils, and has a higher affinity for fibrils than for the soluble form of α-Syn. On the other hand, binding of Ssa1p to α-Syn fibrils does not have a cytoprotective effect, almost certainly due to differences in the amino acid sequences of the peptide binding sites of the two molecular chaperones, which mean that Ssa1p has a lower affinity than Hsc70 for α-Syn fibrils. We stabilized the complex between Ssa1p and α-Syn using chemical cross-linkers, to then map the interaction site between the two proteins. This is indispensable if a “mini” Ssa1p, comprised of only what is necessary and sufficient of Ssa1p, is to be used as a therapeutic agent to decrease the toxicity of α-Syn fibrils. A therapeutic agent based on exogenous protein Ssa1p is less likely to trigger an autoimmune response than for example the endogenous protein Hsc70.

INTRODUÇÃO: A deiscência das anastomoses colorretais continua uma grave complicação da cirurgia colorretal. Pouco se sabe sobre as alterações moleculares que podem influenciar esse processo, mas entende-se que a síntese do colágeno é fundamental para uma adequada cicatrização. As Heat Shock Proteins (HSP) estão expressas em situações de estresse, tais como a hipovolemia, além de participarem da síntese do colágeno. Além disso, há poucas informações sobre a expressão da HSP70 nos processos cicatriciais. OBJETIVOS: Determinar a expressão da HSP70 no intestino grosso e nas anastomoses intestinais de ratos normo e hipovolêmicos, bem como a quantidade do colágeno total e a qualidade das células inflamatórias envolvidas nesse processo, além de correlacioná-las. MÉTODOS: Utilizaram-se 40 ratos Wistar que foram divididos em seis grupos. Submeteram-se 24 animais a cateterização da artéria carótida, seguida de sangramento (20% da volemia); destes, 8 foram submetidos somente à hipovolemia e, os outros 16, à hipovolemia seguida de colectomia (direita e esquerda). Dividiram-se os outros 16 animais em dois grupos: em oito realizou-se colectomia direita e nos outros oito, esquerda. Os espécimes foram avaliados no 7º e no 14º dia de pósoperat ório (PO). Pela coloração de Hematoxilina-eosina, avaliaram-se as células inflamatórias e a presença de necrose nas anastomoses, pelo método de Picrossírius, a quantidade de colágeno e por imunohistoquímica, a expressão da HSP70. RESULTADOS: A expressão da HSP70 no intestino grosso diminuiu com a hipovolemia (p<0,05). Não houve diferença estatística entre as células inflamatórias encontradas nas anastomoses dos grupos estudados. A quantidade de colágeno foi menor no 7º que no 14º dia de pósoperat ório (p<0,05), diferentemente da expressão da HSP70, que foi maior no 14º que no 7º dia (p<0,05). No 7º dia, a quantidade de colágeno estava menor no grupo desafio que no grupo controle (p<0,05), fato que não se repetiu no 14º dia de pós-operatório. Em relação à expressão da HSP70 nas anastomoses intestinais, não houve diferença estatística entre o grupo desafio e o controle, mas no à direita, tanto no 7º quanto no 14º dia houve tendência à diferença significativa. CONCLUSÕES: A expressão da HSP70 no intestino grosso é afetada pela hipovolemia, aumenta na cicatrização das anastomoses e é mais evidente quando a quantidade de colágeno é menor. A expressão da HSP70 pode ser um potencial marcador biológico das complicações das anastomoses intestinais. / INTRODUCTION: The anastomotic leakage remains a serious complication in surgery of large bowel. The influence about the molecular mechanisms involved at this process is barely known, but its well known that collagen synthesis is essential for good healing. The Heat Shock Proteins (HSP) are expressed at stress situations, such as hypovolemia, also they are involved at the process of collagen synthesis. Besides, there are a few information about HSP70 expression at the healing process. OBJECTIVES: To determine the HSP70 expression at large bowel and intestinal anastomoses in normal and hypovolemic rats, as well as the amount of collagen and the quality of the inflammatory cells involved at this process, and correlate them. METHODS: It was used fourty Wistar rats divided into six groups. Twenty-four rats were submitted to catheterization of carotida artery followed by withdraw of 20% of their blood volume; eight of them were underwent just to hypovolemia and the others sixteen to hypovolemia followed by colectomy (right and left). The remaining sixteen rats were divided into two groups: eight of them were underwent to right colectomy and eight to the left. The specimens were analyzed at the seventh and fourteenth postoperative (PO) days. The inflammatory cells were assessed by Hematoxilin-eosin, the amount of collagen was stained by red Picrossirius and the HSP70 expression was determined by immunohistochemical study. RESULTS: The HSP70 expression at the large bowel gets lower when there is hypovolemia (p<0.05). There was no statistic difference on the inflammatory cells found in the groups. The amount of collagen was lower at the seventh than the fourteenth day (p<0.05), unlikely the HSP70 expression, which was the opposite (p<0.05). At the seventh day, the amount of collagen was lower at the study group than at the control group (p<0.05), otherwise at the fourteenth postoperative day. There was no statistic difference in HSP70 expression at the anastomoses of the groups, but at the right colon, there was disposition to significative difference at both days. CONCLUSION: The HSP70 expression at the large bowel is modified by hypovolemia, increases at the anastomoses healing and is more evident when the amount of collagen is lower. The HSP70 expression can be a potential biological marker of the complication of these anastomoses

O conhecimento a respeito de mecanismos de reprodução e geração de diversidade genética de organismos do gênero Leishmania vem sendo alterado com o advento de novas tecnologias. Estudos de laboratório demonstraram ocorrência de reprodução sexuada experimentalmente em organismos do gênero Leishmania. Relatos na literatura mostram a ocorrência de híbridos no ambiente natural, associada a casos de leishmaniose tegumentar americana (LTA). A partir da caracterização fenotípica realizada utilizando anticorpos monoclonais e eletroforese de isoenzimas em linhagens isoladas de pacientes com LTA na região Amazônica do Brasil, sete isolados (M15983, M15984, M15987, M15988, M19672, M19676 e M19697) apresentaram um padrão intermediário entre as espécies L. (V.) shawi shawi e L. (V.) guyanensis. O presente estudo visou realizar a confirmação da identidade híbrida desses parasitas através da clonagem dos isolados, seguida da análise do polimorfismo pela clivagem do produto da reação em cadeia da polimerase (PCR RFLP) tendo como alvo a enzima de choque térmico de 70 quilodaltons (hsp70). O hsp70 PCR RFLP também foi utilizado para análise comparativa da intensidade dos produtos de digestão, para estimar a ploidia dos híbridos e a proporção da herança dos alelos de hsp70. Foi realizada ainda, avaliação do comportamento biológico in vitro e experimental in vivo desses parasitas. A curva de crescimento foi realizada na 4° e 12° passagem após isolamento do parasita. Infecções experimentais de BALB/c foram realizadas para avaliação da evolução da infecção causada pelo isolado híbrido selvagem M19672 e sua comparação com as cepas parentais L. (V.) guyanensis e L. (V.) s. shawi. Foi determinada a carga parasitaria em pele, linfonodo e baço dos animais infectados e análise histopatológica das lesões. Os resultados obtidos a partir da clonagem dos isolados demonstraram que se tratavam de populações homogêneas de parasitas. Três isolados apresentaram padrão heterozigoto de L. (V.) guyanensis/L. (V.) s. shawi e quatro isolados apresentaram padrão homozigoto, apresentando somente os alelos de L. (V.) s. shawi. O ensaio de clivagem de sequências polimórficas amplificadas (SNP-CAPS) mostrou que os homozigotos apresentaram o mesmo número de cópias de hsp70 de L. (V.) s. shawi, enquanto que nos heterozigotos os alelos de L. (V.) guyanensis são mais representativos. Os parasitas híbridos apresentaram uma maior plasticidade fenotípica no crescimento in vitro. A infeção de BALB/c pelos parasitas híbridos apresentou uma evolução rápida tendo seu maior pico entre 14 e 28 dias pós-infecção, com regressão posterior da lesão. Esse período foi acompanhado da maior carga parasitária na pele e linfonodo. A infecção causada pelas cepas parentais mostrou aumento progressivo da lesão a partir do 21° dia de infecção para L. (V.) guyanensis e 35° dia para L. (V.) s.shawi. As alterações histopatológicas da lesão causada pelo híbrido apresentaram um padrão intermediário entre as de L. (V.) s. shawi e L. (V.) guyanensis, guardando, no entanto, mais características desta última. Nossos achados confirmam que as cepas analisadas são híbridas entre L. (V.) guyanensis e L. (V.) s. shawi da região do Baixo Amazonas, estado do Pará, área com alta ocorrência de casos de LTA na Brasil e grande diversidade de espécies do parasita, vetores e reservatórios / Knowledge about the mechanisms of reproduction and generation of genetic diversity of the genus Leishmania organisms has been changed with the advent of new technologies for it studies. Laboratory researches have shown the occurrence of experimentally sexual reproduction in the genus Leishmania. Reports in the literature shows the occurrence of hybrids in the natural environment, associated with cases of american cutaneous leishmaniasis (ACL). From the phenotypic characterization performed using monoclonal antibodies and isozymes in strains isolated from patients with ACL in the Amazon region of Brazil, seven isolates (M15983, M15984, M15987, M15988, M19672, M19676 and M19697) showed an intermediate pattern between species L. (V.) shawi shawi and L. (V.) guyanensis. This study aimed to perform the confirmation of the hybrid identity of these parasites by performing cloning of the isolated, followed by analysis of polymorphism by cleavage of the polymerase chain reaction product (PCR RFLP) targeting heat shock enzyme 70 kilodaltons (hsp70). The hsp70 PCR RFLP was also used for comparative analysis of the intensity of the digestion products to estimate the ploidy of the hybrids and the proportion of the inheritance of hsp70 alleles. It was also observed in this study the biological in vitro behavior of these parasites. The growth curve in vitro was held at 4° and 12° passage after the isolation of mammalian host. Experimental infections of BALB /c mice were carried out to evaluate the evolution of the infection caused by the hybrid wild strain M19672 compared to the parental strains L. (V.) guyanensis and L. (V.) s. shawi. The parasitic load was determined on the skin, lymph nodes and spleen of infected animals and histopathology of the lesions. The results from the cloning of the isolates showed that these are homogeneous populations of parasites. Three isolates presented heterozygous L.(V.) guyanensis/ L.(V.) s. shawi pattern and four presented homozygous pattern with only the L.(V.) s. shawi alleles. The cleavage amplified polymorphic sequences assay (SNP-CAPS) showed that the homozygous had the same number of copies of hsp70 L. (V.) s. shawi, whereas in the heterozygous alleles L. (V.) guyanensis are more representative. Hybrid parasites demonstrated a higher phenotypic plasticity growth in vitro. BALB /c infection by M19762 hybrids showed a rapid evolution with a peak between 14 and 28 days post-infection, with subsequent lesion regression. The period of greatest development of the lesion was accompanied by higher parasite burden in the skin and lymph nodes. The infection caused by parental strains showed a progressive increase in the lesion from the 21th day of infection for L. (V.) guyanensis, and day 35 PI for L. (V.) s. shawi The alteration in the histopathological lesions caused by the hybrid strain showed an intermediate pattern between those of L. (V.) s. shawi and L. (V.) guyanensis, keeping, however, more characteristics of the latter. Our findings confirm that the strains analyzed are true hybrids between L. (V.) guyanensis and L. (V.) s. shawi from Amazon region, Pará state, an area with high occurrence of ACL in Brazil and diversity of parasite species, vectors and reservoirs