Foram utilizadas vinte tartarugas da espécie Trachemys scripta elegans, sendo duas fêmeas jovens, quatro fêmeas adultas, oito machos jovens e seis machos adultos. Inicialmente, retiramos o plastrão, isolamos o coração e, já na aorta descendente, introduzimos uma cânula antes da bifurcação da aorta para injeção de solução de látex corado com pigmento vermelho para identificarmos as artérias com maior precisão. Após a injeção, os animais foram colocados em solução aquosa de formaldeído 20% por período não inferior a 72 horas e, após esse período, dissecamos as artérias responsáveis pela irrigação do baço. Observamos em 30% dos casos, o baço posicionado caudalmente ao cólon transverso e, em 70%, cranialmente ao mesmo, mas sempre apoiado neste segmento intestinal. Com relação à irrigação do baço, observamos que em 95% dos casos, o maior aporte sangüíneo era proveniente da artéria mesentérica cranial, onde apenas 30% dos animais apresentavam irrigação somente pela artéria lienal; já em 40% apresentavam irrigação pela artéria lienal e pequenos ramos da artéria cólica esquerda. Em 5% dos casos era irrigado pela artéria lienal e por um único ramo emitido por uma das artérias jejunais, 5% eram irrigados pela artéria lienal e por um ramo da artéria pancreaticaduodenal cranial e por uma artéria que tinha origem no tronco comum das artérias jejunais; 15% dos animais tinham seu baço irrigado pela artéria lienal e por ramos da artéria pancreaticaduodenal cranial. Em 5% dos animais observamos o baço sendo irrigado apenas por ramificações da artéria cólica esquerda. / This study was conduct using 20 turtles specie Trachemys scripta elegans, which 2 young females, 4 adults females, 8 young males and 6 adults males. Initially, it was took the hoof belly and, isolating the heart to identify more precisely the arteries was injected latex solution with red pigment through aorta descendens with one thin tube before aorta\'s bifurcation. After the latex injection, all the animals were submerged in 20% formaldeid water solution by a period of more than 72 hours. After that period, the arteries responsible by spleen irrigation were dissected. It was found in 30% of the cases the spleen was positioned behind colon transversum; and in 70% in front of colon transversum. Regarding the spleen irrigation, it was observed in 100% of the cases that arteria lienalis had its origin in arteria mesenterica cranialis. Which 30% had irrigation only by arteria lienalis. In 40% the irrigation was done by arteria lienalis and small branches of arteria colica sinistra. In 5% of the cases it was irrigated by arteria lienalis and by na unique branch sent by one of arteriae jejunales. In 5% of the cases the irrigation through arteria lienalis and by one branch of arteria pancreaticaduodenalis cranialis, and also by one artery with origin in the common trunk from arteriae jejunales. In 15% from the animals, irrigation was done by arteria lienalis and by branches from arteria pancreaticaduodenalis cranialis. In 5% of the cases the spleen was irrigated just by ramifications of arteria colica sinistra.

Infusion of the "mononuclear fraction" of human cord blood cells (HUCBC), which is composed of immature blood cells and hematopoietic progenitors, is known to reduce neurobehavioral deficits in rats subject to middle cerebral artery occlusion (MCAO). When MCAO rats are infused with 106 cells 24 hours after the induction of the stroke, their motor function improves. To extend these findings, we first examined the behavioral recovery of MCAO rats in the presence of increasing doses of HUCBC. The recovery in behavioral performance seen with measurements of spontaneous activity and motor deficits, depended on the amount of cells delivered, with 106 HUCBC being the threshold for significant behavioral recovery. Measurements of the ischemic volume revealed an inverse relationship between HUCBC dose and damage volume, which reached significance at the higher HUCBC doses (107 and 3-5x107 cells). Moreover, investigation of the distribution of the intravenously injected cells showed that HUCBC were localized to the injured brain hemisphere and the spleen. Given these findings, we hypothesized that there may be a role of HUCBC in the modulation of the peripheral or brain-localized immune response that is normally evoked after stroke. Results on the effect of HUCBC infusion on splenocytes indicated that HUCBC treatment prevented the alterations in splenocyte type (CD8+ depletion) and function (T-cell suppression) induced by stroke. In particular, examination of cytokine production from splenocyte cultures of HUCBC-treated MCAO rats revealed increased production of IL-10 and decreased production of IFNgamma relative to MCAO rats. Microglia (immunostained with a CD11b antibody) and B cells (identified with the B220 cell marker) that were increased after MCAO were dramatically decreased after HUCBC treatment. Proinflammatory cytokines such as TNF-alpha, IL-1beta and IL-2 were upregulated after MCAO surgery and their expression was abrogated after HUCBC infusion. All these findings indicate that the action of HUCBC may be specifically related to the modulation of the stroke-induced inflammatory response, providing a possible mechanism by which cord blood cells have been repeatedly reported to promote functional recovery from ischemic injury.

Bibliography: leaves 80-106. / 106, [11] leaves, [33] leaves of plates : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Studies the phagocytic function of regenerated splenic tissue to determine its potential for protection against overwhelming post-splenectomy infection, using an in vivo model established in rats. / Thesis (M.D.)--University of Adelaide, Dept. of Surgery, 1996

The circadian system provides living organisms a means to adapt their internal physiology to constantly changing environmental conditions that exists on our rotating planet, Earth. Clocks in peripheral tissues are referred to as peripheral which may participate in tissue-specific functions. The first step to investigating the circadian regulation in the peripheral tissues of avians was to examine for the presence of avian orthologs of core components of the molecular clock using Quantitative real time (qRTPCR) assays. We investigated the avian spleen for daily and circadian control of core clock genes and regulation of the inflammatory response by the spleen clock. The core clock genes, bmal1, bmal2, per2, per3 and clock displayed both daily and circadian rhythms. Proinflammatory cytokines TNFα, IL-1β, IL-6 and IL-18 exhibited daily and circadian rhythmic oscillations. A differential expression of proinflammatory cytokine induction was observed in the spleen undergoing lipopolysaccharide (LPS)-induced acute inflammation. Exogenous melatonin administration during inflammation seems to enhance some and repress a few inflammatory cytokines, implying that melatonin is pleiotropic molecule. To compare and contrast the role of peripheral clocks in regulating energy balance and reproduction in layer vs. broiler chicken, the visceral adipose tissue (VAT), ovary and hypothalamus were examined for the presence of core clock genes were investigated in these two lines of poultry birds. Quantitative RT-PCR was employed to examine daily control of core clock genes in these three peripheral tissues over a 24hr period. The layer hens exhibit rhythmic oscillations in the mRNA abundance of the core clock genes in the VAT, ovary and the hypothalamus. The hypothalamus and VAT of the broiler hens exhibit rhythmic mRNA abundance of the core clock genes. However, the clock genes in the ovary of the broiler pullets exhibit marked reduction in their amplitude and rhythms over a 24hr period. The broiler hens are prone to poor energy balance, obesity and reproductive capacity. In summary, these data provide evidence for a functional link between the circadian clock and the ovary by determining clock gene regulation under conditions of disrupted or eliminated reproductive function vs. normal reproductive output.

published_or_final_version / Medicine / Master / Doctor of Medicine

In dieser Arbeit wurde in einem Maus Tumormodell untersucht, ob durch eine intraoperative Vakzination mit gentechnisch modifizierten autologen Tumorzellen ein antitumoraler Effekt erzielt werden kann. Das Experiment erfolgte mit zwei Tumorzelllinien (B16 Melanom und Lewis Lung Karzinom). Nach Implantation der Tumorzellen in C57/BL 6 Mäuse wurden diese chirurgisch entfernt. Intraoperativ erhielten die Mäuse eine Vakzination. Dazu wurden folgende Impfstoffe verwendet: 1. subletal bestrahlte mIL-12 transfizierte Tumorzellen, 2. subletal bestrahlte pRSC transfizierte Tumorzellen und 3. frostgeschockte Tumorzellen. Die Impfung erfolgte entweder subcutan oder direkt in die Milz. Es wurde die Hypothese aufgestellt, dass eine Injektion in die Milz und eine Modifikation mit IL-12 den besten Effekt erzielt. Eine Kontrollgruppe blieb ohne Vakzin. Beobachtet wurde das Tumorwachstum, der Zeitpunkt bis zum makroskopischen Wiederauftreten eines Tumors, Überlebenszeit und die Metastasierungsrate. Versuchstiere ohne Rezidivtumor erhielten erneut einen Tumor. Es erfolgte eine erneute Evaluation des Tumorwachstums, des Zeitpunktes bis zum makroskopischen Wiederauftreten eines Tumors, der Überlebenszeit und der Metastasierungsrate. In beiden Tumorzelllinien profitierten alle Therapiegruppen nach Tumorresektion und Vakzination bezüglich Tumorrezidivrate, Zeit bis zum makroskopischenWiederauftreten des Tumors, Überlebenszeit, Metastasierungsrate und Tumorwachstumsgeschwindigkeit gegenüber der Kontrollgruppe. Vereinzelt konnten signifikante Vorteile für die direkt in die Milz applizierte Vakzine bezüglich der Tumorwachstumsgeschwindigkeit aufgezeigt werden. Weiterhin ergab sich eine geringere Tumorrezidivrate, wenn IL-12 modifizierte autologe Tumorzellen nach R0 Resektion direkt in die Milz appliziert wurden. Auch nach Tumorreimplantation konnte bezüglich Überlebenszeit und Tumorwachstumsgeschwindigkeit ein Vorteil für alle Therapiegruppen gegenüber der Kontrollgruppe herausgearbeitet werden. Nach Impfung in die Milz zeigte sich tendenziell eine geringere Metastasierungsrate. Intraoperative autologe Tumorzellvakzinationen konnten im Tiermodell in einem adjuvanten Setting einen antitumoralen Effekt auslösen. Möglicherweise kann diese Art der Impfung eine zusätzlich hilfreiche Behandlungsform zu den bisherigen adjuvanten Chemotherapeutika werden.

Currently, the intensive chemotherapy remains the first line treatment for B cell acute lymphoblastic leukemia (B-ALL). Although these regimens have significantly improved patient outcomes, their use is associated with debilitating morbidities and fatal relapses, highlighting the great need in new agents that target essential survival signals in leukemia. Thus, the overall goal of my project was to gain insights into the signaling abnormalities that regulate aberrant proliferation and survival of B-ALL cells in an effort to identify novel targets in this malignancy. This study demonstrated that pre-B cell receptor (pre-BCR)-independent spleen tyrosine kinase (SYK) activity was required for the survival and proliferation of a p53-/-PrkdcSCID/SCID mouse model of B-ALL. I extended this discovery to human disease, demonstrating that SYK was activated in primary B-ALL, independent of the pre-BCR expression. The small molecule SYK inhibitor fostamatinib (fosta) significantly attenuated proliferation of 79 primary diagnostic B-ALL samples at clinically achievable concentrations. Importantly, fosta treatment reduced dissemination of engrafting B-ALL cells into the spleen, liver, kidney and central nervous system (CNS) in a NOD.Prkdcscid/scidIl2rgtm1Wjl/SzJ xenotransplant model of B-ALL. Analysis of signaling abnormalities using a high-throughput phospho-flow cytometry platform demonstrated that pediatric and adult B-ALL samples exhibit variable basal activation of BCR, iii PI3K/AKT/mTOR, MAPK and JAK/STAT pathways. Importantly, we identified that fosta-mediated inhibition of SYK, PLC2, CRKL and EIF4E phosphorylation in B-ALL was predictive of its anti-leukemic activity, and was distinct from the cellular actions of other small molecule inhibitors of key nodal signaling pathways. Examination of molecular mechanism of fosta action by gene expression profiling revealed transcriptional effects of fosta treatment that included, most notably, potent inhibition of pathways involved in lymphocyte activation and inflammation. In conclusion, this study demonstrates that SYK signaling is crucial for B-ALL survival and provides detailed characterization of cellular and molecular mechanisms of fosta action in B-ALL. These data argue in favor of testing small molecule SYK inhibitors in pediatric and adult B-ALL.

Ionising radiation can cause damage to DNA that can result in gene mutations contributing to carcinogenesis. Radiation-protection policy currently estimates cancer risks from exposures to radiation in terms of excess risk per unit dose. At very low radiation dose-rates, where not all cells are absorbing radiation energy, this formula carries the inherent assumption that risk is limited to those cells receiving direct energy depositions. Numerous studies have now called this assumption into question. Such low dose-rates are in the relevant range that the public receives from natural background and man-made sources, and, if this fundamental assumption proves unfounded, current estimations of radiation-induced cancer risk at low doses will be incorrect. Accurate predictions of stochastic cancer risks from low-dose radiation exposures are crucial to evaluating the safety of radiation-based technologies for industry, power generation and the increasing use of radiation for medical diagnostic and screening purposes. This thesis explores phenomena known as radiation-induced bystander effects. The term bystander effects, as used here, describes biological responses to ionising radiation (hitherto observed in vitro) in cells not directly traversed by an ionising track, due to intercellular signals received from neighbouring cells that did receive energy depositions. This study aimed to determine whether radiation effects are communicated between irradiated and unirradiated cells in vivo, and if so, whether this effect alters current estimations of cancer risk following low-dose radiation exposures. In order to answer these questions, an in vivo experimental system for studying bystander effects in mice was developed. The method was based on the adoptive transfer of irradiated splenocytes into unirradiated hosts with simultaneous identification of irradiated donor cells, and biological endpoints in unirradiated bystander cells in situ using fluorescence microscopy and image analysis. Splenocytes from donor mice were radiolabelled with 3H-thymidine or received an acute X-ray dose. The irradiated donor cells, labelled with a fluorescent probe, were then adoptively transferred into unirradiated recipient mice via the tail vein, whilst control mice received sham-irradiated donor cells. A proportion of the cells lodged in the recipient mouse spleens where they remained for a period before the tissues were cryopreserved. The locations of donor cells were identified in frozen spleen sections by the fluorescent probe, and the levels of apoptosis and proliferation were simultaneously evaluated in situ in the surrounding unirradiated bystander cells using fluorescence-based assays. Transgenic pKZ1 recipient mice were also used to quantify chromosomal inversions in bystander cells. Since three-dimensional spatial relationships were preserved, responses could be measured in the local area surrounding irradiated cells as well as further afield. Following the development of the irradiated-cell adoptive transfer protocol and validation of the sensitivity and reproducibility of the biological assays in situ, a series of experiments was performed. In the initial experiments, 500,000 radiolabelled cells (0.33 mBq.cell-1) were injected into recipient mice and the spleen tissues were isolated 22 h later. No changes in apoptosis or proliferation were detected in local bystander spleen cells or throughout the spleen, compared to mice receiving sham-radiolabelled donor cells. In subsequent experiments, the effects of a number of experimental conditions were explored including the injection of tenfold more donor cells, analysis of spleen tissues after three days lodging in vivo, radiolabelling of donor cells with 100-fold higher 3H dose-rate and irradiation of donor cells ex vivo with 0.1 or 1 Gy X-rays. In each case, no changes in apoptosis or proliferation were observed. The in vivo method described here was designed to simulate the conditions of a bystander scenario from low dose-rate exposures relevant to public radiation protection. Contrary to the many reports of bystander effects in vitro, experiments using this sensitive method for examining the in vivo responses of unirradiated cells to neighbouring low-dose irradiated cells, have so far shown no changes in bystander cells in the spleen. This adoptive transfer method is the first in vivo method for examining the effects of known irradiated cells exposed to low radiation doses at low dose-rates, on neighbouring cells in situ that are truly unirradiated. Both the irradiated and bystander cells are normal, non-transformed primary spleen cells functioning in their natural environment. This in vivo experimental system allows the examination of tens of thousands of bystander cells and has shown a remarkable sensitivity, with statistical power to rule out changes in apoptosis &lt10% from the control. The relevance of in vitro bystander findings is unclear. Many reported bystander effects are more analogous to the systemic communication of abscopal effects from highly irradiated tissues. Disagreement between experimental systems and difficulty in reproducing key results between laboratories further complicate the translation of bystander data in vitro to human risk-estimation. The radiation protection community has expressed its need for in vivo validation of the bystander phenomenon before it can be included into the appraisal of carcinogenic risk. This adoptive transfer method is now available to study a range of bystander endpoints and potential signalling mechanisms in vivo, and provides a way to translate the wealth of data previously collected in vitro into findings directly relevant to human risk-estimation.

Schistosomiasis continues to be an important cause of parasitic morbidity and mortality world-wide. Determining the molecular mechanisms regulating the development of schistosome-induced pathology, including granuloma formation, fibrosis and splenomegaly, is essential for understanding how schistosomes influence the immune system of the mammalian host. I report on the first whole genome microarray analysis of the murine liver and spleen during the progression of Schistosoma japonicum infection and of S. japonicum-Soluble Egg Antigen (SEA)-stimulated macrophages. My analyses of the infected liver revealed a distinct temporal relationship between the expression of chemokines and the recruitment of cells to the liver. T-cell and B-cell chemoattractants were up-regulated earlier reflecting the recruitment of these cells to the liver as illustrated by flow cytometry. The later phases of the response corresponded with peak accumulation of eosinophils, neutrophils, macrophages and myofibroblasts/hepatic stellate cells (HSCs) and the expression of chemokines with activity for these cells including CCL11, members of the Monocyte-chemoattractant protein family (CCL7, CCL8, CCL12) and the hepatic stellate cell/myofibroblast chemoattractant CXCL1. Peak expression of macrophage chemoattractants (CCL6, CXCL14) and markers of alternatively-activated macrophages (e.g. Retnla) during this later phase provided further evidence for a role for these cells in schistosome-induced pathology. Additionally, I demonstrated that CCL7 immunolocalises to the fibrotic zone of granulomas. Furthermore, striking up-regulation of neutrophil markers and the localisation of neutrophils and the neutrophil chemokine S100A8 to fibrotic areas suggested the involvement of neutrophils in S. japonicum¬-induced hepatic fibrosis. The transcriptional profile of the spleen was closely related to changes in cellular composition illustrated by flow cytometry and immunohistochemistry. Significant up-regulation of genes associated with progression through the cell cycle, proliferation makers and genes involved in lymphocyte proliferation, paralleled the initial expansion of T-cells and B-cells and the increased cellularity of the spleen overtime. Accumulation of eosinophils, neutrophils and macrophages was paralleled by enhanced expression of markers for these cells and the declining proportion of B- and T-cells in the spleen over time was reflected in the decreased expression of B- and T-cell markers. Significant up-regulation of Chi3l3 and F4/80+ macrophages suggested the presence of alternatively activated macrophages in the spleen, where these cells could play an immunoregulatory role. Comparison of the liver and spleen profiles revealed divergent expression of chemokines and cell adhesion molecules. Expression of lymphocyte chemokines including the homeostatic chemokines, CXCL13, CCL19 and CCL21, were significantly up-regulated in the liver while down regulated in the spleen. Expression of chemokines with activity for eosinophils (CCL11, CCL24), neutrophils (CXCL1) and monocytes (CXCL14, CCL12) and the cell adhesion molecules VCAM1, NCAM1, PECAM1 were up-regulated in the liver while unchanged in the spleen. Chemokines up-regulated in both organs were expressed at significantly higher levels in the liver. Divergent expression of chemokines and cell adhesion molecules likely contributes to the development of a chemotactic signalling gradient that promotes recruitment of effector cells to the liver. The results of liver and spleen microarrays suggested an important role for alternatively activated macrophages in the development of schistosome-induced pathology. This led me to investigate the in vivo transcriptional profile of S. japonicum SEA-stimulated peritoneal macrophages. The transcriptional profile of these cells was characterised by up-regulation of alternatively activated macrophage makers (Chi3l3, Chi3l4, Arg1). Retnla was not significantly induced in these macrophages suggesting that the specific function of these cells may differ to those induced by S. mansoni and other parasites. Other features of the transcriptional profile of these cells included modulated expression of T-cell co-stimulatory molecules and chemokines which may confer immunomodulatory activity. S. japonicum-stimulated alternative activation of macrophages was additionally associated with deactivation of classical activation pathways and altered expression of cell surface receptors and complement components that may alter phagocytic activity. Together these data significantly enhance our understanding of the mechanisms associated with alternative activation of macrophages and provide significant insight into the role of these cells in schistosomiasis japonica. The findings presented in this thesis represent the most comprehensive description to date of the molecular mechanisms, and especially chemotactic signalling pathways, regulating the development of schistosome-induced granulomas, fibrosis, splenomegaly and alternative macrophage activation in the murine host. In summary, my data have revealed that co-ordinated gene expression of chemokines in the liver and spleen regulates the recruitment of cells to the liver during schistosome infection. My results provide additional evidence for a role for neutrophils and alternatively activated macrophages in the development of schistosome-induced pathology and provide further insight to the molecular basis of alternative macrophage activation during infection. Furthermore, my data serve to highlight clear differences in the pathogenesis of schistosomiasis mansoni and schistosomiasis japonica. Together these findings further our understanding of the systemic, local, cellular, and especially, chemokine signalling pathways that regulate the development of S. japonicum-induced pathology and offer correlative insight into the pathogenesis of other chronic inflammatory diseases where fibrosis, splenomegaly and alternative activation of macrophages are common features.

Orientador: Edson Luis Maistro / Resumo: Embora a medicina moderna esteja bem desenvolvida, de acordo com a Organização Mundial da Saúde, cerca de 75 à 95% da população utiliza a medicina popular para alívio de alguma sintomatologia desagradável. A grande preocupação é que mesmo as plantas medicinais sendo muito utilizadas, não se tem um conhecimento completo sobre sua ação no organismo. A planta Crataegus oxyacantha, também conhecida como espinheiro branco ou Hawthorn, é originária da Europa, América do Norte e Ásia, foi levada para outros continentes e vem sendo muito utilizada devido a seus potenciais efeitos farmacológicos, como agente cardiotônico, antioxidante, hipolipidêmico, anti-inflamatório, sedativo, entre outros. Considerando a importância da planta C. oxyacantha como medicamento alternativo natural e a inexistência de estudos envolvendo a toxicidade celular da mesma; o presente estudo teve como objetivo analisar os efeitos morfo-histológicos do extrato de frutos de C. oxyacantha em três grupos de tratamento I (50mg/Kg), II (100 mg/Kg), e III (200 mg/Kg) em células do fígado e baço de camundongos, a fim de avaliar os efeitos citotóxicos do extrato. O fígado dos indivíduos do grupo I não apresentou dano. Os indivíduos do grupo II apresentaram fígado em estágios iniciais de desorganização tissular e citoplasática. Já aqueles do grupo III sofreram maiores alterações histológicas como extensa desorganização citoplasmática, surgimento de vacúolos e capilares sinusóides aumentados e grande quantidade de célula... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Although modern medicine is well developed, according to the World Health Organization, about 75 to 95% of the population uses tradicional medicine to relieve some unpleasant symptoms. The major concern is that even medicinal plants being heavily used, there is no complete knowledge about their action in the body. The plant Crataegus oxyacantha, also known as white hawthorn or Hawthorn, is native to Europe, North America and Asia, was taken to other continents and has been widely used due to its potential pharmacological effects, such as cardiotonic, antioxidant, hypolipidemic, anti-inflammatory, sedative, among others. Considering the importance of the C. oxyacantha plant as a natural alternative medicine and the lack of studies involving its cellular toxicity; the objective of this study was to analyze the morpho-histological effects of C. oxyacantha fruit extract in three treatment groups I (50 mg / kg), II (100 mg / kg) and III (200 mg / kg) in liver and spleen cells of mice, in order to evaluate the cytotoxic effects of the extract. The liver of the individuals in group I showed no damage. Individuals in group II presented liver in the early stages of tissue and cytoplasmic disorganization. Already those of group III suffered major histological alterations such as extensive cytoplasmic disorganization, emergence of vacuoles and increased sinusoidal capillaries and large amount of Kupffer cells. On the other hand, the spleen was not histologically modified after the treat... (Complete abstract click electronic access below) / Mestre