DSpace Community:http://hdl.handle.net/1843/4382024-03-28T19:08:11Z2024-03-28T19:08:11ZEstratégias alternativas de imuno-modulação nas doenças inflamatórias intestinaishttp://hdl.handle.net/1843/597232023-10-19T16:46:52Z2011-09-05T00:00:00ZTitle: Estratégias alternativas de imuno-modulação nas doenças inflamatórias intestinais
Abstract: To maintain intestinal homeostasis, the immune system must faithfully respond to antigens from pathogenic microbes while maintaining a state of tolerance to commensal microorganisms and food antigens that confront it every day. However, disruption of this thin balance can cause inflammatory bowel disease (IBD) in genetic susceptible hosts. IL-10 is a regulatory cytokine that plays a major role in the homeostasis of the gut mucosa and this is illustrated by the fact that IL-10-deficient mice develop spontaneous colitis. In this study, IL-10-/- mice were analyzed for immunological changes accompanying the development of colitis at different ages. Defects in regulatory elements at the gut mucosa such as lower frequency of CD4+CD25+Foxp3+ T cells could be detected since 6 weeks of age. In parallel, there was an increased frequency of activated T cells in the colon. Colitis progression culminates with the reduction in the frequency of TCRγδ+ intraepithelial lymphocytes (IEL) and CD4+LAP+ regulatory T cells in the small and large intestines. Production of IL-17 was higher in the colon at 6 weeks of age but at 16 weeks of age levels of IFN-increased in the colon. TGF-β was increased in the proximal jejunum of mice with colitis. Frequencies of B1 cells were elevated in peritoneum, Peyer´s patches and gut lamina propria. There were alterations in serum immunoglobulin levels and secretory IgA production was increased. Despite all these alterations, 16-week-old IL-10-deficient mice could be rendered tolerant by a continuous feeding protocol of antigen administration. We next tested two therapeutic alternatives for the treatment of IBD. The first one consisted of a diet where proteins were replaced by free aminoacids or short chain peptides. The diet containing free aminoacids aggravated gut inflammation in DSS induced colitis model with reduction in secretory IgA. Likewise, the diet with protein hydrolizates was not able to ameliorate the acute colitis. As a second therapeutic alternative, we used a strain of Lactococcus lactis genetically engineered to deliver Hsp65 in the gut mucosa. Oral administration of Hsp65-producing L. lactis prevented DSSinduced colitis; abolishing weight loss, diarrhea and fecal bleeding. The effectcould not be explained by alterations in secretory IgA or in intestinal permeability. However, mucosal protection was accompanied by diminished levels of TNF-, IL-6, IL-4 and IL-5 and by enhanced IL-10 in colonic mucosa. In addition, mice treated with Hsp65-producing L. lactis had increased frequency Treg cells in the spleen. Moreover, the ratio between effector T cells and Tregs was similar to the one found in control health mice. There was enhanced frequency of dendritic cells expressing CD80, CD86, CD40, CD103, TLR2 e TLR4 in the spleen of mice that received Hsp65-producing L. lactis. Ability of Hsp supernatant in enhancing expression of TLR2 and TLR4 was confirmad in vitro. Moreover, Hsp65-producing L. lactis did not improve the ulcerative colitis in TLR2-/-. In IL-10-/- mice, Hsp65-producing L. lactis was able to reduce the signs of inflammation. Likewise, frequency of CD4+LAP+ Tregs and colonic levels of TGF- increased after treatment with Hsp65-producing L. lactis. Our study provides detailed analysis of changes that precede colitis in genetically susceptible mice and it also suggests that Hsp-65 producing L. lactis could be one alternative treatment to IBD.
Type: Tese2011-09-05T00:00:00ZMorfina inibe corrente mediada por receptores mGluR-I em neurônios do Locus Coeruleushttp://hdl.handle.net/1843/556152023-06-30T15:57:36Z2023-01-01T00:00:00ZTitle: Morfina inibe corrente mediada por receptores mGluR-I em neurônios do Locus Coeruleus
Abstract: Locus Coeruleus (LC) neurons express several types of G-protein coupled
receptors that regulate their cellular excitability and, consequently, the liberation of
noradrenaline throughout the central nervous system. This study presents a
comparison of the relative strength of two of these receptors that have opposite
effects on excitability: Group I mGluR (mGluR-I) receptors and μ-opioid receptors (μ).
As expected, based on many previous studies, morphine (5 μM) hyperpolarized LC
neurons, and this effect was antagonized by the selective μ inverse agonist naloxone.
This well-known effect of morphine is mediated by opioid receptors that activate
KIR-type potassium channels. Subsequent application of the mGluR-I agonist, DHPG
(30 μM) in the presence of morphine completely reversed the inhibitory action of μ.
The possibility that the two receptors exert their opposite effects through a common
mechanism mediated by KIR-type potassium channels was tested in two ways: i) by
examining changes in membrane conductance associated with receptor activation
and ii) by examining the I-V relationship of the underlying currents. Both approaches
failed to provide evidence for a common mechanism. During the experiments, we
observed a surprising functional interaction between the receptors in those currents
evoked by DHPG and measured under voltage clamp were smaller in the presence
of morphine than in control conditions. Further voltage clamp experiments using ramp
protocols again confirmed that this interaction was not mediated by potassium
channels. The results reveal a previously unknown interaction between two receptors
that exert a strong antagonistic modulatory influence on the activity of the Locus
Coeruleus neurons.
Type: Dissertação2023-01-01T00:00:00ZEstudo imuno-histoquímico e molecular de gliomashttp://hdl.handle.net/1843/551082023-06-19T16:20:48Z2014-02-28T00:00:00ZTitle: Estudo imuno-histoquímico e molecular de gliomas
Abstract: Gliomas are the most common primary brain tumor, accounting for over 70% of all primary CNS neoplasms. They are histologically heterogeneous tumors and classified by the World Health Organization to varying degrees, according to their cellular characteristics and its invasiveness. The astrocytomas, oligodendrogliomas and ependymal cell tumors represent the types of gliomas. In gliomas, changes occur in different signaling pathways and from the knowledge of such changes it may be established ways of diagnosis of these tumors; and a better prognosisi can be made. Moreover, these signaling pathways are important as potential therapeutic targets. The molecular alterations present in various types and subtypes of gliomas may be different, and it is important for the understanding of the disease and may be useful for diagnosis. Thus, we studied in this work, molecular and immunohistochemical methods, the frequency of polymorphisms and mutations in PTEN, KRAS, BRAF, IDH1 and IDH2 and the expression of PTEN, BRAF, TGF-β and Ki-67 in association with clinical features of 38 patients with glioma. In sequence analysis, we detected a silent mutation in PTEN in a patient with glioblastoma multiforme, and the R132H mutation in IDH1 in a patient with low-grade astrocytoma. In the remaining genes, we did not detect mutations or polymorphisms. We found a significant difference between the highest Ki-67 expression in high grade tumors compared to low grade tumors, as well as overexpression in BRAF II and grade IV gliomas, when compared to the I grade gliomas In expression analysis TGF-β II receptor, increased expression of the protein observed in the glioma compared to normal tissues. Both TβRII as in PTEN, we found no correlation between the expression and the stages of gliomas. Our data demonstrate that gliomas have superexpression of BRAF, regardless of the presence of BRAFV600E mutation. The change in expression between grades may be associated with malignant progression of these tumors. Gliomas have increased expression of TβRII, irrespective of the histological grade of the tumors, suggesting a role for TGF-β pathway in the pathogenesis of gliomas. The association of PTEN expression and gliomas can not be demonstrated. We did not detect changes in the KRAS and BRAF genes, but we can not rule out the importance of these genes in the formation of gliomas. Studies with larger numbers of samples of gliomas with the same tumor stages are needed to assess the influence of PTEN, KRAS and BRAF in the formation and progression of gliomas.
Type: Tese2014-02-28T00:00:00ZEfeito duradouro da solução hipertônica sobre o tamanho dos quanta na junção neuromuscular de camundongos com deficiência do transportador vesicular de acetilcolinahttp://hdl.handle.net/1843/320202020-01-21T06:32:44Z2015-10-29T00:00:00ZTitle: Efeito duradouro da solução hipertônica sobre o tamanho dos quanta na junção neuromuscular de camundongos com deficiência do transportador vesicular de acetilcolina
Abstract: After application of hypertonic solution to synapses, there is a long
lasting increase in neurotransmitter release, as evidenced by an increase in the
size of miniature endplate potentials (MEPPs). The increase in quantal size was
interpreted as being due to increased incorporation of the acetylcholine into
readily available to release vesicles in a process dependent of the vesicle
acethylcholine transporter (VAChT). This process was called vesicle second
stage loading. Our goal was to test this hypothesis using non pharmacological
tools, in order to study the participation of VAChT in this long lasting changes in
neurotransmission. We used neuromuscular preparations from genetically
modified C57BL male mice with reduced VAChT expression (KDVAChT). We
used animals at two different ages to evaluate the temporal development of
second stage loading. To measure MEPPs, we used the current clamp
technique. We recorded about 100 MEPPs during 300 seconds at five different
fibers of each animal, before and after treatment with hypertonic solution (NaCl
234mM). To measure the MEPCs, we used the voltage clamp technique and a
similar sampling protocol. We did not detect significant differences between WT
and KDVAChT in MEPP size before and after treatment with hypertonic
solution. In WT 3 months old animals hypertonic treatment increased MEPPs
from 1.13 ± 0.19 to 1.55 ± 0.13 mV (n = 9) and in KDVAChT it increased from
0.79 ± 0, 09 mV to 1.21 ± 0.11 mV (n = 5). In 12 months old animals hypertonic
treatment increased MEPP amplitude from 1.29 ± 0.14 mV to 1.69 ± 0.15 mV (n
= 11) and in KDVAChT it increased from 0.98 ± 0, 14 mV to 1.75 ± 0.15 mV (n =
9). We did not observe significant differences in the size of MEPCs between WT
and KDVAChT mice, when recorded before hypertonic stimulation. MEPCs size
in WT animals was 3.02 ± 0.19 nA, n = 3, while in KDVAChT animals it was
2:57 ± 0:23 nA, n = 4. We conclude that reducing VAChT did not change
second stage loading, which suggests that VAChT is not the target of
hypertonic solution effect, or there may be unknown mechanisms to
compensate vesicular filling in reduced VAChT animals.
Type: Dissertação2015-10-29T00:00:00Z