Nonspecific down-regulation of CD8+ T-Cell responses in mice expressing human Papillomavirus Type 16 E7 oncoprotein from the keratin-14 promoter

The E7 oncoprotein of human papillomavirus 16 (HPV16) transforms basal and suprabasal cervical epithelial cells and is a tumor-specific antigen in cervical carcinoma, to which immunotherapeutic strategies aimed at cytotoxic T-lymphocyte (CTL) induction are currently directed. By quantifying major histocompatibility complex class I tetramer-binding T cells and CTL in mice expressing an HPV16 E7 transgene from the keratin-l l (K14) promoter in basal and suprabasal keratinocytes and in thymic cortical epithelium, we show that antigen responsiveness of both E7- and non-E7-specific CD8(+) cells is down-regulation compared to non-E7 transgenic control mice. We show that the effect is specific for E7, and not another transgene, expressed from the K14 promoter, Down-regulation did not involve deletion of CD8(+) T cells of high affinity or high avidity, and T-cell receptor (TCR) VP-chain usage and TCR receptor density were similar in antigen-responsive cells from E7 transgenic and non-E7 transgenic mice. These data indicate that E7 expressed chronically from the K14 promoter nonspecifically down-regulates CD8+ T-cell responses. The in vitro data correlated with the failure of immunized E7 transgenic mice to control the growth of an E7-expressing tumor challenge, We have previously shown that E7-directed CTL down-regulation correlates with E7 expression in peripheral but not thymic epithelium (T, Dean et al., J, Virol. 73:6166-6170, 1999), The findings have implications for the immunological consequences of E7-expressing tumor development and E7-directed immunization strategies. Generically, the findings illustrate a T-cell immunomodulatory function for a virally encoded human oncoprotein.

Chronic hypoxia induced down-regulation of angiotensinogen expression in rat epididymis

The presence of an intrinsic renin-angiotensin system (RAS) in the rat epididymis has been previously established by showing the expression of several key RAS components, and in particular angiotensinogen, the indispensable element for the intracellular generation of angiotensin II. In this study, the possible involvement of this local epididymal RAS in the testicular effects of chronic hypoxia was investigated. Semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR), Western blotting and by in situ hybridization histochemistry of the rat epididymis were used to show changes in localization and expression of angiotensinogen. Results from RT-PCR analysis demonstrated that chronic hypoxia caused a marked decrease (60%) in the expression of angiotensinogen mRNA, when compared with that in the normoxic epididymis. Western blot analysis demonstrated a less decrease (35%) in the expression of angiotensinogen protein. In situ hybridization histochemistry showed that the reduced angiotensinogen mRNA in chronic hypoxia was specifically localized to the epididymal epithelium from the cauda, corpus and caput regions of the epididymis; a distribution similar to that of normoxic rats. It was concluded that chronic hypoxia decreases the transcriptional and translational expression of angiotensinogen, and thus local formation of angiotensin II, in the rat epididymis. (C) 2001 Elsevier Science B.V. All rights reserved.

Cytokine regulation of syndecan-1 and-2 gene expression in human periodontal fibroblasts and osteoblasts

Cell-surface proteoglycans participate in several biological functions including interactions with a variety of growth factors and cytokines. Regulation of syndecan-1 and -2 gene expression was investigated in human periodontal ligament fibroblasts (PDLF), osteoblasts (OB) and gingival fibroblasts (GF), in response to platelet-derived growth factor (PDGF-BB), transforming growth factor (TGF-beta(1)), and interleukin (IL-1beta) by Northern blot analyses. We also compared the effect of PDGF-BB and TGF-beta(1), separately and in combination, in the prolonged presence of IL-1beta on the expression of both syndecan genes. The results demonstrated that the three cell lines regulated the expression of syndecan-1 and -2 in response to growth factors and cytokines in different manners. These cell lines increased syndecan-1 mRNA levels in response to either PDGF-BB or TGF-beta(1) and decreased levels in response to IL-1beta. The effect of IL-1beta on syndecan-1 mRNA synthesis was partially reversed after adding PDGF-BB and TGF-beta(1), separately or in combination, in the presence of IL-1beta. In contrast, syndecan-2 mRNA level was markedly upregulated in response to either TGF-beta(1) or IL-1beta in OB when compared with the other two cell lines. However, the stimulatory effect of TGF-beta(1) on syndecan-2 mRNA production in OB was abolished in the prolonged presence of IL-1beta. These findings lend support to the notion that syndecan-1 and syndecan-2 have distinct functions which correlate with their source and functions within the periodontium.

Ecological regulation of development: Induction of marine invertebrate metamorphosis

In the marine environment a wide range of invertebrates have a pelagobenthic lifecycle that includes planktonic larval and benthic adult phases. Transition between these morphologically and ecologically distinct phases typically occurs when the developmentally competent larva comes into contact with a species-specific environmental cue. This cue acts as a morphogenetic signal that induces the completion of the postlarval/juvenile/adult developmental program at metamorphosis. The development of competence often occurs hours to days after the larva is morphologically mature. In the non-feeding - lecithotrophic - larvae of the ascidian Herdmania curvata and the gastropod mollusc Haliotis asinina, gene expression patterns in pre-competent and competent stages are markedly different, reflecting the different developmental states of these larval stages. For example, the expression of Hemps, an EGF-like signalling peptide required for the induction of Herdmania metamorphosis, increases in competent larvae. Induction of settlement and metamorphosis results in further changes in developmental gene expression, which apparently is necessary for the complete transformation of the larval body plan into the adult form.

The Phox Homology (PX) domain-dependent, 3-phosphoinositide-mediated association of sorting nexin-1 with an early sorting endosomal compartment is required for its ability to regulate epidermal growth factor receptor degradation

Recent studies have shown that phox homology (PX) domains act as phosphoinositide-binding motifs. The majority of PX domains studied show binding to phosphatidylinositol 3-monophosphate (Ptdlns(3)P), an association that allows the host protein to localize to membranes of the endocytic pathway. One issue, however, is whether PX domains may have alternative phosphoinositide binding specificities that could target their host protein to distinct subcellular compartments or allow their allosteric regulation by phosphoinositides other than PtdIns(3)P. It has been reported that the PX domain of sorting nexin 1 (SNX1) specifically binds phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P-3) (Zhong, Q., Lazar, C. S., Tronchere, H., Sato, T., Meerloo, T., Yeo, M., Songyang, Z., Emr, S. D., and Gill, G. N. (2002) Proc. Natl. Acad. Sci. U. S. A. 99,6767-6772). In the present study, we have shown that whereas SNX1 binds PtdIns(3,4,5)P-3 in protein:lipid overlay assays, in liposomes-based assays, binding is observed to PtdIns(3)P and phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P-2) but not to PtdIns(3,4,5)P-3. To address the significance of PtdIns(3,4,5)P-3 binding, we examined the subcellular localization of SNX1 under conditions in which plasma membrane PtdIns(3,4,5)P-3 levels were significantly elevated. Under these conditions, we failed to observe association of SNX1 with this membrane. However, consistent with the binding to PtdIns(3)P and PtdIns(3,5)P-2 being of more physiological significance was the observation that the association of SNX1 with an early endosomal compartment was dependent on a 3-phosphoinositide-binding PX domain and the presence of PtdIns(3)P on this compartment. Finally, we somal association of SNX1 is important for its ability to regulate the targeting of internalized epidermal growth factor receptor for lysosomal degradation.

Fibroblast growth factor receptor 4 (FGFR4) expression in newborn murine calvaria and primary osteoblast cultures

Fibroblast growth factor receptor (FGFR) signalling is important in the initiation and regulation of osteogenesis. Although mutations in FGFR1, 2 and 3 genes are known to cause skeletal deformities, the expression of FGFR4 in bony tissue remains unclear. We have investigated the expression pattern of FGFR4 in the neonatal mouse calvaria and compared it to the expression pattern in cultures of primary osteoblasts. Immunohistochemistry demonstrated that FGFR4 was highly expressed in rudimentary membranous bone and strictly localised to the cellular components (osteoblasts) between the periosteal and endosteal layers. Cells in close proximity to the newly formed osteoid (preosteoblasts) also expressed FGFR4 on both the endosteal and periosteal surfaces. Immunocytochemical analysis of primary osteoblast cultures taken from the same cranial region also revealed high levels of FGFR4 expression, suggesting a similar pattern of cellular expression in vivo and in vitro. RT-PCR and Western blotting for FGFR4 confirmed its presence in primary osteoblast cultures. These results suggest that FGFR4 may be an important regulator of osteogenesis with involvement in preosteoblast proliferation and differentiation as well as osteoblast functioning during intramembranous ossification. The consistent expression of FGFR4 in vivo and in vitro supports the use of primary osteoblast cultures for elucidating the role of FGFR4 during osteogenesis.

Placental growth hormone is not suppressed by oral glucose loading in normal human pregnancy

Placental growth hormone (PGH) progressively replaces pituitary growth hormone in the maternal circulation from mid-gestation onwards in human pregnancy. Our previous investigations have shown that placental growth hormone concentrations correlate well with foetal growth. Despite the apparent correlation between PGH and birthweight, the physiology of its secretion during pregnancy has not been well defined. We investigated the response of maternal serum PCH to oral glucose loading in pregnant women (n = 24) who demonstrated normal glucose tolerance at a mean gestation of 29 weeks. Mean (SEM) fasting PGH concentrations were high (36.9 [6.4] ng/ml). No suppression of PGH was noted at one, two or three hours after a 75 g oral glucose load. Similarly, no changes were noted in growth hormone binding protein or in calculated free PGH over the course of the glucose tolerance test. As expected, insulin concentrations rose sixfold and insulin like growth factor binding protein 1 concentrations fell by 20% with glucose loading. Cot-relation analysis showed maternal weight, BMI, fasting serum glucose serum insulin to be significantly correlated with the babies' birthweight. Our results support the proposition that PGH concentrations in maternal serum are not Suppressed by oral glucose loading in non-diabetic mothers.

Transcriptional regulation of the sodium-sulfate cotransporter NaSi-1 gene

Inorganic sulfate is one of the most abundant anions in mammalian plasma and is essential for proper cell growth and development, as well as detoxification and activation of many biological compounds. To date, little is understood how physiological levels of sulfate are maintained in the body. Our studies, and of others, have identified the NAS(i)-1 protein to be a functional sulfate transporter in the kidney and intestine, and due to this localization, constitutes a strong candidate gene for maintaining body sulfate homeostasis. Several factors, including hormones and metabolic conditions, have been shown to alter NAS(i)-1 mRNA and protein levels in vivo. In this study, we describe the transcriptional regulation of NaSi-1, with a focus on the mouse NaSi-1 gene (Nas1) that was recently cloned in our laboratory. Vitamin D (1,25-(OH)(2)D-3) and thyroid hormone (T-3) led to an increase in Nas1 promoter activity in OK cells. Mutational analysis of the Nas1 promoter resulted in identification of a direct repeat 6-type vitamin-D-responsive element (DR6 VDRE) at -525 to -508 and an imperfect inverted repeat 0-type T-3 responsive element (IRO T3RE) at -426 to -425 which conferred 1,25-(OH)(2)D-3 and T-3 responsiveness respectively. These findings suggest for vitamin D and thyroid hormone regulation of NaSi-1, may provide important clues to the physiological control of sulfate homeostasis.

Targeting lactate transport suppresses in vivo breast tumour growth

Background: Most cancers, including breast cancer, have high rates of glucose consumption, associated with lactate production, a process referred as “Warburg effect”. Acidification of the tumour microenvironment by lactate extrusion, performed by lactate transporters (MCTs), is associated with higher cell proliferation, migration, invasion, angiogenesis and increased cell survival. Previously, we have described MCT1 up-regulation in breast carcinoma samples and demonstrated the importance of in vitro MCT inhibition. In this study, we performed siRNA knockdown of MCT1 and MCT4 in basal-like breast cancer cells in both normoxia and hypoxia conditions to validate the potential of lactate transport inhibition in breast cancer treatment. Results: The effect of MCT knockdown was evaluated on lactate efflux, proliferation, cell biomass, migration and invasion and induction of tumour xenografts in nude mice. MCT knockdown led to a decrease in in vitro tumour cell aggressiveness, with decreased lactate transport, cell proliferation, migration and invasion and, importantly, to an inhibition of in vivo tumour formation and growth. Conclusions: This work supports MCTs as promising targets in cancer therapy, demonstrates the contribution of MCTs to cancer cell aggressiveness and, more importantly, shows, for the first time, the disruption of in vivo breast tumour growth by targeting lactate transport.

Legal and political regulation of higher education in Portugal: an evolutionary perspective on the current policy issues and the problem of order

The higher education system in Europe is currently under stress and the debates over its reform and future are gaining momentum. Now that, for most countries, we are in a time for change, in the overall society and the whole education system, the legal and political dimensions have gained prominence, which has not been followed by a more integrative approach of the problem of order, its reform and the issue of regulation, beyond the typical static and classical cost-benefit analyses. The two classical approaches for studying (and for designing the policy measures of) the problem of the reform of the higher education system - the cost-benefit analysis and the legal scholarship description - have to be integrated. This is the argument of our paper that the very integration of economic and legal approaches, what Warren Samuels called the legal-economic nexus, is meaningful and necessary, especially if we want to address the problem of order (as formulated by Joseph Spengler) and the overall regulation of the system. On the one hand, and without neglecting the interest and insights gained from the cost-benefit analysis, or other approaches of value for money assessment, we will focus our study on the legal, social and political aspects of the regulation of the higher education system and its reform in Portugal. On the other hand, the economic and financial problems have to be taken into account, but in a more inclusive way with regard to the indirect and other socio-economic costs not contemplated in traditional or standard assessments of policies for the tertiary education sector. In the first section of the paper, we will discuss the theoretical and conceptual underpinning of our analysis, focusing on the evolutionary approach, the role of critical institutions, the legal-economic nexus and the problem of order. All these elements are related to the institutional tradition, from Veblen and Commons to Spengler and Samuels. The second section states the problem of regulation in the higher education system and the issue of policy formulation for tackling the problem. The current situation is clearly one of crisis with the expansion of the cohorts of young students coming to an end and the recurrent scandals in private institutions. In the last decade, after a protracted period of extension or expansion of the system, i. e., the continuous growth of students, universities and other institutions are competing harder to gain students and have seen their financial situation at risk. It seems that we are entering a period of radical uncertainty, higher competition and a new configuration that is slowly building up is the growth in intensity, which means upgrading the quality of the higher learning and getting more involvement in vocational training and life-long learning. With this change, and along with other deep ones in the Portuguese society and economy, the current regulation has shown signs of maladjustment. The third section consists of our conclusions on the current issue of regulation and policy challenge. First, we underline the importance of an evolutionary approach to a process of change that is essentially dynamic. A special attention will be given to the issues related to an evolutionary construe of policy analysis and formulation. Second, the integration of law and economics, through the notion of legal economic nexus, allows us to better define the issues of regulation and the concrete problems that the universities are facing. One aspect is the instability of the political measures regarding the public administration and on which the higher education system depends financially, legally and institutionally, to say the least. A corollary is the lack of clear strategy in the policy reforms. Third, our research criticizes several studies, such as the one made by the OECD in late 2006 for the Ministry of Science, Technology and Higher Education, for being too static and neglecting fundamental aspects of regulation such as the logic of actors, groups and organizations who are major players in the system. Finally, simply changing the legal rules will not necessary per se change the behaviors that the authorities want to change. By this, we mean that it is not only remiss of the policy maker to ignore some of the critical issues of regulation, namely the continuous non-respect by academic management and administrative bodies of universities of the legal rules that were once promulgated. Changing the rules does not change the problem, especially without the necessary debates form the different relevant quarters that make up the higher education system. The issues of social interaction remain as intact. Our treatment of the matter will be organized in the following way. In the first section, the theoretical principles are developed in order to be able to study more adequately the higher education transformation with a modest evolutionary theory and a legal and economic nexus of the interactions of the system and the policy challenges. After describing, in the second section, the recent evolution and current working of the higher education in Portugal, we will analyze the legal framework and the current regulatory practices and problems in light of the theoretical framework adopted. We will end with some conclusions on the current problems of regulation and the policy measures that are discusses in recent years.

Regulation of PLK4 levels and activity to ensure centriole number control

Dissertation presented to obtain a Ph.D degree in Cellular Biology

Regulation of the rice gene OsRMC under salt stress: identification and functional characterization of novel transcription factors

Dissertation presented to obtain the Ph.D degree in Biology

Regulation of gene expression by SnRK1 kinases and miRNAs during the plant stress response

Dissertation presented to obtain the Ph.D degree in Plant Physiology

Regulation and function of the cell wall polymer lipoteichoic acid in staphylococcus aureus

A thesis submitted for the Degree of Master in Medical microbiology