Biochemical and spectroscopic properties of Brucella microti glutamate decarboxylase, a key component of the glutamate-dependent acid resistance system

In orally acquired bacteria, the ability to counteract extreme acid stress (pH < 2.5) ensures survival during transit through the animal host stomach. In several neutralophilic bacteria, the glutamate-dependent acid resistance system (GDAR) is the most efficient molecular system in conferring protection from acid stress. In Escherichia coli its structural components are either of the two glutamate decarboxylase isoforms (GadA, GadB) and the antiporter, GadC, which imports glutamate and exports γ-aminobutyrate, the decarboxylation product. The system works by consuming protons intracellularly, as part of the decarboxylation reaction, and exporting positive charges via the antiporter. Herein, biochemical and spectroscopic properties of GadB from Brucella microti (BmGadB), a Brucella species which possesses GDAR, are described. B. microti belongs to a group of lately described and atypical brucellae that possess functional gadB and gadC genes, unlike the most well-known "classical" Brucella species, which include important human pathogens. BmGadB is hexameric at acidic pH. The pH-dependent spectroscopic properties and activity profile, combined with in silico sequence comparison with E. coli GadB (EcGadB), suggest that BmGadB has the necessary structural requirements for the binding of activating chloride ions at acidic pH and for the closure of its active site at neutral pH. On the contrary, cellular localization analysis, corroborated by sequence inspection, suggests that BmGadB does not undergo membrane recruitment at acidic pH, which was observed in EcGadB. The comparison of GadB from evolutionary distant microorganisms suggests that for this enzyme to be functional in GDAR some structural features must be preserved

The Embryonic Protein Nodal Supports Metastatic Phenotypes in Breast Cancer

Metastasis is the process by which tumour cells disseminate to distant organ sites. Aberrant expression of stem cell-associated proteins within tumours is associated with metastasis and poor patient prognosis. One example of a stem cell factor that is associated with cancer progression is Nodal, a member of the TGF-β superfamily. Nodal is normally limited to pluripotent stem cells during embryonic development, and to specialized dynamic adult tissue (such as the cycling endometrium), but is aberrantly re-expressed in multiple cancer types, including melanoma, glioma, prostate cancer, and pancreatic cancer. The central objective of this thesis is to determine the role of Nodal during various aspects of the metastatic cascade in breast cancer. First, I determined that Nodal inhibition in aggressive breast cancer cell lines impairs tumour growth in an orthotopic nude mouse model, concomitant with reduced proliferation and enhanced apoptosis. Furthermore, in an experimental metastasis assay in NOD/SCID/MPSVII mice, I determined that Nodal knockdown prevents the transition from lung micrometastases to macrometastes, by supporting a positive ratio of proliferation to apoptosis. Using numerous animal models, I then discovered that Nodal promotes angiogenesis, and that knocking down its expression in established tumours reduces vascularization and causes necrosis. Notably, Nodal protein was positively correlated with vascular density in human breast cancer lesions. Mechanistically, Nodal induced a pro-angiogenic profile in breast cancer cells by upregulating VEGF and PDGF. Finally, I investigated the role of Nodal in the regulation of EMT and invasion; phenotypes that are classically associated with this morphogen. Specifically, since Nodal is implicated in mammary gland remodeling and placentation, I examined its effects on cellular invasion in these contexts. Nodal overexpression in poorly metastatic breast cancer and choriocarcinoma cell lines enhanced invasion and EMT-associated changes in gene expression, and this effect was in part mediated by ERK signaling. Nodal inhibition in metastatic breast cancer cell lines reduced spontaneous metastasis to the liver (but not the lung) in NOD/SCID/IL2γR- mice. The results presented herein suggest that Nodal promotes several pro-metastatic processes. Given its restriction to embryonic or highly specialized adult contexts, targeting Nodal in breast cancer poses an exciting avenue for therapeutic intervention

Wild birds as carriers of antimicrobial-resistant and ESBL-producing Enterobacteriaceae

open6noopenDotto, G.; Menandro, M.L.; Mondin, A.; Martini, M.; Tonellato, F.R.; Pasotto, D.Dotto, Giorgia; Menandro, MARIA LUISA; Mondin, Alessandra; Martini, Marco; Tonellato, F. R.; Pasotto, Daniel

Nitric Oxide Regulates Neurogenesis in the Hippocampus following Seizures

Hippocampal neurogenesis is changed by brain injury. When neuroinflammation accompanies injury, activation of resident microglial cells promotes the release of inflammatory cytokines and reactive oxygen/nitrogen species like nitric oxide (NO). In these conditions, NO promotes proliferation of neural stem cells (NSC) in the hippocampus. However, little is known about the role of NO in the survival and differentiation of newborn cells in the injured dentate gyrus. Here we investigated the role of NO following seizures in the regulation of proliferation, migration, differentiation, and survival of NSC in the hippocampus using the kainic acid (KA) induced seizuremouse model. We show that NO increased the proliferation of NSC and the number of neuroblasts following seizures but was detrimental to the survival of newborn neurons. NO was also required for the maintenance of long-term neuroinflammation. Taken together, our data show that NO positively contributes to the initial stages of neurogenesis following seizures but compromises survival of newborn neurons.Foundation for Science and Technology (FCT, Portugal); COMPETE; FEDER [PTDC/SAU-NEU/102612/2008, PTDC/NEU-OSD/0473/2012, PEst-C/SAU/LA0001/2013-2014, PEst-OE/EQB/LA0023/2013-2014]; FCT, Portugal [SFRH/BPD/78901/2011, SFRH/BD/77903/2011