O-GlcNAc transferase integrates metabolic pathways to regulate the stability of c-MYC in human prostate cancer cells

Metabolic disruptions that occur widely in cancers offer an attractive focus for generalized treatment strategies. The hexosamine biosynthetic pathway (HBP) senses metabolic status and produces an essential substrate for O-linked β-N-acetylglucosamine transferase (OGT), which glycosylates and thereby modulates the function of its target proteins. Here, we report that the HBP is activated in prostate cancer cells and that OGT is a central regulator of c-Myc stability in this setting. HBP genes were overexpressed in human prostate cancers and androgen regulated in cultured human cancer cell lines. Immunohistochemical analysis of human specimens (n = 1987) established that OGT is upregulated at the protein level and that its expression correlates with high Gleason score, pT and pN stages, and biochemical recurrence. RNA interference-mediated siliencing or pharmacologic inhibition of OGT was sufficient to decrease prostate cancer cell growth. Microarray profiling showed that the principal effects of OGT inhibition in prostate cancer cells were related to cell-cycle progression and DNA replication. In particular, c-MYC was identified as a candidate upstream regulator of OGT target genes and OGT inhibition elicited a dose-dependent decrease in the levels of c-MYC protein but not c-MYC mRNA in cell lines. Supporting this relationship, expression of c-MYC and OGT was tightly correlated in human prostate cancer samples (n = 1306). Our findings identify HBP as a modulator of prostate cancer growth and c-MYC as a key target of OGT function in prostate cancer cells.

c-Met inhibition in a HOXA9/Meis1 model of CN-AML

BACKGROUND: Hematopoiesis is a paradigm for developmental processes, hierarchically organized, with stem cells at its origin. Hematopoietic stem cells (HSCs) replenish progenitor and precursor cells of multiple lineages, which normally differentiate into short-lived mature circulating cells. Hematopoiesis has provided insight into the molecular basis of tissue homeostasis and malignancy. Malignant hematopoiesis, in particular acute myeloid leukemia (AML), results from impaired development or differentiation of HSCs and progenitors. Co-overexpression of HOX and TALE genes, particularly the HOXA cluster and MEIS1, is associated with AML. Clinically relevant models of AML are required to advance drug development for an aging patient cohort.

RESULTS: Molecular analysis identified altered gene, microRNA, and protein expression in HOXA9/Meis1 leukemic bone marrow compared to normal controls. A candidate drug screen identified the c-Met inhibitor SU11274 for further analysis. Altered cell cycle status, apoptosis, differentiation, and impaired colony formation were shown for SU11274 in AML cell lines and primary leukemic bone marrow.

CONCLUSIONS: The clonal HOXA9/Meis1 AML model is amenable to drug screening analysis. The data presented indicate that human AML cells respond in a similar manner to the HOXA9/Meis1 cells, indicating pre-clinical relevance of the mouse model.

Optimisation of an aptamer-biosensor to detect C-reactive protein

Dissertação de Mestrado, Engenharia Biológica, Faculdade de Engenharia de Recursos Naturais, Universidade do Algarve, 2009

Deteção de proteínas virais com nanossensores óticos

O desenvolvimento de tecnologias para a deteção de vírus é um tema de grande interesse em medicina de diagnóstico. Neste contexto, os biossensores têm-se revelado importantes ferramentas bioanalíticas, na medida em que permitem efetuar deteções moleculares de forma rápida, específica, reprodutível e com baixo custo associado. No âmbito desta dissertação, estabeleceram-se metodologias para a deteção fluorescente do fator de infetividade viral (Vif) do VIH através de um nanossensor ótico funcionalizado com anticorpos recombinantes anti-Vif, fragmentos variáveis de cadeia simples 4BL. Quantum dots carboxílicos conjugados a proteínas Vif são excitados a um comprimento de onda de 405 nm, transmitindo luz fluorescente a 605 nm a um fotodetetor de silício amorfo hidrogenado com um filtro de fluorescência integrado. Para implementar um sistema de deteção sensível e eficiente, estudaram-se estratégias de ativação de superfície em chips de vidro. A variabilidade das condições experimentais de um protocolo de silanização com (3-mercaptopropil)-trimetoxisilano (MPTS) foi avaliada através da análise de ângulos de contacto e densidades moleculares de fluoresceína funcionalizada com maleimida. Estabeleceu-se que, uma incubação dos substratos por 4 h com 5% (w/v) MPTS, seguida de cura a 110 ºC por 2 h, e redução com 10 mM ditiotreitol por 30 min, promove a formação de camadas de organosilanos de qualidade com grupos tiol reativos bem orientados. Estratégias de imobilização do elemento recetor foram testadas em substratos de vidro ativados e em sistemas de microfluídica vidro/PDMS, tomando por base o tag de afinidade, glutationa S-transferase (GST), do anticorpo recombinante. Vidros funcionalizados com glutationa e anti-GST mostraram-se igualmente reativos ao anticorpo GST-4BL, resultando uma capacidade de ligação do antigénio de aproximadamente 4.76 x 1010 moléculas/cm2 de QD-Vif, o que corresponde a 15% da cobertura máxima de superfície. Dos ensaios em sistemas de microfluídica resultaram sinais fracos e poucos reprodutíveis, destacando a necessidade de desenvolver metodologias de funcionalização mais apropriadas.

Lancefield group C and G streptococci in human infection : molecular typing, virulence and antimicrobial susceptibility

Tese de doutoramento, Ciências e Tecnologias da Saúde (Microbiologia), Universidade de Lisboa, Faculdade de Medicina, 2014

Arte rupestre do Vale do Tejo. Um ciclo artístico-cultural pré e proto-histórico

Dissertação apresentada para cumprimento dos requisitos necessários à obtenção do grau de Doutor em História, especialidade Arqueologia

Remyelination in vitro following protein kinase C activator-induced demyelination.

In previous work we found that mezerein, a C kinase activator, as well as basic fibroblast growth factor (FGF-2) induce demyelination and partial oligodendrocyte dedifferentiation in highly differentiated aggregating brain cell cultures. Here we show that following protein kinase C activator-induced demyelination, effective remyelination occurs. We found that mezerein or FGF-2 caused a transient increase in DNA synthesis following a pronounced decrease of the myelin markers myelin basic protein and 2',3'-cyclic nucleotide 3'-phosphohydrolase. Both oligodendrocytes and astrocytes were involved in this mitogenic response. Within 17 days after demyelination, myelin was restored to the level of the untreated controls. Transient mitotic activity was indispensable for remyelination. The present results suggest that myelinating oligodendrocytes retain the capacity to reenter the cell cycle, and that this plasticity is important for the regeneration of the oligodendrocyte lineage and remyelination. Although it cannot be excluded that a quiescent population of oligodendrocyte precursor cells was present in the aggregates and able to proliferate, differentiate and remyelinate, we could not find evidence supporting this view.

Hematopoietic stem cell function and survival depend on c-Myc and N-Myc activity.

Myc activity is emerging as a key element in acquisition and maintenance of stem cell properties. We have previously shown that c-Myc deficiency results in accumulation of defective hematopoietic stem cells (HSCs) due to niche-dependent differentiation defects. Here we report that immature HSCs coexpress c-myc and N-myc mRNA at similar levels. Although conditional deletion of N-myc in the bone marrow does not affect hematopoiesis, combined deficiency of c-Myc and N-Myc (dKO) results in pancytopenia and rapid lethality. Interestingly, proliferation of HSCs depends on both myc genes during homeostasis, but is c-Myc/N-Myc independent during bone marrow repair after injury. Strikingly, while most dKO hematopoietic cells undergo apoptosis, only self-renewing HSCs accumulate the cytotoxic molecule Granzyme B, normally employed by the innate immune system, thereby revealing an unexpected mechanism of stem cell apoptosis. Collectively, Myc activity (c-Myc and N-Myc) controls crucial aspects of HSC function including proliferation, differentiation, and survival.

Functional studies of the deubiquitinating enzymes USP19, USP4 and UCH-L1

El marcaje de proteínas con ubiquitina, conocido como ubiquitinación, cumple diferentes funciones que incluyen la regulación de varios procesos celulares, tales como: la degradación de proteínas por medio del proteosoma, la reparación del ADN, la señalización mediada por receptores de membrana, y la endocitosis, entre otras (1). Las moléculas de ubiquitina pueden ser removidas de sus sustratos gracias a la acción de un gran grupo de proteasas, llamadas enzimas deubiquitinizantes (DUBs) (2). Las DUBs son esenciales para la manutención de la homeostasis de la ubiquitina y para la regulación del estado de ubiquitinación de diferentes sustratos. El gran número y la diversidad de DUBs descritas refleja tanto su especificidad como su utilización para regular un amplio espectro de sustratos y vías celulares. Aunque muchas DUBs han sido estudiadas a profundidad, actualmente se desconocen los sustratos y las funciones biológicas de la mayoría de ellas. En este trabajo se investigaron las funciones de las DUBs: USP19, USP4 y UCH-L1. Utilizando varias técnicas de biología molecular y celular se encontró que: i) USP19 es regulada por las ubiquitin ligasas SIAH1 y SIAH2 ii) USP19 es importante para regular HIF-1α, un factor de transcripción clave en la respuesta celular a hipoxia, iii) USP4 interactúa con el proteosoma, iv) La quimera mCherry-UCH-L1 reproduce parcialmente los fenotipos que nuestro grupo ha descrito previamente al usar otros constructos de la misma enzima, y v) UCH-L1 promueve la internalización de la bacteria Yersinia pseudotuberculosis.

APM/CD13 and FOS in the hypothalamus of monosodium glutamate obese and food deprived rats

Protein (western blotting) and gene (PCR) expressions, catalytic activity of puromycin-insensitive membrane-bound neutral aminopeptidase (APM/CD13) and in situ regional distribution of CD13 and FOS immunoreactivity (it) were evaluated in the hypothalamus of monosodium glutamate obese (MSG) and/or food deprived (FD) rats in order to investigate their possible interplay with metabolic functions. Variations in protein and gene expressions of CD13 relative to controls coincided in the hypothalamus of MSG and MSG-FD (decreased 2- to 17-fold). Compared with controls, the reduction of hypothalamic CD13 content reflected a negative balance in its regional distribution in the supraoptic, paraventricular, periventricular and arcuate nuclei. CD13-ir increased in the supraoptic nucleus in MSG (2.5-fold) and decreased in the paraventricular nucleus (2-fold) together with FOS-ir (1.5-fold) in FD. In MSG-FD. FOS-ir decreased (7-fold) in the paraventricular nucleus, while CD13-ir decreased in the periventricular (5.6-fold) and the arcuate (3.7-fold) nuclei. It was noteworthy that all these changes of CD13 were not related to catalytic activity of APM. Data suggested that hypothalamic CD13 plays a role in the regulation of energy metabolism not by means of APM enzyme activity. (c) 2010 Elsevier B.V. All rights reserved.

Male and female odors induce Fos expression in chemically defined neuronal population

Olfactory information modulates innate and social behaviors in rodents and other species. Studies have shown that the medial nucleus of the amygdala (MEA) and the ventral premammillary, nucleus (PMV) are recruited by conspecific odor stimulation. However, the chemical identity of these neurons is not determined. We exposed sexually inexperienced male rats to female or male odors and assessed Fos immunoreactivity (Fos-ir) in neurons expressing NADPH diaphorase activity (NADPHd, a nitric oxide synthase), neuropeptide Urocortin 3, or glutamic acid decarboxylase rnRNA (GAD-67, a GABA-synthesizing enzyme) in the MEA and PMV. Male and female odors elicited Fos-ir in the MEA and PMV neurons, but the number of Fos-immunoreactive neurons was higher following female odor exposure, in both nuclei. We found no difference in odor induced Fos-ir ill the MEA and PMV comparing fed and fasted animals. Ill the MEA, NADPHd neurons colocalized Fos-ir only in response to female odors. In addition, Urocortin 3 neurons comprise a distinct population and they do not express Fos-ir after conspecific odor stimulation. We found that 80% of neurons activated by male odors coexpressed GAD-67 mRNA. Following female odor, 50% of Fos neurons coexpressed GAD-67 rnRNA. The PMV expresses very little GAD-67, and virtually no colocalization with Fos was observed. We found intense NADPHd activity in PMV neurons, some of which coexpressed Fos-ir after exposure to both odors. The majority of the PMV neurons expressing NADPHd colocalized cocaine-and amphetamine-regulated transcript (CART). Our findings suggest that female and male odors engage distinct neuronal populations in the MEA, thereby inducing contextualized behavioral responses according to olfactory cues. In the PMV, NADPHd/CART neurons respond to male and female odors, suggesting a role in neuroendocrine regulation in response to olfactory cues. (C) 2009 Elsevier Inc. All rights reserved.

A modified rat model of neonatal anoxia: Development and evaluation by pulseoximetry, arterial gasometry and Fos immunoreactivity

Neonatal anoxia is a worldwide clinical problem that has serious and lasting consequences. The diversity of models does not allow complete reproducibility, so a standardized model is needed. In this study, we developed a rat model of neonatal anoxia that utilizes a semi-hermetic system suitable for oxygen deprivation. The validity of this model was confirmed using pulse oximetry, arterial gasometry, observation of skin color and behavior and analysis of Fos immunoreactivity in brain regions that function in respiratory control. For these experiments, 87 male albino neonate rats (Rattus norvegicus, lineage Wistar) aged approximate 30 postnatal hours were divided into anoxia and control groups. The pups were kept in an euthanasia polycarbonate chamber at 36 +/- 1 degrees C, with continuous 100% nitrogen gas flow at 3 L/min and 101.7 kPa for 25 min. The peripheral arterial oxygen saturation of the anoxia group decreased 75% from its initial value. Decreased pH and partial pressure of oxygen and increased partial pressure of carbon dioxide were observed in this group, indicating metabolic acidosis, hypoxia and hypercapnia. respectively. Analysis of neuronal activation showed Fos immunoreactivity in the solitary tract nucleus, the lateral reticular nucleus and the area postrema, confirming that those conditions activated areas related to respiratory control in the nervous system. Therefore, the proposed model of neonatal anoxia allows standardization and precise control of the anoxic condition, which should be of great value in indentifying both the mechanisms underlying neonatal anoxia and novel therapeutic strategies to combat or prevent this widespread public health problem. (C) 2011 Elsevier B.V. All rights reserved.

Tectonothermal evolution and exhumation history of the Paleozoic Proto-Andean Gondwana margin crust: The Famatinian Belt in NW Argentina

We studied the P-T-t evolution of a mid-crustal igneous-metamorphic segment of the Famatinian Belt in the eastern sector of the Sierra de Velasco during its exhumation to the upper crust. Thermobarometric and geochronological methods combined with field observations permit us to distinguish three tectonic levels. The deepest Level I is represented by metasedimentary xenoliths and characterized by prograde isobaric heating at 20-25 km depth. Early/Middle Ordovician granites that contain xenoliths of Level I intruded in the shallower Level II. The latter is characterized by migmatization coeval with granitic intrusions and a retrograde isobaric cooling P-T path at 14-18 km depth. Level II was exhumed to the shallowest supracrustal Level III, where it was intruded by cordierite-bearing granites during the Middle/Late Ordovician and its host-rock was locally affected by high temperature-low pressure HT/LP metamorphism at 8-10 km depth. Level III was eventually intruded by Early Carboniferous granites after long-term slow exhumation to 6-7 km depth. Early/Middle Ordovician exhumation of Level II to Level III (Exhumation Period I,0.25-0.78 mm/yr) was faster than exhumation of Level III from the Middle/Late Ordovician to the Lower Carboniferous (Exhumation Period II, 0.01-0.09 mm/yr). Slow exhumation rates and the lack of regional evidence of tectonic exhumation suggest that erosion was the main exhumation mechanism of the Famatinian Belt. Widespread slow exhumation associated with crustal thickening under a HT regime suggests that the Famatinian Belt represents the middle crust of an ancient Altiplano-Puna-like orogen. This thermally weakened over-thickened Famatinian crust was slowly exhumed mainly by erosion during similar to 180 Myr. (C) 2010 International Association for Gondwana Research. Published by Elsevier B.V. All rights reserved.

Efeito da anoxia e da recuperação sobre o metabolismo intermediário do caranguejo Chasmagnathus granulata alimentado com dieta rica em carboidratos ou proteínas

O caranguejo Chasmagnathus granulata (Crustacea, Decapoda, Grapsidae) apesar de ter evoluído de formas marinhas é uma espécie tipicamente estuarina, sendo dificilmente encontrada no mar. Em seu hábitat, o caranguejo permanece longos períodos fora da água, sendo considerado um animal tipicamente semiterrestre. As respostas fisiológicas às variações das concentrações de O2 incluem alterações significativas na ventilação, na circulação, no metabolismo e nas propriedades de afinidade da hemocianina pelo O2. Estudos anteriores comprovaram que em alguns tecidos específicos da carpa Carassius carassius ocorre redução significativa da síntese protéica mas no cérebro não ocorre a mesma resposta metabólica, sugerindo ser esta uma das estratégias de conservação de energia. Os resultados obtidos no presente estudo mostram que, após uma hora de anoxia, a glicose hemolinfática aumentou 3 e 4 vezes em animais alimentados com dieta rica em carboidratos (RC) e rica em proteínas (RP), respectivamente. Após três horas de recuperação, os níveis glicêmicos retornaram aos valores semelhantes aos do grupo controle em ambos os tratamentos alimentares. Após uma hora de anoxia, a concentração de glicose livre nas brânquias anteriores (BA) reduziu significativamente em animais alimentados com dieta RC (70%) e RP (60%). Nas brânquias posteriores (BP) de animais alimentados com dieta RC e dieta RP, a anoxia reduziu 64% e 54% (p<0,05), respectivamente. Estes valores mantiveram-se reduzidos durante o período de recuperação, tanto nas BA como nas BP. Em animais alimentados com dieta RP, as concentrações de glicogênio reduziram somente após três horas de recuperação, tanto em BA como em BP, entretanto,somente nas BA de animais alimentados com dieta RP houve um aumento de 118% (p<0,05) dos níveis de ATP na anoxia e 116% na recuperação aeróbica No hepatopâncreas, a síntese proteica reduziu 78% e 65% (p<0,05) na anoxia e recuperação em animais alimentados com dieta RP, respectivamente. Já nos animais alimentados com dieta RC, a síntese protéica no hepatopâncreas aumentou em 42%, na recuperação. No músculo de animais que receberam este tratamento alimentar, ocorreu um aumento de 213% (p<0,05) na síntese protéica durante o período de anoxia e uma redução de 12% durante o período de recuperação aeróbica. Em caramguejos alimentados com dieta RP, a síntese protéica reduziu na anoxia 57% nas BA e 36% nas BP. Na dieta RC houve um aumento de 13% da síntese protéica nas brânquias anteriores e 8% nas brânquias posteriores durante a recuperação. Portanto, a capacidade de síntese protéica no caranguejo C. granulata, está relacionada com a característica funcional e a capacidade metabólica do tecidos submetidos à anoxia e à recuperação aeróbica. Os resultados do presente estudo demonstram que durante a anoxia e a recuperação aeróbica no caranguejo C. granulata ocorrem ajustes metabólicos diferenciados. Estes ajustes também dependem dos tecidos analisados e das dietas administradas.