RESUMO CAPÍTULO I The araticum (Annona crassiflora Mart.) is a typical brazilian plant found in cerrados of Brazil. This plant contains acetogenins that presents cytotoxic, antitumorigenic, antiparasitic and antimicrobial properties. Its leaves, barks, fruits and seeds are used by the population as therapeutic medicine to treat several diseases as diarrhoea, rheumatism and syphilis. In the present study we have evaluated the mutagenic activity of the crude ethanolic extract of leaves of araticum though quantification of micronuclei induced in mice bone marrow. Doses of 10 mg/kg (5% of LD50), 20 mg/kg (10% of LD50), 50 mg/kg (25% of LD50), 100 mg/kg (50% of LD50) and 160 mg/kg (80% of LD50) were applied i.p. in mice Mus musculus, in groups of 5 (five) animals for each dose. The cytological preparations were made according to Heddle`s methodology. For all the applied doses, frequency of micronucleated polychromatic erythrocytes (MNPCE) was evaluated after 24, 48 and 72 h of treatment. The cytotoxicity was evaluated by ratio the between numbers of polychromatic and normochromatic erythrocytes (PCE/NCE). Ours results indicated an absence of significantly increased of micronuclei for all the doses tested (p>0,01). Thus, we concluded that the phytoterapic araticum did not present mutagenic activity under our experimental conditions. However, the toxic activity was observed upon analysis of LD50 and PCE/NCE ratio. RESUMO CAPÍTULO II Annona crassiflora Mart. (araticum) is a typical brazilian plant found in cerrados of Brazil. This plant contains acetogenins that presents cytotoxic, antitumorigenic, antiparasitc and antimicrobial properties. Its leaves, barks, fruits and seeds are used by the population as therapeutic medicine to treat several diseases as diarrhoea, rheumatism and syphilis. In the present study we have evaluated the antimutagenic activity of the crude ethanolic extract of leaves of araticum through quantification of micronuclei induced in mice bone marrow. Doses of the extract (10mg/kg, 20mg/kg, 50mg/kg, 100mg/kg and 160 mg/kg) and MMC (4mg/kg) were co-applied i.p in mice Mus musculus in groups of 5 (five) animals for each dose. The cytological preparations were made in according to Heddle s methodology. For all the applied doses, frequency of micronucleated polychomatic erythrocytes (MNPCE) was evaluated after 36 h of treatment. The obtained results indicated that the crude ethanol extract from leaves ay doses of 20 mg/kg, 50 mg/kg and 100 mg/kg inhibited significantly reduced frequencies of micronuclei (P<0.01). Thus, we concluded that the phytoterapic araticum exerted an antimutagenic effect.

Esse estudo buscou investigar o papel do estresse oxidativo e nitrosativo no enfisema pulmonar induzido por elastase. Foram utilizados camundongos machos C57BL/6 submetidos a dois modelos de indução do enfisema por elastase pancreática suína (PPE): intratraqueal (i.t.) e intranasal (i.n.). No modelo intratraqueal a PPE foi instilada nas doses de 0,05 U ou 0,5 U/camundongo para avaliação temporal do enfisema 7, 14 e 21 dias após instilação de PPE. Em outra etapa, o papel da iNOS foi avaliado através da sua inibição farmacológica por aminoguanidina (AMG) 1% na água de beber ou pela sua exclusão genética em camundongos deficientes em iNOS que tiveram o enfisema induzido por 0,5 U PPE i.t. após 21 dias. No modelo intranasal a dose de PPE foi 3 U/camundongo para avaliação temporal do enfisema (1, 7, 14 e 21 dias após PPE). O papel do estresse oxidativo e nitrosativo foi avaliado com diferentes tratamentos antioxidantes na água de beber: tempol, apocinina+alopurinol, n-acetilcisteína, vitamina C+E, e aminoguanidina durante os 21 dias de indução do enfisema. Os grupos controles foram submetidos à instilação de salina. Lavado broncoalveolar, imunoensaios, análises bioquímicas de estresse oxidativo e ensaios morfométricos foram realizados nos pulmões dos animais. O enfisema foi histologicamente alcançado em 21 dias após 0,5 U PPE i.t., evidenciado pelo aumento do diâmetro alveolar médio Lm e da densidade de volume dos espaços alveolares - Vvair em comparação ao grupo controle. TNF-α foi aumentado em 7 e 14 dias após 0,5 U PPE comparados ao controle, concomitante com a redução de IL-10 nos mesmos períodos, comparados ao controle. O estresse oxidativo foi observado na fase inicial do enfisema, com aumento dos níveis de nitrito, TBARS e superóxido dismutase no grupo 7 dias após 0,5 U PPE (i.t.) quando comparados ao controle ao passo que no modelo intranasal as alterações típicas do estresse foram vistas no grupo 1 dia após 3 U de PPE. Atividade da glutationa peroxidase foi aumentada em todos os grupos PPE (i.t.). A exposição à 0,5 U PPE induziu o aumento da iNOS, eNOS e nitrotirosina, sendo revertido no grupo PPE+AMG. Os animais tratados com AMG 1% e os deficientes em iNOS tiveram o enfisema atenuado histologicamente, mantendo o Lm e o Vvair semelhantes ao grupo controle. Os grupos tratados com n-acetilcisteína e aminoguanidina no modelo i.n. tiveram redução do Lm quando comparados ao grupo PPE. Esses resultados sugerem que as vias de estresse oxidativo e nitrosativo são disparadas pela produção de óxido nítrico via iNOS no enfisema pulmonar. A modulação da iNOS parece uma estratégia promissora no estabelecimento do enfisema pulmonar

Diabetes is a metabolic disease caused by abnormal insulin secretion or action. In the present study, the effects of betulinic acid (BA, a triterpene) are evaluated on glucose, α-amylase and plasma insulin levels, insulin resistance and the histopathology of pancreatic islets in streptozotocin-nicotinamide (STZNA) diabetic mice. Seventy adult male NMRI mice were randomly divided into seven groups: control, sham, diabetic, diabetic treated with BA (10, 20 and 40 mg/kg) and diabetic treated with metformin (200 mg/kg). Diabetes was induced in mice by intraperitoneal injection of streptozotocin 50 mg/kg after a dose of nicotinamide 120 mg/kg. Two weeks after treatment with BA, blood samples were collected for measuring glucose, α-amylase and insulin levels, and the pancreas was isolated for histopathology evaluation. Diabetes reduced the number and diameter of pancreatic islets, and increased α-amylase and insulin resistance. BA treatment reduced blood glucose, α-amylase and improved insulin sensitivity as well as pancreas histopathology. In addition, BA showed stronger effects on the pancreatic histology and insulin resistance compared to the metformin group.

Arteriogenesis requires growth of pre-existing arteriolar collateral networks and determines clinical outcome in arterial occlusive diseases. Factors responsible for the development of arteriolar collateral networks are poorly understood. The Notch ligand Deltalike 4 (Dll4) promotes arterial differentiation and restricts vessel branching. We hypothesized that Dll4 may act as a genetic determinant of collateral arterial networks and functional recovery in stroke and hind limb ischemia models in mice. Genetic lossand gain-of-function approaches in mice showed that Dll4-Notch signaling restricts pial collateral artery formation by modulating arterial branching morphogenesis during embryogenesis. Adult Dll4+/− mice showed increased pial collateral numbers, but stroke volume upon middle cerebral artery occlusion was not reduced compared with wild-type littermates. Likewise, Dll4+/− mice showed reduced blood flow conductance after femoral artery occlusion, and, despite markedly increased angiogenesis, tissue ischemia was more severe. In peripheral arteries, loss of Dll4 adversely affected excitation-contraction coupling in arterial smooth muscle in response to vasopressor agents and arterial vessel wall adaption in response to increases in blood flow, collectively contributing to reduced flow reserve. We conclude that Dll4-Notch signaling modulates native collateral formation by acting on vascular branching morphogenesis during embryogenesis. Dll4 furthermore affects tissue perfusion by acting on arterial function and structure. Loss of Dll4 stimulates collateral formation and angiogenesis, but in the context of ischemic diseases such beneficial effects are overruled by adverse functional changes, demonstrating that ischemic recovery is not solely determined by collateral number but rather by vessel functionality.

The motor features in Parkinson's disease (PD) are associated with the degeneration of dopaminergic cells in the substantia nigra in the brain. Thus, the gold-standard in PD therapeutics still consists of dopamine replacement with levodopa. However, as the disease progresses, this therapeutic option becomes less effective and can be accompanied by levodopa-induced complications. On the other hand, several other neuronal pathways have been implicated in the pathological mechanisms of PD. In this context, the development of alternative therapeutic options that modulate non-dopaminergic targets is emerging as a major goal in the field. In a phenotypic-based screen in a zebrafish model of PD, we identified tapentadol as a candidate molecule for PD. The therapeutic potential of an agent that modulates the opioid and noradrenergic systems has not been explored, despite the implication of both neuronal pathways in parkinsonism. Therefore, we assessed the therapeutic properties of this µ-opioid receptor agonist and norepinephrine reuptake inhibitor in the 6-hydroxydopamine mouse model of parkinsonism. We further submitted 6-hydroxydopamine-lesioned mice to chronic treatment with levodopa and evaluated the effects of tapentadol during levodopa OFF states and on levodopa-induced dyskinesia. Importantly, we found that tapentadol halted the aggravation of dyskinesia and improved the motor impairments during levodopa OFF states. Altogether, our findings raise the hypothesis that concomitant modulation of µ-opioid receptor and norepinephrine transporter might constitute relevant intervention strategies in PD and that tapentadol holds therapeutic potential that may be translated into the clinical practice.

A mortalidade embrionária precoce em humanos, que ocorre frequentemente devido a erros antes, durante ou imediatamente após a implantação, é uma preocupação mundial com grandes implicações sociais e económicas. A via das prostaglandinas (PGs) é uma das vias de sinalização mais estudada com influência nos processos de implantação e de espaçamento dos embriões. A via Wnt canónica desempenha um papel importante em vários eventos do desenvolvimento embrionário e na preparação do útero para a receção do embrião. Desta forma, o objetivo deste trabalho foi inferir sobre uma possível interação entre estas duas vias de sinalização, já reportada noutros sistemas biológicos, e caracterizar o padrão de transcrição dos elementos da via de produção das PGs no embrião murino ao longo do desenvolvimento embrionário pré-implantação. Em relação à transcrição dos genes associados à via das PGs nos embriões, verificou-se um padrão crescente do nível de transcrição da COX-1 ao longo do desenvolvimento embrionário e ausência de transcrição de COX-2 em todos os estadios analisados. Observou-se ainda um aumento significativo (p<0,05) na transcrição da cPGES e mPGES-2 nos blastocistos eclodidos, e da mPGES-1 e PGFS nos blastocistos expandidos. No que diz respeito ao bloqueio da via Wnt pelo Dkk1, um inibidor da via Wnt canónica, não foram registadas diferenças estatisticamente significativas (p>0,05) entre os grupos estudados para nenhum dos genes ou dos estadios de desenvolvimento analisados. O padrão de transcrição dos genes associados à via das PGs no embrião murino foi, tanto quanto sabemos, descrito pela primeira vez neste trabalho, tendo-se concluído que a PGE2 parece ser a PG com maior nível de transcrição nos estadios estudados, indicando a sua importância no desenvolvimento embrionário pré-implantação, no processo de eclosão e na aquisição de competência do embrião para a implantação. Além disso, parece existir nesta espécie uma preferência pela via COX-1/PGES para a síntese de PGE2, tanto no embrião como no endométrio. A análise da transcrição nos locais de implantação, também evidenciou a PGE2 como a PG com maior importância. Por fim, não foi encontrada nenhuma interação entre as vias de sinalização Wnt-PG no desenvolvimento e competência do embrião para a implantação.

EDEM3 encodes a protein that converts Man8GlcNAc2 isomer B to Man7-5GlcNAc2. It is involved in the endoplasmic reticulum-associated degradation pathway, responsible for the recognition of misfolded proteins that will be targeted and translocated to the cytosol and degraded by the proteasome. In this study, through a combination of exome sequencing and gene matching, we have identified seven independent families with 11 individuals with bi-allelic protein-truncating variants and one individual with a compound heterozygous missense variant in EDEM3. The affected individuals present with an inherited congenital disorder of glycosylation (CDG) consisting of neurodevelopmental delay and variable facial dysmorphisms. Experiments in human fibroblast cell lines, human plasma, and mouse plasma and brain tissue demonstrated decreased trimming of Man8GlcNAc2 isomer B to Man7GlcNAc2, consistent with loss of EDEM3 enzymatic activity. In human cells, Man5GlcNAc2 to Man4GlcNAc2 conversion is also diminished with an increase of Glc1Man5GlcNAc2. Furthermore, analysis of the unfolded protein response showed a reduced increase in EIF2AK3 (PERK) expression upon stimulation with tunicamycin as compared to controls, suggesting an impaired unfolded protein response. The aberrant plasma N-glycan profile provides a quick, clinically available test for validating variants of uncertain significance that may be identified by molecular genetic testing. We propose to call this deficiency EDEM3-CDG.

There is currently limited understanding of the role played by haemodynamic forces on the processes governing vascular development. One of many obstacles to be overcome is being able to measure those forces, at the required resolution level, on vessels only a few micrometres thick. In this paper, we present an in silico method for the computation of the haemodynamic forces experienced by murine retinal vasculature (a widely used vascular development animal model) beyond what is measurable experimentally. Our results show that it is possible to reconstruct high-resolution three-dimensional geometrical models directly from samples of retinal vasculature and that the lattice-Boltzmann algorithm can be used to obtain accurate estimates of the haemodynamics in these domains. We generate flow models from samples obtained at postnatal days (P) 5 and 6. Our simulations show important differences between the flow patterns recovered in both cases, including observations of regression occurring in areas where wall shear stress (WSS) gradients exist. We propose two possible mechanisms to account for the observed increase in velocity and WSS between P5 and P6: (i) the measured reduction in typical vessel diameter between both time points and (ii) the reduction in network density triggered by the pruning process. The methodology developed herein is applicable to other biomedical domains where microvasculature can be imaged but experimental flow measurements are unavailable or difficult to obtain.

Formation of blood vessel networks by sprouting angiogenesis is critical for tissue growth, homeostasis and regeneration. How endothelial cells arise in adequate numbers and arrange suitably to shape functional vascular networks is poorly understood. Here we show that YAP/TAZ promote stretch-induced proliferation and rearrangements of endothelial cells whilst preventing bleeding in developing vessels. Mechanistically, YAP/TAZ increase the turnover of VE-Cadherin and the formation of junction associated intermediate lamellipodia, promoting both cell migration and barrier function maintenance. This is achieved in part by lowering BMP signalling. Consequently, the loss of YAP/TAZ in the mouse leads to stunted sprouting with local aggregation as well as scarcity of endothelial cells, branching irregularities and junction defects. Forced nuclear activity of TAZ instead drives hypersprouting and vascular hyperplasia. We propose a new model in which YAP/TAZ integrate mechanical signals with BMP signaling to maintain junctional compliance and integrity whilst balancing endothelial cell rearrangements in angiogenic vessels.