Bioengineering of Lactococcus lactis through modulation of its major glucose transporter

Dissertation to obtain the Master Degree in Biotechnology

Ocular Pulse Amplitude and Doppler Waveform Analysis in Glaucoma Patients

PURPOSE: To determine the correlation between ocular blood flow velocities and ocular pulse amplitude (OPA) in glaucoma patients using colour Doppler imaging (CDI) waveform analysis. METHOD: A prospective, observer-masked, case-control study was performed. OPA and blood flow variables from central retinal artery and vein (CRA, CRV), nasal and temporal short posterior ciliary arteries (NPCA, TPCA) and ophthalmic artery (OA) were obtained through dynamic contour tonometry and CDI, respectively. Univariate and multiple regression analyses were performed to explore the correlations between OPA and retrobulbar CDI waveform and systemic cardiovascular parameters (blood pressure, blood pressure amplitude, mean ocular perfusion pressure and peripheral pulse). RESULTS: One hundred and ninety-two patients were included [healthy controls: 55; primary open-angle glaucoma (POAG): 74; normal-tension glaucoma (NTG): 63]. OPA was statistically different between groups (Healthy: 3.17 ± 1.2 mmHg; NTG: 2.58 ± 1.2 mmHg; POAG: 2.60 ± 1.1 mmHg; p < 0.01), but not between the glaucoma groups (p = 0.60). Multiple regression models to explain OPA variance were made for each cohort (healthy: p < 0.001, r = 0.605; NTG: p = 0.003, r = 0.372; POAG: p < 0.001, r = 0.412). OPA was independently associated with retrobulbar CDI parameters in the healthy subjects and POAG patients (healthy CRV resistance index: β = 3.37, CI: 0.16-6.59; healthy NPCA mean systolic/diastolic velocity ratio: β = 1.34, CI: 0.52-2.15; POAG TPCA mean systolic velocity: β = 0.14, CI 0.05-0.23). OPA in the NTG group was associated with diastolic blood pressure and pulse rate (β = -0.04, CI: -0.06 to -0.01; β = -0.04, CI: -0.06 to -0.001, respectively). CONCLUSIONS: Vascular-related models provide a better explanation to OPA variance in healthy individuals than in glaucoma patients. The variables that influence OPA seem to be different in healthy, POAG and NTG patients.

Schistosoma mansoni in mice: modulation of granulomatous response after reinfection and chemotherapeutic treatment

Mice previously infected with Schistosoma mansoni, and cured by specific treatment (400mg/kg oxamniquine, p. o.) in the chronic phase of the disease, were reinfected 20 days after treatment to assess their capacityfor modulation ofthe granulomatous response. Histopathologic examination of the animals ' liver, at 60 days after reinfection, evidenced the presence of typical granulomas of the chronic phase in most animals. This infer that the capacity for modulation of the granulomatous response had been maintained, thus preventing a new acute phase of the disease. Conversely, a group of previously infected mice, untreated and submitted to reinfection, showed reactivation of the granulomatous response in 50% of the animals. The possible implications of these findings in human schistosomiasis mansoni are discussed.

Modulation of α-synuclein aggregation and toxicity

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Immunodiagnosis of chronic Chagas' disease using the Tc 46 and Tc 58 antigens

The polypeptides of 46 and 58kDa were recognized in different T. cruzi strains (Y, WSL and Colombiana) by serum of all chagasic patients studied. These polypeptides were isolated from T. cruzi Y strain and used in ELISA. The sensitivity and specificity were 97.6% [CI 95%: 86-100%] and 100% [CI 95%: 89.3-100%], respectively when Tc 46 was used. When Tc 58 was used the sensitivity and specificity were 100% [CI 95%: 89.6-100%] and 90.2% [CI 95%: 75.9-96.8%], respectively.

Evaluation of motor neuron excitability by CMAP scanning with modulated current

It is important to have better evaluation and understanding of the motor neuron physiology, with the goal to early and objectively diagnose and treat patients with neurodegenerative pathologies. The Compound Muscle Action Potential (CMAP) scan is a non-invasive diagnosis technique for neurodegenerative pathologies, such as ALS, and enables a quick analysis of the muscle action potentials in response to motor nerve stimulation. This work aims to study the influence of different pulse modulated waveforms in peripheral nerve excitability by CMAP scan technique on healthy subjects. A total of 13 healthy subjects were submitted to the same test. The stimuli were applied in the medium nerve on the right wrist and electromyography signal collected on the Abductor Pollicis Brevis (APB) muscle surface on the right thumb. Stimulation was performed with an increasing intensities range from 4 to 30 mA, with varying steps, 3 stimuli per step. The procedure was repeated 4 times per subject, each repetition using a different single pulse stimulation waveform: monophasic square, monophasic triangular, monophasic quadratic and biphasic square. Results were retrieved from the averaging of the stimuli on each current intensity step. The square pulse needs less current intensity to generate the same response amplitude regarding the other waves and presents a more steep curve slope and this effect is gradually decreasing for the triangular and quadratic pulse,respectively, being the difference even more evident regarding the biphasic pulse. The control of the waveform stimulation pulse allows varying the stimulusresponse curve slope.

Dose de radiação e risco para a saúde em doentes com Linfoma não-Hodgkin submetidos a estudos de TC

RESUMO - A exposição a radiações ionizantes em tomografia computorizada (TC) pode constituir-se como um risco para a saúde dos utentes. A TC é utilizada no diagnóstico e follow-up de doentes com Linfoma não-Hodgkin, subtipo Linfoma Difuso das Grandes Células B (LDGCB). O objetivo deste estudo foi determinar a dose efetiva acumulada e o risco de segundas neoplasias nestes doentes, diagnosticados em 2011 no IPOLFG e seguidos na consulta de hematologia até 2013. Foram avaliados retrospetivamente 70 doentes com base nos registos de dose do “Patient Protocol” das TC efetuadas. Em média cada doente fez 12 TC e a dose efetiva acumulada foi de 64,76 mSv (percentil 75). Três doentes foram expostos a doses de radiação superiores 90 mSv e um atingiu 111,72 mSv. Os resultados demonstram ser necessário rever os parâmetros e protocolos de exames de TC: (i) TC crânio (DLP= 777 mGycm) e TC abdominal-pélvico (DLP= 628 mGycm). O aumento do número de exames de TC efetuados e a consequente dose parece corresponder a um aumento do risco de segundas neoplasias e risco de morte por doenças neoplásicas durante a vida destes doentes. Os resultados são aparentemente mais significativos para as mulheres, que apresentam o dobro do risco de cancro do pulmão e risco de mortalidade superior em 14% para todas as doenças neoplásicas. O elevado número de exames de TC realizados por cada doente contribui para o aumento da probabilidade de efeitos deletérios e também para o aumento dos níveis de dose efetiva coletiva na população em geral.

Modulation of the secretome of hBMSCs by tailoring the macromolecular gradient in hydrogels to generate tissue-to-tissue interfaces

Tissue-to-tissue interfaces are commonly present in all tissues exhibiting structural, biological and chemical gradients serving a wide range of physiological functions. These interfaces are responsible for mediation of load transfer between two adjacent tissues. They are also important structures in sustaining the cellular communications to retain tissueâ s functional integration and homeostasis. [1] All cells have the capacity to sense and respond to physical and chemical stimulus and when cultured in three-dimensional (3D) environments they tend to perform their function better than in two-dimensional (2D) environments. Spatial and temporal 3D gradient hydrogels better resemble the natural environment of cells in mimicking their extracellular matrix. [2] In this study we hypothesize that differential functional properties can be engineered by modulation of macromolecule gradients in a cell seeded threedimensional hydrogel system. Specifically, differential paracrine secretory profiles can be engineered using human Bone Marrow Stem Cells (hBMSCâ s). Hence, the specific objectives of this study are to: assemble the macromolecular gradient hydrogels to evaluate the suitablity for hBMSCâ s encapsulation by cellular viability and biofunctionality by assessing the paracrine secretion of hBMSCâ s over time. The gradient hydrogels solutions were prepared by blend of macromolecules in one solution such as hyaluronic (HA) acid and collagen (Col) at different ratios. The gradient hydrogels were fabricated into cylindrical silicon moulds with higher ratio solutions assembled at the bottom of the mould and adding the two solutions consecutively on top of each other. The labelling of the macromolecules was performed to confirm the gradient through fluorescence microscopy. Additionally, AFM was conducted to assess the gradient hydrogels stiffness. Gradient hydrogels characterization was performed by HA and Col degradation assay, degree of crosslinking and stability. hBMSCâ s at P3 were encapsulated into each batch solution at 106 cells/ml solution and gradient hydrogels were produced as previously described. The hBMSCâ s were observed under confocal microscopy to assess viability by Live/Dead® staining. Cellular behaviour concerning proliferation and matrix deposition was also performed. Secretory cytokine measurement for pro-inflammatory and angiogenesis factors was carried out using ELISA. At genomic level, qPCR was carried out. The 3D gradient hydrogels platform made of different macromolecules showed to be a suitable environment for hBMSCâ s. The hBMSCâ s gradient hydrogels supported high cell survival and exhibited biofunctionality. Besides, the 3D gradient hydrogels demonstrated differentially secretion of pro-inflammatory and angiogenic factors by the encapsulated hBMSCâ s. References: 1. Mikos, AG. et al., Engineering complex tissues. Tissue Engineering 12,3307, 2006 2. Phillips, JE. et al., Proc Natl Acad Sci USA, 26:12170-5, 2008

Tensile stress–crack width law for steel fibre reinforced self-compacting concrete obtained from indirect (splitting) tensile tests

In this work, the fracture mode I parameters of steel fibre reinforced self-compacting concrete (SFRSCC) were derived from the numerical simulation of indirect splitting tensile tests. The combined experimental and numerical research allowed a comparison between the stress-crack width (σ - w) relationship acquired straightforwardly from direct tensile tests, and the σ - w response derived from inverse analysis of the splitting tensile tests results. For this purpose a comprehensive nonlinear 3D finite element (FE) modeling strategy was developed. A comparison between the experimental results obtained from splitting tensile tests and the corresponding FE simulations confirmed the good accuracy of the proposed strategy to derive the σ – w for these composites. It is concluded that the post-cracking tensile laws obtained from inverse analysis provided a close relationship with the ones obtained from the experimental uniaxial tensile tests.

A model to simulate the moment-rotation and crack width of FRC members reinforced with longitudinal bars

The present work describes a model for the determination of the moment–rotation relationship of a cross section of fiber reinforced concrete (FRC) elements that also include longitudinal bars for the flexural reinforcement (R/FRC). Since a stress–crack width relationship (σ–w)(σ–w) is used to model the post-cracking behavior of a FRC, the σ–w directly obtained from tensile tests, or derived from inverse analysis applied to the results obtained in three-point notched beam bending tests, can be adopted in this approach. For a more realistic assessment of the crack opening, a bond stress versus slip relationship is assumed to simulate the bond between longitudinal bars and surrounding FRC. To simulate the compression behavior of the FRC, a shear friction model is adopted based on the physical interpretation of the post-peak compression softening behavior registered in experimental tests. By allowing the formation of a compressive FRC wedge delimited by shear band zones, the concept of concrete crushing failure mode in beams failing in bending is reinterpreted. By using the moment–rotation relationship, an algorithm was developed to determine the force–deflection response of statically determinate R/FRC elements. The model is described in detail and its good predictive performance is demonstrated by using available experimental data. Parametric studies were executed to evidence the influence of relevant parameters of the model on the serviceability and ultimate design conditions of R/FRC elements failing in bending.

Light-controlled resistive switching in laser-assisted annealed Ba0.8Sr0.2TiO3 thin films

In this work, Ba0.8Sr0.2TiO3 (BST)/ITO structures were grown on glass substrate and laser assisted annealing (LAA) was performed to promote the crystallization of BST. Atomic force microscopy and X-ray diffraction studies confirm the crack free and polycrystalline perovskite phase of BST. White light controlled resistive switching (RS) effect in Au/BST/ITO device is investigated. The device displays the electroforming-free bipolar RS characteristics and are explained by the modulationof the width and height of barrier at the BST/ITO interface via ferroelectric polarization. Moreover, the RS effect is signifi- cantly improved under white light illumination compared to that in the dark. The enhanced RS and photovoltaic effects are explained by considering depolarization field and charge distribution at the interface. The devices exhibit stable retention characteristics with low currents (mA), which make them attractive for non volatile memory devices.

Flood pulse and spatial dynamics of mercury in sediments in Puruzinho lake, Brazilian Amazon

The study was conducted in Puruzinho lake (Humaitá, AM) considering seasonal periods of rainy and dry in way to elucidate the flood pulse importance in the deposition, remobilization and distributions of mercury and organic matter in bottom sediments in the Madeira River Basin (Brazilian Amazon). Bottom sediments and soils samples were analyzed for total mercury and organic matter. Mercury concentrations obtained in bottom sediment were 32.20-146.40 ng g-1 and organic matter values were 3.5 - 18.0%. The main region for accumulation of mercury and organic matter was in the central and deepest lake area In the rainy season there was a greater distribution of Hg and organic matter, mainly controlled by means of income of the Madeira river water during flooding, while the predominant process in the dry season was the remobilization of total Hg due to the resuspension of bottom sediments.

Exploring the role of O-Mannosylation in the modulation of E-Cadherin function in Gastric Cancer cell line models

Dissertação de mestrado em Bioquímica Aplicada – Biomedicina

Pulse wave velocity distribution in a cohort study: from arterial stiffness to early vascular aging

BACKGROUND: By contrast with other southern European people, north Portuguese population registers an especially high prevalence of hypertension and stroke incidence. We designed a cohort study to identify individuals presenting accelerated and premature arterial aging in the Portuguese population. METHOD: Pulse wave velocity (PWV) was measured in randomly sampled population dwellers aged 18-96 years from northern Portugal, and used as a marker of early vascular aging (EVA). Of the 3038 individuals enrolled, 2542 completed the evaluation. RESULTS: Mean PWV value for the entire population was 8.4?m/s (men: 8.6?m/s; women: 8.2?m/s; P??10?m/s). Logistic regression models indicated gender differences concerning the risk of developing large artery damage, with women having the same odds of PWV above 10?m/s 10 years later than men. CONCLUSION: The population PWV values were higher than expected in a low cardiovascular risk area (Portugal). High prevalence rates of EVA and noteworthy large artery damage in young ages were found.

Autonomic Modulation of the Heart in Systemic Arterial Hypertension

OBJECTIVE: To analyze the heart rate variability in patients with mild to moderate systemic arterial hypertension. METHODS: Thirty-two healthy (group I) and 70 systemic arterial hypertensive (group II) individuals, divided according to age (40 to 59 and 60 to 80 years old, respectively) and with a similar distribution by sex were studied. Thirty-one had left ventricular hypertrophy (LVH), 22 were overweight, and 16 had Type II diabetes mellitus. Smoking, alcohol ingestion, and sedentary habits were the same between groups. Variability in heart rate was analyzed in the time domain, using standard deviations of normal RR intervals (SDNN) and the differences between maximal brady- and tachycardia (D-BTmax) during sustained inspiration. Analysis of the frequency band of the power spectrum between 0.05 and 0.40 Hz at rest and during controlled respiration was chosen for analysis of the frequency domain. RESULTS: In both time and frequency domains, variables were lower in group II than in group I. Within groups, statistically significant variables were only found for individuals in the 40 to 59 year old group. The presence of LVH, overweight, or diabetes mellitus did not influence the variability in heart rate to a significant extent. CONCLUSION: Variability in heart rate was a valuable instrument for analyzing autonomic modulation of the heart in arterial hypertension. The autonomic system undergoes significant losses in cardio-modulatory capacity, more evident in subjects between 40 and 59 years old. In those over 60 years old, reduced variability in heart rate imposed by aging was not significantly influenced by the presence of systemic arterial hypertension.