Development and validation of gold nanoprobes for human SNP detection towards commercial application

Conventional molecular techniques for detection and characterization of relevant nucleic acid (i.e. DNA) sequences are, nowadays, cumbersome, expensive and with reduced portability. The main objective of this dissertation consisted in the optimization and validation of a fast and low-cost colorimetric nanodiagnostic methodology for the detection of single nucleotide polymorphisms (SNPs). This was done considering SNPs associated to obesity of commercial interest for STAB VIDA, and subsequent evaluation of other clinically relevant targets. Also, integration of this methodology into a microfluidic platform envisaging portability and application on points-of-care (POC) was achieved. To warrant success in pursuing these objectives, the experimental work was divided in four sections: i) genetic association of SNPs to obesity in the Portuguese population; ii) optimization and validation of the non-cross-linking approach for complete genotype characterization of these SNPs; iii) incorporation into a microfluidic platform; and iv) translation to other relevant commercial targets. FTO dbSNP rs#:9939609 carriers had higher body mass index (BMI), total body fat mass, waist perimeter and 2.5 times higher risk to obesity. AuNPs functionalized with thiolated oligonucleotides (Au-nanoprobes) were used via the non-cross-linking to validate a diagnostics approach against the gold standard technique - Sanger Sequencing - with high levels of sensitivity (87.50%) and specificity (91.67%). A proof-of-concept POC microfluidic device was assembled towards incorporation of the molecular detection strategy. In conclusion a successful framework was developed and validated for the detection of SNPs with commercial interest for STAB VIDA, towards future translation into a POC device.

Development and modulation of magnetic responsive supported ionic liquid membranes

The work presented in this thesis aims at developing a new separation process based on the application of supported magnetic ionic liquid membranes, SMILMs, using magnetic ionic liquids, MILs. MILs have attracted growing interest due to their ability to change their physicochemical characteristics when exposed to variable magnetic field conditions. The magnetic responsive behavior of MILs is thus expected to contribute for the development of more efficient separation processes, such as supported liquid membranes, where MILs may be used as a selective carrier. Driven by the MILs behavior, these membranes are expected to switch reversibly their permeability and selectivity by in situ and non-invasive adjustment of the conditions (e.g. intensity, direction vector and uniformity) of an external applied magnetic field. The development of these magnetic responsive membrane processes were anticipated by studies, performed along the first stage of this PhD work, aiming at getting a deep knowledge on the influence of magnetic field on MILs properties. The influence of the magnetic field on the molecular dynamics and structural rearrangement of MILs ionic network was assessed through a 1H-NMR technique. Through the 1H-NMR relaxometry analysis it was possible to estimate the self-diffusion profiles of two different model MILs, [Aliquat][FeCl4] and [P66614][FeCl4]. A comparative analysis was established between the behavior of magnetic and non-magnetic ionic liquids, MILs and ILs, to facilitate the perception of the magnetic field impact on MILs properties. In contrast to ILs, MILs show a specific relaxation mechanism, characterized by the magnetic dependence of their self-diffusion coefficients. MILs self-diffusion coefficients increased in the presence of magnetic field whereas ILs self-diffusion was not affected. In order to understand the reasons underlying the magnetic dependence of MILs self-diffusion, studies were performed to investigate the influence of the magnetic field on MILs’ viscosity. It was observed that the MIL´s viscosity decreases with the increase of the magnetic field, explaining the increase of MILs self-diffusion according to the modified Stokes- Einstein equation. Different gas and liquid transport studies were therefore performed aiming to determine the influence of the magnetic behavior of MILs on solute transport through SMILMs. Gas permeation studies were performed using pure CO2 andN2 gas streams and air, using a series of phosphonium cation based MILs, containing different paramagnetic anions. Transport studies were conducted in the presence and absence of magnetic field at a maximum intensity of 1.5T. The results revealed that gas permeability increased in the presence of the magnetic field, however, without affecting the membrane selectivity. The increase of gas permeability through SMILMs was related to the decrease of the MILs viscosity under magnetic field conditions.(...)

Systematics, functional morphology and distribution of a bivalve (Apachecorbula muriatica gen. et sp. nov.) from the rim of the ‘Valdivia Deep’ brine pool in the Red Sea

The deep brine pools of the Red Sea comprise extreme, inhospitable habitats yet house microbial communities that potentially may fuel adjacent fauna. We here describe a novel bivalve from a deep-sea (1525 m) brine pool in the Red Sea, where conditions of high salinity, lowered pH, partial anoxia and high temperatures are prevalent. Remotely operated vehicle (ROV) footage showed that the bivalves were present in a narrow (20 cm) band along the rim of the brine pool, suggesting that it is not only tolerant of such extreme conditions but is also limited to them. The bivalve is a member of the Corbulidae and named Apachecorbula muriatica gen. et sp. nov. The shell is atypical of the family in being modioliform and thin. The semi-infaunal habit is seen in ROV images and reflected in the anatomy by the lack of siphons. The ctenidia are large and typical of a suspension feeding bivalve, but the absence of guard cilia and the greatly reduced labial palps suggest that it is non-selective as a response to low food availability. It is proposed that the low body mass observed is a consequence of the extreme habitat and low food availability. It is postulated that the observed morphology of Apachecorbula is a result of paedomorphosis driven by the effects of the extreme environment on growth but is in part mitigated by the absence of high predation pressures.

Development and psychometric properties of ECPICID-AVC to measure informal caregivers’ skills when caring for older stroke survivors at home

Introduction: Informal caregivers provide a significant part of the total care needed by dependent older people poststroke. Although informal care is often the preferred option of those who provide and those who receive informal care, informal caregivers often report lack of preparation to take care of older dependent people. This article outlines the development and psychometric testing of informal caregivers’ skills when providing care to older people after a stroke – ECPICID-AVC. Design: Prospective psychometric instrument validation study. Methods: Eleven experts participated in a focus group in order to delineate, develop and validate the instrument. Data were gathered among adult informal caregivers (n = 186) living in the community in Northern Portugal from August 2013 to January 2014. Results: The 32-item scale describes several aspects of informal caregiver’s skills. The scale has eight factors: skill to feed/hydrate by nasogastric feeding, skill to assist the person in personal hygiene, skill to assist the person for transferring, skill to assist the person for positioning, skill to provide technical aids, skill to assist the person to use the toilet, skill to feed/hydrate and skill to provide technical aids for dressing/undressing. Analysis demonstrated adequate internal consistency (Cronbach’s alpha = 0.83) and good temporal stability 0.988 (0.984–0.991). Conclusion: The psychometric properties of the measurement tool showed acceptable results allowing its implementation in clinical practice by the nursing community staff for evaluating practical skills in informal caregivers when providing care to older stroke survivors living at home.

Density, size and distribution of stomata in 35 rainforest tree species in Central Amazonia

Stomata are turgor-operated valves that control water loss and CO2 uptake during photosynthesis, and thereby water relation and plant biomass accumulation is closely related to stomatal functioning. The aims of this work were to document how stomata are distributed on the leaf surface and to determine if there is any significant variation in stomatal characteristics among Amazonian tree species, and finally to study the relationship between stomatal density (S D) and tree height. Thirty five trees (>17 m tall) of different species were selected. Stomatal type, density (S D), size (S S) and stomatal distribution on the leaf surface were determined using nail polish imprints taken from both leaf surfaces. Irrespective of tree species, stomata were located only on the abaxial surface (hypostomaty), with large variation in both S D and S S among species. S D ranged from 110 mm-2 in Neea altissima to 846 mm-2 in Qualea acuminata. However, in most species S D ranges between 271 and 543 mm-2, with a negative relationship between S D and S S. We also found a positive relationship between S D and tree height (r² = 0.14, p < 0.01), but no correlation was found between S D and leaf thickness. The most common stomatal type was anomocytic (37%), followed by paracytic (26%) and anisocytic (11%). We conclude that in Amazonian tree species, stomatal distribution on the leaf surface is a response most likely dependent on the genetic background of every species, rather than a reaction to environmental changes, and that somehow S D is influenced by environmental factors dependent on tree height.

Structure and distribution of glandular trichomes in three species of Bignoniaceae

Glandular trichomes play a major role in the morphological characterization of the Bignoniaceae. Due to their great diversity of forms and functions, this study aimed to inventory the glandular trichomes present in the aerial vegetative axis of Amphilophium magnoliifolium, Martinella obovata and Stizophyllum riparium, analyze their structure and register the participation of ants in these plants. Fresh samples from the nodal region, petiole and from medium to apical regions of the leaflet blade were fixed and processed according to usual methods in light and scanning electron microscopies. The glandular trichomes found were: peltate, capitate, stipitate, and patelliform/cupular. Peltate trichomes are the most abundant ones and present the most uniform distribution. Patelliform/cupular trichomes occur at specific regions, such as prophylls, leaflet blade and nodal regions. Martinella obovata is the only species that presents capitate and stipitate trichomes, which are widely distributed along the entire aerial vegetative axis. Ants were found in all species, mainly at nodal regions. The occurrence of the capitate-type trichome is reported for the first time to the genus.

Feature Nets: behavioural modelling of software product lines

Software product lines (SPL) are diverse systems that are developed using a dual engineering process: (a)family engineering defines the commonality and variability among all members of the SPL, and (b) application engineering derives specific products based on the common foundation combined with a variable selection of features. The number of derivable products in an SPL can thus be exponential in the number of features. This inherent complexity poses two main challenges when it comes to modelling: Firstly, the formalism used for modelling SPLs needs to be modular and scalable. Secondly, it should ensure that all products behave correctly by providing the ability to analyse and verify complex models efficiently. In this paper we propose to integrate an established modelling formalism (Petri nets) with the domain of software product line engineering. To this end we extend Petri nets to Feature Nets. While Petri nets provide a framework for formally modelling and verifying single software systems, Feature Nets offer the same sort of benefits for software product lines. We show how SPLs can be modelled in an incremental, modular fashion using Feature Nets, provide a Feature Nets variant that supports modelling dynamic SPLs, and propose an analysis method for SPL modelled as Feature Nets. By facilitating the construction of a single model that includes the various behaviours exhibited by the products in an SPL, we make a significant step towards efficient and practical quality assurance methods for software product lines.

Development and characterization of lipid-based nanosystems for controlled release of bioactive compounds

Tese de Doutoramento em Engenharia Química e Biológica.

Desarrollo e interrupción de la generatividad en ámbitos familiares, comunitarios y políticos. Development and interruption of the generatividad in familiar, communitarian and political ambients.

El presente proyecto está centrado en tres crisis vitales del desarrollo humano: la identidad, la generatividad y la integridad del yo (Erikson, 1961). Se indagará la relación existente entre estos momentos del desarrollo psicosocial con las vicisitudes y crisis sociales y políticas que atraviesan el desarrollo personal. El sentido de lo político, la participación comunitaria y social se pondrán en tensión con la historia de acontecimientos que le sucedieron a las personas a lo largo de su vida y se plasman en las narrativas vitales.La tarea de actualizar las identificaciones tempranas, fundirse en un proyecto personal, de pareja, social y laboral implica la consolidación de la identidad y prepara a las personas para la etapa más fructífera de la adultez: la intimidad y la generatividad. Esta tarea implica involucrarse en la guía de otras generaciones más jóvenes a la vez que se sostiene el cuidado personal y el propio desarrollo y expansión del self.Sin embargo, existen dificultades en el desarrollo adulto que, sin conformarse en patologías, obturan la expresión de estos componentes egosintónicos y se manifiestan otros componentes egodistónicos o debilidades del yo. Asimismo, crisis sociales y eventos históricos significativos atraviesan la vida de las personas marcando sus metas de desarrollo y transformando el sentido de sí mismos y el significado de ser en el mundo con otros. Las expresiones personales y familiares de la generatividad tienen diferentes manifestaciones que se expresan en el campo social, comunitario y político y en el ejercicio de distintos roles sociales (voluntariado, participación política, inclusión en organizaciones comunitario, liderazgo de instituciones sociales, gremiales, partidarias o religiosas). En cualquier caso interesa conocer y determinar qué factores del desarrollo temprano y adulto establecen diferentes niveles de compromiso, interés e involucramiento. Los objetivos se centran en determinar los componentes de la generatividad y sus expresiones agénticas y comunitarias. Se utilizará un abordaje combinado de metodología cuantitativa y cualitativa. Se desarrollará una escala de generatividad aportando las evidencias de confiabilidad y validez requeridos en el desarrollo psicométrico. Posteriormente se administrará una entrevista pautada semidirigida, que permita valorar cualitativamente las diferencias centrales en las historias de vida entre sujetos con diferentes niveles de generatividad y grupos u organizaciones específicamente vinculadas al cuidado de los otros (derechos humanos, organizaciones comunitarias y ONG's vinculados al desarrollo infantil y cuidado de la vejez). De esta forma se valorará el impacto de crisis vitales y familiares, tanto como adversidades sociales y políticas (violencia, represión) en la generatividad, la madurez en adultez y la participación social y comunitaria.Se espera, así, identificar los aspectos egosintónicos comunes que son esenciales para desarrollar y consolidar fortalezas, secuencias de sentido, de redención y plenitud vital y compromiso político y comunitario a pesar de las adversidades en poblaciones diversas. La contrastación de las narrativas vitales permitirá elucidar qué potencialidades y recursos psicológico se ponen en juego, no sólo para la recuperación tras la adversidad (factores resilientes), sino para el desarrollo de una madurez comprometida con causas sociales y humanitarias que de forma reparatoria, restauran una porción de la realidad en la que se sufrió la injusticia y la crueldad, no ya para sí mismos sino para el resto de la sociedad.

Rol de la infección bacteriana en el desarrollo y progresión del cáncer prostático. Influence of bacterial infection in the development and progression of prostatitic cancer.

En la patogénesis del cáncer, factores microambientales como la inflamación están estrechamente vinculados al desarrollo y crecimiento tumoral, sustentando la clásica hipótesis de Virchow del origen del cáncer en sitios de inflamación crónica, la cual incitaría a carcinogénesis por múltiples factores. Estudios previos en este laboratorio evidenciaron en un modelo de prostatitis bacteriana agudo con E. coli, profundos cambios estromales semejantes al "estroma reactivo", con predominio de miofibroblasto, que se genera en el cáncer. En correlación, existe abundante evidencia obtenida en modelos experimentales animales confirmando que el microambiente estromal en el cual se desarrollan los tumores epiteliales influencia profundamente la progresión tumoral. El rol protagónico del estroma del huésped en el crecimiento neoplásico, también se ha demostrado inoculando la misma línea tumoral en diferentes tejidos y analizando su comportamiento en comparación con su implantación en el sitio anatómico original del tumor (implante ortotópico); otro factor clave en la repuesta del huésped al tumor está dado por el infiltrado de células inmunes que puede favorecer o limitar el crecimiento tumoral de acuerdo al perfil de citoquinas que secreten. Teniendo en cuenta estos antecedentes, este proyecto tiene como Objetivo General estudiar la influencia de la infección bacteriana crónica en la inducción y evolución del cáncer prostático. Para ello trabajaremos in vivo con dos formas de formas de Tumores Prostáticos, un Tumor Inducido por combinación del carcinógeno N-methyl-N-nitrosourea (MNU) y testosterona; el segundo mediante Transplante Ortotópico de células tumorales prostáticas MAT-LU. En ambos modelos se inducirá previamente una prostatitis bacteriana, a fin de estudiar los efectos de la prostatitis en la inducción del tumor en el primer modelo, y en la implantación de las células tumorales en el segundo. También se inducirá prostatitis después de establecido el tumor por ambos procedimientos, a fin de determinar si la prostatitis bacteriana modula la progresión neoplásica. Finalmente, proponemos un modelo in vitro que permita estudiar la interacción tumor/estroma separado de la influencia del sistema inmune. A tal fin se utilizarán co-cultivos combinando células tumorales con estromales modificadas de diferente modo. La Inducción de Tumores Prostáticos se realizará en ratas de la cepa Wistar adultas, en las cuales se inducirán lesiones displásicas y neoplásicas siguiendo protocolos de carcinogénesis prostática por MNU, para lo cual es necesario el tratamiento previo con acetato de ciprosterona y propionato de testosterona, seguido por administración crónica de testosterona. Los estudios con Transplante Ortotópico de células tumorales se realizarán en ratas Copenhagen. La influencia de la infección bacteriana en el desarrollo tumoral será investigada inyectando E. coli intraprostáticamente: Se realizará Análisis Macroscópico, de Parámetros Morfológicos de las lesiones tumorales, grado de malignidad, extensión e invasión de las lesiones de acuerdo a consensos internacionales, y Bioquímicos mediante análisis de la expresión, por IHQ y WB, de fosfatasa ácida, citoqueratina 8, Prostatic Binding Protein, PTEN (gen supresor tumoral) y el receptor de Andrógenos, todos parámetros de actividad y de transformación celular. También se evaluará apoptosis por TUNEL y proliferación celular. Los cambios del compartimiento estromal en respuesta al implante tumoral y la influencia de la inflamación bacteriana se evaluarán mediante análisis morfológico e inmunocitoquímico, caracterizando el fenotipo de las poblaciones celulares con a-actina, vimentina, calponina y tenascina. Se espera que los resultados aporten evidencias acerca de las interacciones bidireccionales entre células neoplásicas prostáticas y su entorno estromal, que en un futuro puedan servir como base para establecr estrategias para prevenir y/o modificar el crecimiento neoplásico.

Algæ. Vol. I. Myxophyceæ, Peridinieæ, Bacillarieæ, Chlorophyceæ, together with a brief summary of the occurrence and distribution of freshwat4er Algæ. By G.S. West.

v.1 (1916)

Algæ. Vol. I. Myxophyceæ, Peridinieæ, Bacillarieæ, Chlorophyceæ, together with a brief summary of the occurrence and distribution of freshwat4er Algæ. By G.S. West.

v.1 (1916)