Evaluation of antimicrobial strategies against biomaterial-associated infections: impact of the surrounding environmental conditions

Dissertação de mestrado em Bioengenharia

Characterization of microbial populations in a wastewater treatment plant focusing on Staphylococcus aureus and Pseudomonas aeruginosa

Dissertação de mestrado em Bioengenharia

Aspectos bioquímicos y celulares de la reproducción de los vectores de la enfermedad de Chagas: regulación e importancia fisiológica. Biochemical and cellular aspects of the reproduction of Chagas’ disease vectors: regulation and physiologycal impact

En la hipótesis de trabajo del presente proyecto se considera la importancia del metabolismo de lípidos y proteínas en los insectos hematófagos, en particular en los vectores de la enfermedad de Chagas, para afrontar exitosamente la demanda energética de la reproducción. Las hembras de estas especies pueden ingerir una comida de sangre abundante en lípidos y proteínas, los que son modificados en el intestino para su utilización y posterior almacenamiento en estructuras organizadas en el tejido ovárico, sustentando así el rápido crecimiento de los ovocitos. Estos aspectos resultan críticos para el ciclo de vida del insecto y para el mantenimiento de la cadena epidemiológica de la enfermedad. En estas especies, recientemente hemos caracterizado a nivel bioquímico y celular la interacción entre lipoproteínas y tejidos [Fruttero y col., Insect Biochem. Mol. Biol. 39: 322-331 (2009); Fruttero y col. Biocel 33 (3): 260 (2009)] y las fases del ciclo reproductivo [Aguirre y col., J. Insect Physiol. 54: 393-402 (2008)]. No obstante, los factores que participan en su regulación son aún escasamente conocidos. En este contexto, el estudio propone emplear dos especies de triatominos con el objeto de: (1) caracterizar los factores involucrados en la formación y regulación de reservas nutricionales en los ovocitos; (2) analizar los eventos que participan en la regresión del tejido ovárico: atresia folicular y mecanismos de muerte celular. (3) evaluar el impacto de productos naturales (ureasas vegetales y péptidos derivados) en el desarrollo del tejido ovárico. Para la ejecución de los objetivos se llevarán a cabo ensayos in vivo e in vitro con trazadores fluorescentes, fraccionamiento subcelular, estudios de expresión de proteínas (mRNA y proteína), estudios histo-morfológicos, ultraestructurales e inmunocitoquímicos, microscopía láser confocalizada, ensayos de actividad enzimática, ELISA, western-blot, electroforesis bidimensional, espectrometria de masas en tándem, etc. También se evaluarán los mecanismos de muerte celular (apoptosis/autofagia) mediante microscopía electrónica, detección de apoptosis in situ (TUNEL), inmunofluorescencia, etc. Los resultados obtenidos permitirán un mejor conocimiento sobre la fisiología y bioquímica de estos vectores, los que resultan indispensables en el diseño de nuevas estrategias para su control. Debido a la carencia de un tratamiento específico para la enfermedad y a la falta de métodos preventivos (vacuna), el control del vector es una de las vías más importantes para reducir la incidencia de la enfermedad. Actualmente, la situación socio-económica que sufren amplios núcleos de nuestra población propicia condiciones de vida que facilitan la reproducción de los vectores y la transmisión vectorial del parásito. El estudio permitirá además explorar aspectos bioquímicos y celulares básicos, generando conocimientos que podrían ser extensivos a otros insectos de importancia económica en la ganadería y/o agricultura. The aim of this project is to analyze the biochemical and cellular events involved in the lipid and protein metabolism in Chagas' disease vectors, and to evaluate their impact on the physiology of reproduction, particularly in the formation of nutritional resources in developing oocytes. At present, little is known about these critical aspects for the life cycle of the insect and for the epidemiology of the disease. The experimental approaches, which will be carried out using two species of triatomines, were designed: (1) to characterize factors involved in the formation and regulation of nutritional resources in developing oocytes; (2) to analyze the biochemical and cellular events that play a role during the regression of ovarian tissue, including the processes of oocyte resorption and programmed cell death. (3) to evaluate the impact of natural products (ureases from jackbean and related peptides) in the development of ovarian tissue. Methods and techniques involved in the project are: in vivo and in vitro assays with fluorescent tracers, ELISA, chemical assays, enzyme activities, western-blot; protein expression (mRNA), histological techniques, immunohistochemical and ultrastructural studies. Cell death will be analyzed by detection of apoptosis in situ (TUNEL), immunofluorescence (for autophagy), among others. The results obtained from the study will offer the opportunity to explore important aspects in the biology and physiology of Chagas' disease vectors that could be of potential utility in designing alternative strategies for the control of the insect.

Groundwater vulnerability and protection in County Tipperary (South Riding), Ireland

The aim of the project was to determine the extent and quality of the groundwater in Tipperary South Riding with a view to developing a groundwater protection plan which would allow the Local Authority to manage, protect and develop the groundwater as efficiently as possible. The geology of the area varies with topography. The low-lying areas of the county comprise mainly Carboniferous limestones while the elevated regions consist of sandstones and shales of Upper Carboniferous, Devonian and Silurian ages. Deformation of these rocks decreases in magnitude moving northwards over the area; the Southern Synclines having suffered the effects of the Hercynian orogeny and the northern region exhibiting Caledonian orogenic trends. Quaternary (subsoil) deposits are found throughout the area and are of variable thickness and permeability. Till is the most widespread deposit with discontinuous pockets of sand and gravel in various proportions, and some marl, alluvium and peat in places. The principal aquifers of the area are the Kiltorcan sandstone formation and various limestone units within the Carboniferous succession. 50 % of south Tipperary constitutes either regionally or locally important aquifers. Secondary permeabilities created by structural deformation, dolomitisation, karstification and weathering processes create high transmissivities and often have large well yields. Specific baseflow analysis highlighted the complexity of the aquifers and proved that the lower part of the Suir river system is a major groundwater resource region. The hydrochemistry and water quality of the local authority groundwater sources was examined briefly. The majority of south Tipperary is underlain by limestone or Quaternary deposits derived from limestone and, consequently, calcium/magnesium bicarbonate waters predominate. The quality of the groundwater in south Tipperary demonstrates that the main concern originates from the presence of E.coli, and Total coliforms. The primary sources of contamination are from farmyard wastes and septic tanks. The vulnerability of groundwater to diffuse and point sources of pollution has been found to be dependent on the overlying soil, subsoil and the thickness of the unsaturated zone. A conceptual rather than quantitative approach is used and it is found that approximately 60% of south Tipperary is designated as being extremely or highly vulnerable. The groundwater protection plan was devised subsequent to an understanding of the aquifer systems, an assessment of the vulnerability, and a review of the Irish planning system and environmental law. It is recommended that the plan be integrated into the county development plan for legislative purposes. A series of acceptability matrices were devised to restrict potentially polluting activities in vulnerable areas while maintaining a balance between protection of the groundwater resource and the need to site essential developments.

Green furniture: sustainable design guidelines for the Irish furniture and wood products industry.

This thesis presents the research and development of sustainable design guidelines for the furniture and wood products industry, suitable for sustainably enhancing design, manufacturing and associated activities. This sustainable guideline is based on secondary research conducted on subject areas such as ‘eco’ design, ‘green’ branding and ‘green’ consumerism, as well as an examination of existing certifications and sustainable tools techniques and methodologies, national and international drivers for sustainable development and an overview of sustainability in the Irish furniture manufacturing context. The guideline was further developed through primary research. This consisted of a focus group attended by leading Irish designers, manufacturers and academics in the area of furniture and wood products. This group explored the question of ‘green branding’ saturation in the market and the viability of investing in sustainability just yet. Participants stated that they felt the market for ‘green’ products is evolving very slowly and that there is no metric or legal framework present to audit whether or not companies are producing products that really embody sustainability. All the participants believed that developing and introducing a new certification process to incorporate a sustainable design process was a viable and necessary solution to protecting Irish furniture and wood manufacturers going forward. For the purposes of the case study, the author investigated a ‘sustainable’ design process for Team woodcraft, Ltd., through the design and development of a ‘sustainable’ children’s furniture range. The case study followed a typical design and development process; detailing customer design specifications, concept development and refinement and cumulating in final prototype, as well as associated engineering drawings. Based on this primary and secondary research, seven fundamental core principles for this sustainable guideline have been identified by the author. The author then used these core principles to expand into guidelines for the basis of proposed new Irish sustainable design guidelines for the furniture and wood products industry, the concept of which the author has named ‘Green Dot’. The author suggests that the ‘Green Dot’ brand or logo could be used to market an umbrella network of Irish furniture designers and manufactures who implement the recommended sustainable techniques.

Coalition formation in a contest game with three heterogeneous players

We analyze the incentives for cooperation of three players differing in their efficiency of effort in a contest game. We concentrate on the non-cooperative bargaining foundation of coalition formation, and therefore, we adopt a two-stage model. In the first stage, individuals form coalitions following a bargaining protocol similar to the one proposed by Gul (1989). Afterwards, coalitions play the contest game of Esteban and Ray (1999) within the resulting coalition structure of the first stage. We find that the grand coalition forms whenever the distribution of the bargaining power in the coalition formation game is equal to the distribution of the relative efficiency of effort. Finally, we use the case of equal bargaining power for all individuals to show that other types of coalition structures may be observed as well.

On the regional impact of public capital formation in Spain

The objective of this paper is to investigate, in a methodologically consistent manner, the regional effects of public capital formation and the possible existence of regional spillover effects in Spain. The empirical results are based on VAR estimates at both the aggregate and regional levels using output, employment, and private capital, as well as different measures of public capital. Empirical results suggest that public capital affects output positively at the aggregate level as well as in all but one region. For most regions, the effects of public capital installed in the region itself are important but the spillover effects induced from public capital installed elsewhere are also very important. In fact, the spillover effects account for over half of the total effects of public capital formation in Spain. Furthermore, these spillover effects have a clear geographical pattern in that they tend to be more important in the peripheral regions of the country. We also find that relative to their share of the Spanish output, the biggest beneficiaries of public capital formation are the largest regions in the country. This suggests that public capital formation has contributed to concentration of output in these regions. Finally, in terms of the effects of public capital formation on the private inputs we find that both private capital and employment are affected positively at the aggregate level as well as for most of the regions. Nevertheless, the effects on private capital seem to be larger. Also, the spillover effects are very important for private capital but not for employment. This reflects a great degree of dynamism and mobility in the capital markets as opposed to the labor markets.

The European Union Police Mission: The Beginning of a New Future for Bosnia and Herzegovina?

The creation, reform and/or restructuring of the police in post-conflict societies remains one of the key challenges for practitioners and scholars in the contemporary fields of peace and security, particularly due to the changing nature of conflicts. Since the 1990s the world has witnessed a proliferation of international police missions, with regional organisations gradually acquiring a prominent role. This paper analyses the 2003-2005 period of the European Union Police Mission (EUPM) in Bosnia and Herzegovina. Much is at stake in this mission, both in terms of the development of the EU´s external identity but also for Bosnia and Herzegovina’s road to EU membership and sustainable peace. This paper will argue that by 2005 the balance sheet was mixed. EUPM fell short of fulfilling its overall goal of ‘Europeanising’ Bosnian police services, and of its desire to be seen as providing that additional ingredient in police matters that would set it apart from the earlier UN mission. Nevertheless, despite its shortcomings, the Mission did not merit the harsh criticisms it was faced with. Its lack of success was not entirely the Mission’s doing. The paper focuses on three aspects: political and economic viability and sustainability, security levels in Bosnia and Herzegovina, and institution and capacity building. The explanatory framework used in this paper is based on the democratic policing discourse. In doing so the argument developed here will also shed light on the nature of so-called “best European practices” in police matters.

Chondrodysplasia and Abnormal Joint Development Associated with Mutations in IMPAD1, Encoding the Golgi-Resident Nucleotide Phosphatase, gPAPP.

We used whole-exome sequencing to study three individuals with a distinct condition characterized by short stature, chondrodysplasia with brachydactyly, congenital joint dislocations, cleft palate, and facial dysmorphism. Affected individuals carried homozygous missense mutations in IMPAD1, the gene coding for gPAPP, a Golgi-resident nucleotide phosphatase that hydrolyzes phosphoadenosine phosphate (PAP), the byproduct of sulfotransferase reactions, to AMP. The mutations affected residues in or adjacent to the phosphatase active site and are predicted to impair enzyme activity. A fourth unrelated patient was subsequently found to be homozygous for a premature termination codon in IMPAD1. Impad1 inactivation in mice has previously been shown to produce chondrodysplasia with abnormal joint formation and impaired proteoglycan sulfation. The human chondrodysplasia associated with gPAPP deficiency joins a growing number of skeletoarticular conditions associated with defective synthesis of sulfated proteoglycans, highlighting the importance of proteoglycans in the development of skeletal elements and joints.

Post-translational modifications regulate distinct functions of CARMA1 and BCL10.

Activation of the transcription factor nuclear factor (NF)-kappaB is essential for the normal functioning of the immune system. Deregulated NF-kappaB signalling in lymphocytes can lead to immunodeficiency, but also to autoimmunity or lymphomas. Many of the signalling components controlling NF-kappaB activation in lymphocytes are now known, but it is less clear how distinct molecular components of this pathway are regulated. Here, we summarize recent findings on post-translational modifications of intracellular components of this pathway. Phosphorylation of the CARMA1 and BCL10 proteins and ubiquitylation of BCL10 affect the formation and stability of the CARMA1-BCL10-MALT1 (CBM) complex, and also control negative feedback regulation of the NF-kappaB signalling pathway. Moreover, the study of BCL10 phosphorylation isoforms has revealed a new mechanism controlling BCL10 nuclear translocation and an unexpected role for BCL10 in the regulation of the actin cytoskeleton.

Ectodysplasin has a dual role in ectodermal organogenesis: inhibition of Bmp activity and induction of Shh expression.

Ectodermal organogenesis is regulated by inductive and reciprocal signalling cascades that involve multiple signal molecules in several conserved families. Ectodysplasin-A (Eda), a tumour necrosis factor-like signalling molecule, and its receptor Edar are required for the development of a number of ectodermal organs in vertebrates. In mice, lack of Eda leads to failure in primary hair placode formation and missing or abnormally shaped teeth, whereas mice overexpressing Eda are characterized by enlarged hair placodes and supernumerary teeth and mammary glands. Here, we report two signalling outcomes of the Eda pathway: suppression of bone morphogenetic protein (Bmp) activity and upregulation of sonic hedgehog (Shh) signalling. Recombinant Eda counteracted Bmp4 activity in developing teeth and, importantly, inhibition of BMP activity by exogenous noggin partially restored primary hair placode formation in Eda-deficient skin in vitro, indicating that suppression of Bmp activity was compromised in the absence of Eda. The downstream effects of the Eda pathway are likely to be mediated by transcription factor nuclear factor-kappaB (NF-kappaB), but the transcriptional targets of Edar have remained unknown. Using a quantitative approach, we show in cultured embryonic skin that Eda induced the expression of two Bmp inhibitors, Ccn2/Ctgf (CCN family protein 2/connective tissue growth factor) and follistatin. Moreover, our data indicate that Shh is a likely transcriptional target of Edar, but, unlike noggin, recombinant Shh was unable to rescue primary hair placode formation in Eda-deficient skin explants.

The p53 gene expression and its developmental regulation in schistosomes

We have studied the gene expression, especially of the oncoproteins, and its regulation in schistosomes. Schistosomes have a complex life cycle with defined dimorphic lifestyle. The parasite are so far unique in biology in expressing oncogene products in their adult stage. In order to characterize the expression and developmental regulation, a lambda gt 11 cDNA library and lambda EMBL4 genomic DNA library of each growth stage of Schistosoma mansoni and S. japonicum was constructed, and was screened with various monoclonal antibodies against ongogene products. One positive plaque reacted to anti-p53 antibody (Ab-2, Oncogene Science, Inc.) was further analyzed. This fusion protein was about 120 KDa in molecular weights, and expressed as 1.4 Kb RNA in the adult stage. P53 gene is well-known as the negative regulator of the cell cicle, and the mutations in the gene are turning out to be the most common genetic alterations in human cancers. The comparison of the gene structure among species and stages were being conducted. Chromosome structures, C-band formation, and the results of in situ hybridization using the phage probe would be discussed.

Variation in the intensity of inbreeding depression among successive life-cycle stages and generations in gynodioecious Silene vulgaris (Caryophyllaceae).

Inbreeding depression is one of the hypotheses explaining the maintenance of females within gynodioecious plant populations. However, the measurement of fitness components in selfed and outcrossed progeny depends on life-cycle stage and the history of inbreeding. Comparative data indicate that strong inbreeding depression is more likely to occur at later life-cycle stages. We used hermaphrodite individuals of Silene vulgaris originating from three populations located in different valleys in the Swiss Alps to investigate the effect of two generations of self- and cross-fertilization on fitness components among successive stages of the life cycle in a glasshouse experiment. We detected significant inbreeding depression for most life-cycle stages including: the number of viable and aborted seeds per fruit, probability of germination, above ground biomass, probability of flowering, number of flowers per plant, flower size and pollen viability. Overall, the intensity of inbreeding depression increased among successive stages of the life cycle and cumulative inbreeding depression was significantly stronger in the first generation (delta approximately 0.5) compared with the second generation (delta approximately 0.35). We found no evidence for synergistic epistasis in our experiment. Our finding of more intense inbreeding depression during later stages of the life cycle may help to explain the maintenance of females in gynodioecious populations of S. vulgaris because purging of genetic load is less likely to occur.

PGC1alpha expression is controlled in skeletal muscles by PPARbeta, whose ablation results in fiber-type switching, obesity, and type 2 diabetes.

Mice in which peroxisome proliferator-activated receptor beta (PPARbeta) is selectively ablated in skeletal muscle myocytes were generated to elucidate the role played by PPARbeta signaling in these myocytes. These somatic mutant mice exhibited a muscle fiber-type switching toward lower oxidative capacity that preceded the development of obesity and diabetes, thus demonstrating that PPARbeta is instrumental in myocytes to the maintenance of oxidative fibers and that fiber-type switching is likely to be the cause and not the consequence of these metabolic disorders. We also show that PPARbeta stimulates in myocytes the expression of PGC1alpha, a coactivator of various transcription factors, known to play an important role in slow muscle fiber formation. Moreover, as the PGC1alpha promoter contains a PPAR response element, the effect of PPARbeta on the formation and/or maintenance of slow muscle fibers can be ascribed, at least in part, to a stimulation of PGC1alpha expression at the transcriptional level.

Efficient gene delivery and selective transduction of astrocytes in the mammalian brain using viral vectors.

Astrocytes are now considered as key players in brain information processing because of their newly discovered roles in synapse formation and plasticity, energy metabolism and blood flow regulation. However, our understanding of astrocyte function is still fragmented compared to other brain cell types. A better appreciation of the biology of astrocytes requires the development of tools to generate animal models in which astrocyte-specific proteins and pathways can be manipulated. In addition, it is becoming increasingly evident that astrocytes are also important players in many neurological disorders. Targeted modulation of protein expression in astrocytes would be critical for the development of new therapeutic strategies. Gene transfer is valuable to target a subpopulation of cells and explore their function in experimental models. In particular, viral-mediated gene transfer provides a rapid, highly flexible and cost-effective, in vivo paradigm to study the impact of genes of interest during central nervous system development or in adult animals. We will review the different strategies that led to the recent development of efficient viral vectors that can be successfully used to selectively transduce astrocytes in the mammalian brain.