From micro to nanostructure: an investigation into the connective tissue of the ageing human optic nerve head

The purpose of this thesis was to investigate the micro- and nanostructural changes of the optic nerve head (ONH) regions, namely lamina cribrosa (LC), peripapillary sclera (ppsclera) and region of insertion (RoIns) as a function of age and glaucoma. The microstructure of ageing and glaucomatous ONHs have been explored following nonlinear microscopy [two-photon excited fluorescence (TPEF) and second harmonic generation (SHG)]. Small-angle (SAXS) and wide-angle (WAXS) X-ray diffraction have been used to explore into the nanoarchitecture of collagen and elastic fibre in the ageing ONH. X-ray microtomography (XMT) was used to assess microstructural changes of the ONH in age and glaucoma. Micro- and nanomechanics have been explored following Brillouin microscopy and the combination of SAXS/WAXS, respectively. In the ageing ppsclera, collagen crimp period increased from 19.64 ± 6.41 µm at the age 2 to 22.04 ± 10 µm at the age of 88 years (p=0.004). Elastic fibre content significantly increased from 0% in the 2 years old up to 29% in the 88 years old (p=0.003) in the LC and from 0% up to 49% (p=0.019) in the region of insertion. Significant increase in collagen fibril diameter [from 98.98 nm ± 0.9 at 22 years old to 113.01 nm ± 1.5 at the age of 85 (p=0.026)] and intermolecular Bragg spacing [from 1.44 nm ± 0.12 to 1.53 nm ± 0.32 (p=0.022)] were found along with a decreased interfibrillar spacing [from 37.63 nm ± 7.22 to 12.6 nm ± 4.54 (p=0.003)]. Collagen crimp significantly increased in the LC and region of insertion in moderate (p=0.013, p=0.013) and advanced (p=0.021, p=0.033) glaucoma. XMT enabled the discrimination of the connective tissue from the surrounding neuronal pores and 3D reconstruction of XMT dataset showed altered LC and pore shape in glaucomatous ONH. In conclusion, changes in the connective tissue of the ageing and glaucomatous ONH at both micro- and nanostructure aided in a better understanding on how the ONH responds to intraocular pressure elevation

Establishment of long-term ostracod epidermal culture

Primary crustacean cell culture was introduced in the 1960s, but to date limited cell lines have been established. Skogsbergia lerneri is a myodocopid ostracod, which has a body enclosed within a thin, durable, transparent bivalved carapace, through which the eye can see. The epidermal layer lines the inner surface of the carapace and is responsible for carapace synthesis. The purpose of the present study was to develop an in vitro epidermal tissue and cell culture method for S. lerneri. First, an optimal environment for the viability of this epidermal tissue was ascertained, while maintaining its cell proliferative capacity. Next, a microdissection technique to remove the epidermal layer for explant culture was established and finally, a cell dissociation method for epidermal cell culture was determined. Maintenance of sterility, cell viability and proliferation were key throughout these processes. This novel approach for viable S. lerneri epidermal tissue and cell culture augments our understanding of crustacean cell biology and the complex biosynthesis of the ostracod carapace. In addition, these techniques have great potential in the fields of biomaterial manufacture, the military and fisheries, for example, in vitro toxicity testing

La valoración de los servicios ecosistémicos en los ecosistemas forestales: Un caso de estudio en Los Alpes Italianos

The values of goods and services by natural resources are not included in the political decision making process concerning natural resources management. This gap is due to the fact that many ecosystem services are not marketed. In order to overcome this gap in the management of natural resources, it is necessary to apply some environmental economic methods to capture the total economic value of marketed and non-marketed ecosystem services. Ecosystem services are not homogeneous across landscapes but they are heterogeneous in space. Consequently, mapping ecosystem services is considered as a fundamental requirement for landscape planning. The aim of this paper is to show a method for the economic valuation and mapping of the forest ecosystem services. The method was applied to a case study characterized by a high importance of ecosystem services and involved in the recharge.green project (Alpine Space Programme): the Gesso-Vermenagna valley in Italy. Results show that the highest economic values are for regulating services (from 11 € ha-1to 4.300 € ha-1per year) and for provisioning services (from 6 € ha-1to 1.980 € ha-1per year), while the lowest values are for cultural services (from 6 € ha-1to 627 € ha-1per year). This study can provide useful information to decision makers in order to improve the management of natural resources at local level

Mapping the value of ecosystem services: A case study from the Austrian Alps

Ecosystem services (ES) are considered the conditions and processes through which natural ecosystems sustain and fulfil human life. In the decision making process related to the natural resources management, two key-aspects must be taken into account: the economic value of the benefits provided by ES (provisioning, regulating and cultural services) and the spatial distribution of these benefits. The purpose of the paper is to develop a method aimed to capture the economic value of the benefits provided by ES, in order to support the decision makers in the natural resources management. ES are not homogeneous across landscapes nor in space. Consequently, gaining knowledge on the spatial distribution of the economic relevance of ES is a fundamental information to target management actions. This method was tested in a case study in the Austrian Alps (Leiblachtal area in Vorarlberg region), characterized by a high importance of forests and grasslands. The results show that the highest economic values could be achieved for the provisioning services with a range between 200 €/ha year and 1,400€/ha year. The regulating services have also important economic values, but strongly localized in specific areas (i.e. protective forests against natural hazards). The economic values of the cultural services are influenced by the preferences of tourists and varies between 5 €/ha year to 60 €/ha year. The method allowed us to reveal the spatial heterogeneity of provisioning, regulating and cultural ES, depending on the ecological characteristics of the site