Aging-related tau astrogliopathy (ARTAG):harmonized evaluation strategy

Pathological accumulation of abnormally phosphorylated tau protein in astrocytes is a frequent, but poorly characterized feature of the aging brain. Its etiology is uncertain, but its presence is sufficiently ubiquitous to merit further characterization and classification, which may stimulate clinicopathological studies and research into its pathobiology. This paper aims to harmonize evaluation and nomenclature of aging-related tau astrogliopathy (ARTAG), a term that refers to a morphological spectrum of astroglial pathology detected by tau immunohistochemistry, especially with phosphorylation-dependent and 4R isoform-specific antibodies. ARTAG occurs mainly, but not exclusively, in individuals over 60 years of age. Tau-immunoreactive astrocytes in ARTAG include thorn-shaped astrocytes at the glia limitans and in white matter, as well as solitary or clustered astrocytes with perinuclear cytoplasmic tau immunoreactivity that extends into the astroglial processes as fine fibrillar or granular immunopositivity, typically in gray matter. Various forms of ARTAG may coexist in the same brain and might reflect different pathogenic processes. Based on morphology and anatomical distribution, ARTAG can be distinguished from primary tauopathies, but may be concurrent with primary tauopathies or other disorders. We recommend four steps for evaluation of ARTAG: (1) identification of five types based on the location of either morphologies of tau astrogliopathy: subpial, subependymal, perivascular, white matter, gray matter; (2) documentation of the regional involvement: medial temporal lobe, lobar (frontal, parietal, occipital, lateral temporal), subcortical, brainstem; (3) documentation of the severity of tau astrogliopathy; and (4) description of subregional involvement. Some types of ARTAG may underlie neurological symptoms; however, the clinical significance of ARTAG is currently uncertain and awaits further studies. The goal of this proposal is to raise awareness of astroglial tau pathology in the aged brain, facilitating communication among neuropathologists and researchers, and informing interpretation of clinical biomarkers and imaging studies that focus on tau-related indicators

NMR-Untersuchungen zu dynamischen Umfaltungsprozessen in RNA-Molekülen

The following thesis is concerned with the elucidation of structural changes of RNA molecules during the time course of dynamic processes that are commonly denoted as folding reactions. In contrast to the field of protein folding, the concept of RNA folding comprises not only folding reactions itself but also refolding- or conformational switching- and assembly processes (see chapter III). The method in this thesis to monitor these diverse processes is high resolution liquid-state NMR spectroscopy. To understand the reactions is of considerable interest, because most biological active RNA molecules function by changing their conformation. This can be either an intrinsic property of their respective sequence or may happen in response to a cellular signal such as small molecular ligand binding (like in the aptamer and riboswitch case), protein or metal binding. The first part of the thesis (chapters II & III) provides a general overview over the field of RNA structure and RNA folding. The two chapters aim at introducing the reader into the current status of research in the field. Chapters II is structured such that primary structure is first described then secondary and tertiary structure elements of RNA structure. A special emphasis is given to bistable RNA systems that are functionally important and represent models to understand fundamental questions of RNA conformational switching. RNA folding in vitro as well as in vivo situations is discussed in Chapter III. The following chapters IV and V also belong to the introduction part and review critically the NMR methods that were used to understand the nature and the dynamics of the conformational/structural transitions in RNA. A general overview of NMR methods quantifying dynamics of biomolecules is provided in chapter IV. A detailed discussion of solvent exchange rates and time-resolved NMR, as the two major techniques used, follows. In the final chapter V of the first part the NMR parameters used in structure calculation and structure calculation itself are conferred. The second part of the thesis, which is the cumulative part, encompasses the conducted original work. Chapter VI reviews the general NMR techniques applied and explains their applicability in the field of RNA structural and biochemical studies in several model cases. Chapter VII describes the achievement of a complete resonance assignment of an RNA model molecule (14mer cUUCGg tetral-loop RNA) and introduces a new technique to assign quaternary carbon resonances of the nucleobases. Furthermore, it reports on a conformational analysis of the sugar backbone in this RNA hairpin molecule in conjunction with a parameterization of 1J scalar couplings. Achievements: • Establishment of two new NMR pulse-sequences facilitating the assignment of quaternary carbons in RNA nucleobases • First complete (99.5%) NMR resonance assignment of an RNA molecule (14mer) including 1H, 13C, 15N, 31P resonances • Description of RNA backbone conformation by a complete set of NMR parameters • Description of the backbone conformational dependence in RNA of new NMR parameters (1J scalar couplings) Chapters VII & VIII summarize the real-NMR studies that were conducted to elucidate the conformational switching events of several RNA systems. Chapter VIII gives an overview on the experiments that were accomplished on three different bistable RNAs. These molecules where chosen to be good model systems for RNA refolding reactions and so consequently served as reporters of conformational switching events of RNA secondary structure elements. Achievements: • First kinetic studies of RNA refolding reactions with atomic resolution by NMR • Application of [new] RT-NMR techniques either regarding the photolytic initiation of the reaction or regarding the readout of the reaction • Discovery of different RNA refolding mechanisms for different RNA molecules Deciphering of a general rule for RNA refolding methodology to conformational switching processes of RNA tertiary structure elements. The models for these processes were a) the guanine-dependent riboswitch RNA and b) the minimal hammerhead ribozyme. Achievements: • NMR spectroscopic assignment of imino-resonances of the hypoxanthine bound guanine-dependent riboswitch RNA • Application of RT-NMR techniques to monitor the ligand induced conformational switch of the aptamer domain of the guanine-dependent riboswitch RNA at atomic resolution • Translation of kinetic information into structural information • Deciphering a folding mechanism for the guanine riboswitch aptamer domain • Application of RT-NMR techniques to monitor the reaction of the catalytically active mHHR RNA at atomic resolution In the appendices the new NMR pulse-sequences and the experimental parameters are described, which are not explicitly treated in the respective manuscripts.Die vorliegende Doktorarbeit beschäftigt sich mit den strukturellen Änderungen in RNA Molekülen während dynamischer konformationeller Änderungen, die gemeinhin als RNA-Faltung bezeichnet werden. Im Gegensatz zur Proteinfaltung sind RNA-Faltungsprozesse nicht exklusiv als die Faltung einer definierten Konformation aus einem Ensemble an ungefalteten, d.h. ausgehend von unstrukturierten Molekülen, zu verstehen. RNA-Faltung beinhaltet vielmehr die strukturelle Umwandlung verschiedener stabiler Konformationen (die als RNA-Umfaltung benannt wird) und den Aufbau von molekularen Komplexen aus mehreren Molekülen (siehe Kapitel III). Die experimentelle Technik, die hier zur Untersuchung dieser Prozesse genutzt wurde, ist die hochauflösende Flüssig-NMR-Spektroskopie. Das Verständnis der strukturellen und biophysikalischen Grundlagen solcher Umfaltungsreaktionen von RNA ist essentiell, da solche konformationellen Änderungen die biologische Funktion der Moleküle modulieren. Dabei ist zu bemerken, dass eine Umfaltungsreaktion eine intrinsische Eigenschaft einer gegebenen RNA-Sequenz sein kann oder die Antwort auf ein externes zelluläres Signal, wie die Bindung eines niedermolekularen Liganden (z.B. in Aptameren und in Riboswitch RNAs), eines Proteins oder eines Metall-Ions. Der erste Teil dieser Doktorarbeit (Kapitel I & II) hält einen Überblick über die Themengebiete RNA-Struktur und RNA-Faltung bereit. Beide Kapitel führen in den derzeitigen Stand der Forschung ein. Kapitel II führt dabei entlang der hierarchischen Ordnung von RNA Molekülen und diskutiert die Eigenschaften von Primär-, Sekundär- und Tertiär-Strukturelementen. Ein besonderes Augenmerk wird dabei auf bistabile RNA Systeme gelegt; ihre wichtige biologische Funktionalität wird dargestellt, ebenso wird das Potential ausgeleuchtet, diese funktionale Klasse von RNA Molekülen als Modellsysteme zu nutzen, um fundamentale Fragen zu konformationellen Übergängen in RNA zu beantworten. In Kapitel III folgt sodann die Diskussion über RNA-Faltung in in vitro Experimenten als auch im zellulären Kontext (in vivo). Die Kapitel IV und V besprechen die NMR-spektroskopischen Techniken, die genutzt werden, um die Art und die dynamischen Eigenschaften von konformationellen/strukturellen Umwandlungen in RNA zu untersuchen. Hierbei wird der Schwerpunkt auf die verwendeten Techniken des Wasseraustauschs an labilen Protonen und der zeitaufgelösten NMR-Spektroskopie gelegt. Der zweite Teil der Doktorarbeit fasst kumulativ die durchgeführten Studien zusammen. Kapitel VI bespricht hierbei die grundlegenden NMR Techniken, die zur Strukturaufklärung von RNA Molekülen angewendet werden und zeigt deren Anwendungsmöglichkeiten an unterschiedlichen Beispielen von strukturellen und biochemischen Studien. Das folgende Kapitel VII beschreibt die komplette Resonanzzuordnung eines RNA Modell-Moleküls (14mer cUUCGg tetra-loop RNA) und stellt eine neue Pulstechnik vor, die zur Zuordnung der Resonanzen von quatären Kohlenstoffen in Purinbasen benützt werden kann. Weiterhin schließt sich ein Report an, wie die Konformation des Zuckerrückgrates in RNA-Molekülen bestimmt wird und schlägt mittels einer an oben genanntem Modellsystem durchgeführte Parametrisierung 1J skalare Kopplungen als neue Strukturparameter vor. Kapitel VII & VIII fassen die hierzu durchgeführten RT-NMR Studien zusammen. Kapitel VIII gibt hierbei einen Überblick über die Untersuchungen an drei bistabilen RNA-Systemen. Diese Moleküle wurden ausgewählt, da sie als Modelle für RNA-Umfaltungsreakionen dienen. Das finale Kapitel IX behandelt die Anwendung der oben ausgeführten neuen Methodologie auf konformationelle Umwandlungen von RNA Tertiär-Strukturelementen: a) Guanin-abhängige Riboswitch RNA (GSW) und b) Minimales "hammerhead" Ribozym (mHHR)

Computing genomic science: bioinformatics and standardisation in proteomics

Science is divided and compartmentalised into distinct areas of research. As science develops new research areas emerge and nurture new technologies, new methodological approaches, new disciplines and new research communities. These demarcations are socially constructed spaces that impose a sense of order on science by authenticating the new forms of knowledge that surface. Simply stated, the specific research areas and the social relations contained within them, enable science to progress in a proficient, communal, and sometimes cumulative manner. In this sense the constructed boundaries can be viewed as a set of ordering devices. The mapping of the Human Genome was a significant technical event that reordered biological activity by creating a number of these new socially constructed spaces. This celebrated scientific achievement helped yield a number of emerging 'omic' disciplines, numerous innovative high-throughput technologies, and a myriad of embryonic scientific communities, each with its own distinct identity. In this thesis the Human Genome Project is viewed as the genomic stage of the omic revolution or stage one. The period directly after the sequencing has been coined the post-genomic era and this is described in the thesis as stage two of the social reorganisation of biology. Underpinning the whole thesis is the understanding that omic science is driven by a systems biology (SB) approach to twenty-first century biology. The realisation of this will constitute stage three. Computational biologists are also using a similar model of scientific practice in order to map, trace and direct future scientific practice. However in using this developmental model, the organisation of scientific practice may turn messy when boundaries need to be permeated, re-aligned and re-ordered in the movement from post-genomic science to systems biology science. Consequently the specific aim of this research is to trace how two of these maturing research areas, 'proteomics' and 'bioinformatics', are emerging and stabilising within stage two of the omic model, and to explore some of the social issues that are being reordered within their infrastructure. Drawing upon thirty-one interviews the research provides valuable insight into the social construction of post-genomic knowledge and adds to the growing literature in the field of science and technology studies (STS) by revealing how socially constructed knowledges are translated and transferred within and between newly created scientific communities. This is achieved through an examination of scientific identity, interdisciplinary expertise and community-based standardisation

Innovative approaches to understanding and limiting the public health risks of Cryptosporidium in animals in Australian drinking water catchments

Cryptosporidium is the most important waterborne pathogen due to its resistance to chlorine in drinking water. The contribution of Cryptosporidium to waterborne diseases in Australia is however, unknown. The level of faecal contamination of drinking water catchments with this parasite was assessed by longitudinal analysis of faecal samples collected from marsupials, sheep, cattle and rabbits (n = 5,774) from eleven drinking water catchments across three states; New South Wales (NSW), Queensland (QLD) and Western Australia (WA). Faecal samples were screened by quantitative PCR (qPCR) and typed at two loci using Sanger sequencing. The overall prevalence of Cryptosporidium in faecal samples was 18.3% (1,054/5,774; 95% CI, 17.3-19.3). Of these, 873 samples produced clean Sanger sequencing chromatograms, and the remaining 181 samples, which initially produced chromatograms suggesting the presence of multiple different sequences, were re-analysed by Next Generation Sequencing (NGS) to resolve the presence of Cryptosporidium and the species composition of mixed infections. The overall prevalence of mixed infection was 1.7% (98/5,774), and in the remaining 83 samples, NGS detected only one species of Cryptosporidium. Of the 17 Cryptosporidium species and four genotypes detected (Sanger sequencing combined with NGS), 13 are capable of infecting humans; C. parvum, C. hominis, C. ubiquitum, C. cuniculus, C. meleagridis, C. canis, C. felis, C. muris, C. suis, C. scrofarum, C. bovis, C. erinacei and C. fayeri. Sewage (influent) samples across these states were also collected (n = 730) and screened by qPCR and typed using next generation sequencing (NGS). In sewage samples, the overall Cryptosporidium prevalence was 11.4% (83/730); 14.3% (3/21) in NSW, 10.8% (51/470); in QLD and 12.1% (29/239) in WA, and a total of 17 Cryptosporidium species and 6 genotypes were detected by NGS, including some of the same zoonotic species detected in animal faecal samples. This study highlights the public health importance of continued identification of the sources/carriers of human pathogenic strains for accurate risk assessment and optimal catchment management

Study on the molecular mechanisms at the basis of photosynthesis toward microalgae domestication

Oxygenic photosynthesis sustains life on Earth, being the process through which sunlight is used to drive carbon fixation into biomass releasing oxygen as by-product. Photosynthetic organisms convert light energy into chemical energy thanks to the activity of pigment-binding multiproteic complexes known as photosystems and their relative light-harvesting antenna complexes. More than often, light availability is non-optimal, either too scarce or in excess compared to the photosynthetic capacity of the organism operating photosynthesis, to the point it may result dangerous. Among the most sensitive complexes of the photosynthetic chain, photosystem II is the main site of Reactive Oxygen Species (ROS) formation. These detrimental compounds may lead to a compromised growth and losses in biomass productivity. For this reason, photosynthetic organisms evolved different photoprotection mechanisms, among which Non-Photochemical Quenching (NPQ) is one of the most important and investigated. Microalgae dissipate most of the absorbed energy through this safe-valve mechanism, even when conditions are not dangerous, determining a consequent reduction of biomass accumulation. Moving towards an optimized photosynthetic yield is one of the main objectives in microalgae domestication, which itself is an ever-growing interest, because sustainable microalgal industrial applications have the potential to satisfy many global demands. Among the most important, is the use of algal biomass as food and feed, bio-fuels production, extraction of high-value nutraceuticals and pharmaceuticals, recovery of wastewaters, and carbon capture. However, realization of this potential requires a decrease of the current production costs. To achieve profitability, identification of limiting factors is fundamental. Particularly, light-to-biomass conversion efficiency is a key bottleneck that needs to be addressed to achieve domestication. With this focus, the aim of this thesis was to study microalgal photosynthetic behavior upon different growth conditions and the relative activated energy dissipation processes as a possible target to improve productivity, thus enabling the use of microalgae as green cell factories. All the studies herein presented were conducted on the model organism for green algae, Chlamydomonas reinhardtii, for which extensive literature, mutants\u2019 libraries, and genetic tools are available

Structural Determinants in the folding of epidermal growth factor (EGF)-Like domains

Master'sMASTER OF SCIENC