Towards defining the role of glycans as hardware in information storage and transfer: Basic principles, experimental approaches and recent progress

The term `code' in biological information transfer appears to be tightly and hitherto exclusively connected with the genetic code based on nucleotides and translated into functional activities via proteins. However, the recent appreciation of the enormous coding capacity of oligosaccharide chains of natural glycoconjugates has spurred to give heed to a new concept: versatile glycan assembly by the genetically encoded glycosyltransferases endows cells with a probably not yet fully catalogued array of meaningful messages. Enciphered by sugar receptors such as endogenous lectins the information of code words established by a series of covalently linked monosaccharides as fetters for example guides correct intra- and intercellular routing of glycoproteins, modulates cell proliferation or migration and mediates cell adhesion. Evidently, the elucidation of the structural frameworks and the recognition strategies within the operation of the sugar code poses a fascinating conundrum. The far-reaching impact of this recognition mode on the level of cells, tissues and organs has fueled vigorous investigations to probe the subtleties of protein-carbohydrate interactions. This review presents information on the necessarily concerted approach using X-ray crystallography, molecular modeling, nuclear magnetic resonance spectroscopy, thermodynamic analysis and engineered ligands and receptors. This part of the treatise is flanked by exemplarily chosen insights made possible by these techniques. Copyright (C) 2001 S. Karger AG, Basel

AI Solutions for MDS: Artificial Intelligence Techniques for Misuse Detection and Localisation in Telecommunication Environments

This report considers the application of Articial Intelligence (AI) techniques to the problem of misuse detection and misuse localisation within telecommunications environments. A broad survey of techniques is provided, that covers inter alia rule based systems, model-based systems, case based reasoning, pattern matching, clustering and feature extraction, articial neural networks, genetic algorithms, arti cial immune systems, agent based systems, data mining and a variety of hybrid approaches. The report then considers the central issue of event correlation, that is at the heart of many misuse detection and localisation systems. The notion of being able to infer misuse by the correlation of individual temporally distributed events within a multiple data stream environment is explored, and a range of techniques, covering model based approaches, `programmed' AI and machine learning paradigms. It is found that, in general, correlation is best achieved via rule based approaches, but that these suffer from a number of drawbacks, such as the difculty of developing and maintaining an appropriate knowledge base, and the lack of ability to generalise from known misuses to new unseen misuses. Two distinct approaches are evident. One attempts to encode knowledge of known misuses, typically within rules, and use this to screen events. This approach cannot generally detect misuses for which it has not been programmed, i.e. it is prone to issuing false negatives. The other attempts to `learn' the features of event patterns that constitute normal behaviour, and, by observing patterns that do not match expected behaviour, detect when a misuse has occurred. This approach is prone to issuing false positives, i.e. inferring misuse from innocent patterns of behaviour that the system was not trained to recognise. Contemporary approaches are seen to favour hybridisation, often combining detection or localisation mechanisms for both abnormal and normal behaviour, the former to capture known cases of misuse, the latter to capture unknown cases. In some systems, these mechanisms even work together to update each other to increase detection rates and lower false positive rates. It is concluded that hybridisation offers the most promising future direction, but that a rule or state based component is likely to remain, being the most natural approach to the correlation of complex events. The challenge, then, is to mitigate the weaknesses of canonical programmed systems such that learning, generalisation and adaptation are more readily facilitated

Aerospace medicine and biology: A continuing bibliography with indexes (supplement 333)

This bibliography lists 122 reports, articles and other documents introduced into the NASA Scientific and Technical Information System during January, 1990. Subject coverage includes: aerospace medicine and psychology, life support systems and controlled environments, safety equipment, exobiology and extraterrestrial life, and flight crew behavior and performance

Development of multidimensional fluorescence imaging technology with a view towards the imaging of signalling at the immunological synapse

This thesis describes the development and application of multidimensional fluorescence imaging to signalling events at the Natural Killer cell immunological synapse. The primary techniques used in this work are intensity imaging, ratiometric spectral fluorescence imaging and fluorescence lifetime imaging, which have been applied to live and fixed cells. It is shown that although protein accumulation at the immunological synapse can simply be determined by intensity imaging, the presence of protein does not indicate that signalling events are occuring. Signalling at the inhibitory synapse as determined by KIR2DL1 receptor phosphorylation is imaged by means of confocal FLIM. The resolution achievable using this technique is then improved upon by the use of optical tweezers for cell reorientation. A comparison of the sectioning abilities of single point confocal and multiphoton microscopy with multipoint spinning disk based systems is made and a means of achieving an increased rate of imaging for the gold standard of FLIM methods, TCSPC FLIM, is proposed. The proposed multifocal multiphoton TCSPC FLIM system is first simulated and then implemented, with a comparison to widefield time-gated FLIM being carried out. The system is then used to image test samples, and to acquire cell-level metabolic information with the highest time resolution achieved to date via autofluorescence imaging of NADH. Membrane order at activating and inhibitory NK cell immunological synapses is examined by means of ratiometric imaging of a lipid phase-sensitive dye, and software is developed for the analysis of NK cell spreading patterns, and this software was used to demonstrate that the spreading behaviour of NK cells is affected by the type ofligands encountered in terms of the symmetry and dynamics of spreading

Bioengineering strategies for cancer therapy and modelling

Tese de doutoramento em Engenharia de Tecidos, Medicina Regenerativa e Células EstaminaisCancer is a global pandemic with a high incidence among the world population and effective treatments are for the most part elusive. The tumor microenvironment is a highly complex and heterotypic mixture of cells that interact to regulate central control mechanisms, driving immunosuppression and promoting both survival and invasion of cancer cells into surrounding tissues. It has been this complexity that has made finding effective therapeutics such a demanding task and therefore cancer still remains a burden worldwide in health as well as in economic terms. While the progression in the field of cancer research has been clear over the years, there are still several challenges that need to be addressed. Herein, two different sides to this disease are explored: treatment and in vitro models. Adoptive T cell therapy has shown impressive results, however not without its limitations. The use of the T cell mitogen IL-2 within culture systems is known to lead to early exhaustion of T cell subsets while high density of co-stimulating molecules has been linked to undesired immune responses. As an alternative, a nanoparticle system based on the natural polymer gellan-gum was proposed, with tailorable surface functionalization with co-stimulatory molecules. High levels of T cell expansion were observed over the studied period, with secreted IL-2 levels overcoming those of commercial alternatives. With this system, increased expression of cytotoxic molecules Granzyme B and Perforin were also detected in vitro. On the other spectrum, 3D cancer models have sustained a great number of developments observed by an increase in similarity towards native tissues; however, a requirement for even more complex architectures capable of better mimicking cellular interactions is still present. Therefore, an assembloid-based approach was proposed to develop a 3D in vitro melanoma model to further study cellular interactions. These heterotypic tumor assembloids presented a complex architecture capable of sustaining endothelial cell function as well as a high expression of stemness-related markers. These models were subjected to functionality assays where they showed a capacity for “cooperative invasion” which was coincident with an observed increased production of MMP-2. To further unravel the role of stromal cells in the invasive potential of cancer cells a 3D chemotaxis chamber was developed to study cellular interactions observed in the tumor microenvironment, where stem cells and fibroblasts showed to have a crucial role. Ultimately, this thesis allowed to explore biomedical engineering approaches to further contribute to the knowledge in the field opening new doors to be explored in the future.O cancro é uma pandemia global com uma elevada incidência e cujo desenvolvimento de tratamentos eficazes continua a ser difícil. O microambiente tumoral é uma mistura altamente complexa e heterotípica de células que interagem para regular mecanismos centrais que provocam imunossupressão promovendo a sobrevivência e invasão de células tumorais para os tecidos circundantes. É esta complexidade que tem tornado desafiante encontrar terapias eficazes, tornando esta doença um fardo a nível global em termos de saúde e economia. Enquanto a progressão na área da investigação oncológica tem sido clara ao longo dos anos, existem ainda vários desafios que precisam de serem encarados para permitir futuros desenvolvimentos. Aqui, foram exploradas duas vertentes diferentes desta doença: o tratamento e os modelos in vitro. A terapia celular adotiva tem demonstrado resultados impressionantes, no entanto não sem as suas limitações. O uso do mitogénio IL-2 nestes sistemas de cultura é conhecido por levar rapidamente à exaustão das células T, enquanto o uso de moléculas co-estimulatórias em elevadas densidades está associado a respostas imunes não desejadas. Como alternativa, foi proposto um sistema de nanopartículas baseado no polímero natural goma gelana e funcionalizado com moléculas co estimulatórias. Foram observados elevados níveis de expansão de células T e quantidade de IL-2 secretada superior à de alternativas comerciais. Foi ainda verificado in vitro um aumento de expressão das moléculas citotóxicas Grazima B e Perforina. No outro espectro, têm sido desenvolvidos modelos tumorais 3D com uma cada vez maior similaridade para tecidos nativos; no entanto, a necessidade de arquiteturas ainda mais complexas capazes de melhor representar interações celulares persiste. Assim, foi proposta uma abordagem baseada em “assemblóides” para obter modelos 3D in vitro de melanoma para estudar interações celulares. Estes “assemblóides” tumorais heterotípicos apresentaram uma arquitetura complexa capaz de suportar a função de células endoteliais, bem como a elevada expressão de marcadores de pluripotência. Estes modelos foram sujeitos a ensaios de funcionalidade onde mostraram a capacidade de “invasão cooperativa” que foi coincidente com uma produção aumentada de MMP-2. Para tornar mais claro o papel das células estaminais no potencial invasivo de células tumorais, uma câmara 3D de quimiotaxia foi desenvolvida para estudar as interações celulares observadas no microambiente tumoral onde as células estaminais e fibroblastos mostraram ter um papel determinante. Em última análise, esta tese permitiu explorar abordagens da engenharia biomédica de forma a contribuir para o conhecimento da área e abrir novas portas a serem exploradas no futuro

Bacterial detection using an anharmonic acoustic aptasensor

Infectious diseases are currently, one of the greatest global challenges in medicine. Rapid and precise diagnosis and identification of pathogen is important for timely initiation of appropriate antimicrobial therapy. However, many patients with infectious diseases receive empirical treatment rather than appropriate pathogen-directed therapy. As a result antimicrobials have been overused and/or misused, which has ultimately led to antimicrobial resistance (AMR). AMR is broadly considered as the most significant public health threat facing the world today. Policy makers from all over the world have recognised the urgent need for rapid point-of-care (POC) diagnostics that would not only identify pathogens but also provide antimicrobial susceptibility profiles in meaningful timeframe to initiate appropriate antimicrobial therapy and thereby, prevent AMR. Traditional culture-dependent diagnostic methods are still considered as gold standard methods. But they are very slow and generally require 18 to 48 hours with further 8 to 48 hours to perform antibiotic susceptibility test. Among culture-independent methods, PCR and ELISA are label-based, costly, laborious and require specialised equipment and trained personnel to operate them. Lateral flow assays (LFAs) that are low-cost, simple, rapid and paper-based portable detection platforms are very popular, as they can be applied at the POC. [Continues.

Python-Based Analysis to Segment Bone and Soft Tissue in a Healing Callus

The main objective of this study was to produce a Python script that would help facilitate the segmentation process of both bone and soft tissue. The proposed script, in tangent with ImageJ and Mimics, was successful in producing viable results when the bone and soft tissue sample was placed near hydroxyapatite (HA) phantoms during the image acquisition process. It was important to acquire both the HA phantoms and the sample within the same image sequence as the script functioned by analyzing the statistical distribution of the different HA regions to locate the most ideal thresholding ranges to determine the bone mineral density (BMD) percent composition. When the sample and HA phantoms were in the same set of images, they were both subject to the same type of noise and attenuation, thus allowing for better results to be produced. The script was successful in processing input images and was able to calculate the overall volume and surface area of both the bone and soft tissue, as well as determining the overall bone mineral density of bone. It was also attempted to process bone and soft tissue samples separate of the HA phantoms, but the results were inconclusive