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The design and development of fluorescent reporters focussed on highly sensitive, specific, and selective imaging of cancer targets is described. These novel optical molecular probes were synthesised with the aim of creating bio-imaging breakthroughs that will aid the clinical analysis of cancer. A specific target of the project was to develop fluorescent reporters for Caspases; intracellular endopeptidases that play an essential role in apoptosis. Lack of activation of the ‘Caspase Cascade’ causes uncontrolled proliferation of cells and has been deemed a ‘Hallmark of Cancer’. In particular, low Caspases-3/7 activities have been associated with a range of cancers, thus molecular detection of Caspases-3/7 activities could therefore lead to advances in oncology. A 14-member FRET library, based upon Caspases-3/7 specific peptide sequences, was initially developed. The cleavage rates and KM values were evaluated for Caspases-3/7, along with the cleavage rates for Cathepsin B, to determine the peptide with the greatest affinity and specificity for Caspase-3. Also developed was a set of internally quenched activity based molecular reporters constructed by attaching fluorophores to a tribranched dendron through the Caspase specific peptide, developed from the FRET Library. The KM values of the dendron probes with Caspase-3 were also evaluated. Furthermore, the dendron reporters were attached to cell penetrating peptides to enable delivery to intracellular Caspase and allow in situ detection of activated Caspase-3 within live cells. In addition, a new labelling moiety was developed enabling dual detection of reporters through fluorescence and MRI imaging. To achieve this, a perfluoro tag (C8F17) was tethered to a Cy5 dye to enable dual detection. The dual 19F-MRI/Cy5 dye was conjugated onto to a cell penetrating peptide to enable in vivo detection of the probe by 19F-MRI and fluorescent imaging.

Solid-state nanopores are an emerging class of single-molecule detectors that provide information about molecular identity via the analysis of transient fluctuations in the ionic current flowing across a nanoscale pore in a thin membrane. The transport of biomolecules across a pore is a key step in nanopore-based sensing of DNA, RNA and proteins. The dynamics of biomolecular transport are complex and depend on the strength of many interactions, which can be tuned with temperature. However, temperature is rarely controlled during solid-state nanopore experiments because of the added electrical noise from the temperature control and measurement systems, greatly reducing the signal-to-noise ratio when detecting individual molecules. So far, the use of electric-based heating and cooling strategies has limited the recording bandwidth to the kHz range, restricting the studies to long polymers translocating via the pore relatively slowly. Yet, many molecules translocate through the pore orders of magnitude faster. This research presents the development and testing of an instrument to allow low-noise electrical recording of nanopore signals at MHz bandwidth as a function of temperature. Initial experiments using this custom-built instrument for the study of linear DNA polymers confirm previously observed translocation behaviours, while providing a higher temporal resolution. Overall results show that high-speed nanopore experiments are possible while controlling the temperature up to 70 °C, opening up exciting opportunities to study the unfolding of proteins toward single-molecule protein sequencing and the passage of DNA nanostructures for different bioassays. Future work will focus on realizing microfluidic flow cells and nanopore performance at higher temperature for longer recording times.

Electrospinning is a technique to achieve nanometer scale fibers. Similar to the conventional spin methods of making fabric, the viscous polymer solution is ejected from a spinneret; stretched and solidified in the air, the solution forms the fibers. The different part of electrospinning among others is that the fibers are driven by the electrostatic force, which induces the repulsion inside the liquid and further reduces the diameter. The resultant product is a non-woven membrane, which is porous; and the pore size is around several nanometers to a micrometer wide. In this work, the relationship between the diameter of electrospun silica fibers, experimental parameters such as concentration and voltage, and between pore size of the fiber membrane and experimental time were studied. Materials used in the process are Polyvinylpyrrolidone (PVP), butanol and spin-on-glass coating solution, which act as polymer carrier, solvent, and silica-precursor, respectively. Polymer/silica precursor composite fibers were ejected from the needle of a plastic syringe when an electrical field, as high as several kV/cm, was applied. Then silica fibers were achieved by baking the composite ones at 773 oK for 12 h. Electrospun silica nanofibers were characterized as a function of polymer solution parameters. The calcined fibers were examined by using a field emission scanning electron microscope. The results showed that the fiber diameters decrease with decreasing proportion of polymer and silica precursor, and increase with a higher electric field. Pore sizes, defined as the grid areas enclosed by fibers on nearby layers, were also examined and showed no time-dependent tendency when the electrospin time was between 1-5 min. Fiber membranes were then used as the platform for protein detection. The results were compared with the control, which used glass slides as the platform. The results make it possible to make a more sensitive biosensing device.

Human norovirus (NoV) causes gastroenteritis worldwide and has been associated with a number of produce related outbreaks. The design of effective inactivation and prevention procedures requires an understanding of virus survival in environments applicable to the production and processing of fresh produce. To evaluate the extent of NoV risk from farm to fork, the survival of murine norovirus (MNV), a surrogate for human NoV, was studied on stainless steel disks, soil and in bottled water for 42 days and on lettuce for 15 days in the laboratory. Stability experiments were then conducted on farm during one lettuce planting/harvest cycle, for 4 weeks. MNV stability was tested at room temperature in the laboratory or under ambient conditions on the farm. A one log reduction in virus titre was achieved after 30 days in water, 4 days on lettuce, 15 days on stainless steel disks, 12 days on loamy and sandy soil. For farm testing, infectious virus was recovered from both soil and lettuce on the day of inoculation. Although infectious virus was not recovered at later time points, the viral genomes were detected for up to four weeks. The observed long-term persistence of NoV, under both laboratory and field conditions, provides valuable information for developing risk assessments and control procedures to limit the possibility for NoV transmission in the food supply.

Human norovirus (NoV) causes gastroenteritis worldwide and has been associated with a number of produce related outbreaks. The design of effective inactivation and prevention procedures requires an understanding of virus survival in environments applicable to the production and processing of fresh produce. To evaluate the extent of NoV risk from farm to fork, the survival of murine norovirus (MNV), a surrogate for human NoV, was studied on stainless steel disks, soil and in bottled water for 42 days and on lettuce for 15 days in the laboratory. Stability experiments were then conducted on farm during one lettuce planting/harvest cycle, for 4 weeks. MNV stability was tested at room temperature in the laboratory or under ambient conditions on the farm. A one log reduction in virus titre was achieved after 30 days in water, 4 days on lettuce, 15 days on stainless steel disks, 12 days on loamy and sandy soil. For farm testing, infectious virus was recovered from both soil and lettuce on the day of inoculation. Although infectious virus was not recovered at later time points, the viral genomes were detected for up to four weeks. The observed long-term persistence of NoV, under both laboratory and field conditions, provides valuable information for developing risk assessments and control procedures to limit the possibility for NoV transmission in the food supply.

Human norovirus (NoV) causes gastroenteritis worldwide and has been associated with a number of produce related outbreaks. The design of effective inactivation and prevention procedures requires an understanding of virus survival in environments applicable to the production and processing of fresh produce. To evaluate the extent of NoV risk from farm to fork, the survival of murine norovirus (MNV), a surrogate for human NoV, was studied on stainless steel disks, soil and in bottled water for 42 days and on lettuce for 15 days in the laboratory. Stability experiments were then conducted on farm during one lettuce planting/harvest cycle, for 4 weeks. MNV stability was tested at room temperature in the laboratory or under ambient conditions on the farm. A one log reduction in virus titre was achieved after 30 days in water, 4 days on lettuce, 15 days on stainless steel disks, 12 days on loamy and sandy soil. For farm testing, infectious virus was recovered from both soil and lettuce on the day of inoculation. Although infectious virus was not recovered at later time points, the viral genomes were detected for up to four weeks. The observed long-term persistence of NoV, under both laboratory and field conditions, provides valuable information for developing risk assessments and control procedures to limit the possibility for NoV transmission in the food supply.

Human norovirus (NoV) causes gastroenteritis worldwide and has been associated with a number of produce related outbreaks. The design of effective inactivation and prevention procedures requires an understanding of virus survival in environments applicable to the production and processing of fresh produce. To evaluate the extent of NoV risk from farm to fork, the survival of murine norovirus (MNV), a surrogate for human NoV, was studied on stainless steel disks, soil and in bottled water for 42 days and on lettuce for 15 days in the laboratory. Stability experiments were then conducted on farm during one lettuce planting/harvest cycle, for 4 weeks. MNV stability was tested at room temperature in the laboratory or under ambient conditions on the farm. A one log reduction in virus titre was achieved after 30 days in water, 4 days on lettuce, 15 days on stainless steel disks, 12 days on loamy and sandy soil. For farm testing, infectious virus was recovered from both soil and lettuce on the day of inoculation. Although infectious virus was not recovered at later time points, the viral genomes were detected for up to four weeks. The observed long-term persistence of NoV, under both laboratory and field conditions, provides valuable information for developing risk assessments and control procedures to limit the possibility for NoV transmission in the food supply.

Η επιπεφυκίτιδα (φλεγμονή του επιπεφυκότα) είναι η πιο συχνή ασθένεια των οφθαλμών, η οποία εκδηλώνεται σε παγκόσμια κλίμακα με τη μορφή σποραδικών κρουσμάτων ή επιδημίας. Μπορεί να είναι λοιμώδους (βακτήρια, ιοί, παράσιτα) ή μη λοιμώδους αιτιολογίας. Η κύρια αιτία της οξείας ιογενούς αιτιολογίας επιπεφυκίτιδας είναι οι αδενοϊοί. Περίπου το 15- 70% του συνόλου των κρουσμάτων της επιπεφυκίτιδας οφείλονται στους αδενοϊούς. Σκοπός της μελέτης είναι η χρήση επιδημιολογικών δεδομένων προκειμένου να πραγματοποιηθεί επιδημιολογική παρακολούθηση των κρουσμάτων επιπεφυκίτιδας στο Πανεπιστημιακό Γενικό Νοσοκομείο Πατρών (ΠΓΝΠ) από ασθενείς οι οποίοι επισκέφθηκαν την οφθαλμιατρική κλινική και τα εξωτερικά ιατρεία του νοσοκομείου τη χρονική περίοδο 2 Ιανουαρίου – 29 Ιουλίου 2012 (εβδομάδες 1- 30), ο καθορισμός της συχνότητας της ιογενούς αιτιολογίας επιπεφυκίτιδας και ο εντοπισμός πιθανής ύπαρξης επιδημίας. Ταυτόχρονα, πραγματοποιήθηκε μοριακή ανίχνευση και τυποποίηση αδενοϊών από ασθενείς με κλινική εικόνα ιογενούς επιπεφυκίτιδας το χρονικό διάστημα μεταξύ 27 Φεβρουαρίου και 17 Ιουνίου. Όλα τα κρούσματα καταγράφηκαν από τα ιατρικά αρχεία του ΠΓΝΠ για το χρονικό διάστημα Ιανουαρίου- Ιουλίου του 2012 και για το ίδιο χρονικό διάστημα το προηγούμενο έτος (2011). Καταγράφηκαν 231 κρούσματα επιπεφυκίτιδας (47,1% άνδρες και 52,8% γυναίκες), από τα οποία τα 205 ήταν ιογενούς αιτιολογίας, τα 4 βακτηριογενούς αιτιολογίας και 22 ήταν απροσδιόριστης αιτιολογίας από τους ιατρούς. Για την ίδια χρονική περίοδο το προηγούμενο έτος (2011), σύμφωνα με τα αρχεία του ΠΓΝΠ καταγράφηκε ένα σύνολο από 156 κρούσματα επιπεφυκίδας (38,5% άνδρες και 61,5% γυναίκες), από τα οποία τα 135 ήταν ιογενούς αιτιολογίας, τα 3 βακτηριογενούς αιτιολογίας και 18 ήταν απροσδιόριστης αιτιολογίας. Ο αριθμός κρουσμάτων επιπεφυκίτιδας τους δυο πρώτους μήνες καθώς και τον Ιούλιο του 2012 ήταν στα ίδια επίπεδα με τους αντίστοιχους μήνες το 2011 και παρατηρείται επιδημία που πραγματοποιήθηκε μεταξύ Μαρτίου- Ιουνίου 2012. Οι ασθενείς κατανέμονταν σε όλες τις ηλικίες και στα δυο φύλα. Το χρονικό διάστημα μεταξύ 27 Φλεβάρη- 17 Ιουνίου του 2012 (εβδομάδες 9- 24), 48 επιχρίσματα επιπεφυκότα ασθενών με κλινική εικόνα αδενικής επιπεφυκίτιδας συλλέχθηκαν από τους ιατρούς του ΠΓΠΝ και μεταφέρθηκαν υπό κατάλληλες συνθήκες στο εργαστήριο Υγιεινής της Ιατρικής Σχολής του Πανεπιστημίου Πατρών. Ταυτόχρονα συμπληρώθηκε από τους ιδίους ερωτηματολόγιο με δημογραφικά και κλινικά στοιχεία. Το DNA του ιού απομονώθηκε με Qiagen και ενισχύθηκε με nested PCR. Τα θετικά αποτελέσματα επιβεβαιώθηκαν με αλληλούχιση του PCR προϊόντος. Για τον προσδιορισμό της συγγένειας μεταξύ των διαφόρων απομονωμένων αλληλουχιών του DNA, φυλογενετική ανάλυση πραγματοποιήθηκε. Από το σύνολο των δειγμάτων που αναλύθηκαν με μοριακές τεχνικές, DNA αδενοϊού ανιχνεύθηκε σε 40 δείγματα (83%). Στα σαράντα θετικά δείγματα καθορίστηκε η αλληλουχία του DNA τους, από τα οποία τα 29 (72,5%) προσδιορίστηκαν ως τύπος HAdV17 και τα 5 (12,5%) ως τύπος HAdV-54. Σε 6 θετικά δείγματα (15%) ο τύπος του ιού δεν προσδιορίστηκε. Τέλος, με τη βοήθεια των μοριακών τεχνικών προκύπτει το συμπέρασμα ότι το στέλεχος αδενοϊού 17 (Αd 17) είναι η αιτία της εμφάνισης επιδημίας μεταξύ Μαρτίου- Ιουνίου 2012. Η έρευνα αυτή είναι από τις λίγες πιυ έχουν πραγματοποιηθεί στον Ελλαδικό χώρο σε κρούσματα επιπεφυκίτιδας με αποτέλεσμα να εμπλουτίζει τα φτωχά επιδημιολογικά και μοριακά δεδομένα για την συγκεκριμένη ασθένεια και το συγκεκριμένο τύπο ιών. Παράλληλα μέσω της έρευνας υπογραμμίζεται η ανάγκη για εθνικό σύστημα επιτήρησης της επιπεφυκίτιδας. / Conjunctivitis (inflammation of the conjunctiva) is the most common eye disease that occurs worldwide in both sporadic and epidemic form. There are infectious conjunctivitis, which is caused by a variety of microorganisms (such as bacteria, viruses and parasites) and noninfectious conjunctivitis, which is caused by an allergic reaction. The leading cause of acute viral conjunctivitis in clinical practice includes human adenoviruses (HAdVs). About 15- 70% of all conjunctivitis cases worldwide are associated with AdVs. The aim of the study is the performance of epidemiological surveillance of cases of conjunctivitis using epidemiological data from patients who visited the ophthalmic clinic and the outpatient ophthalmic department of the University General Hospital of Patras (UGHP) in the period from January 2nd to July 29th in 2011 and 2012 (weeks 1st-30th), in order to determine the frequency of viral conjunctivitis and to determine a potential epidemic. An additional task of the study is the molecular detection and typing of adenoviruses for cases of patients with clinical viral conjunctivitis in the period from February 27th to June 17th 2012. All conjunctivitis cases referred to UGHP in the period between January and July 2012 as well as between January and July 2011 were ascertained using medical records. 231 conjunctivitis cases were reported (47.1% male and 52.8% female), in which 205 were virological conjunctivitis, 4 bacteriological conjunctivitis and 22 were undefined conjunctivitis. For the same period the previous year (2011), according to the records of UGHP recorded a total of 156 conjunctivitis cases (38.5% male and 61.5% female), of which 135 were of viral origin, 3 bacteriogenic orogin and 18 were undetermined etiology. The number of conjunctivitis cases in the first two months and in July 2012 was at the same level as the corresponding period in 2011 and there is an epidemic that took place between March and June 2012. Patients were allocated to all age groups and both sexes. In the period from February 27th ,2012 to June 17th , 2012 (weeks 9th – 24th), 48 conjunctival swabs were collected from cases which were clinically suspected of having adenoviral conjunctivitis and transported under appropriate conditions to the laboratory of Hygiene, Medical School, University of Patras. At the same time, the patients were asked to answer a structured questionnaire with demographic and clinical data. The viral DNA was isolated with Qiagen and amplified by nested PCR. The positive results were confirmed by sequencing the PCR product. To determine the relatedness between the different isolated sequences, a phylogenetic analysis was constructed. Of the total samples, which were analyzed with molecular techniques, adenovirus DNA was detected in 40 samples (83%). Of the positive samples which were confirmed by sequencing, 29 samples (72.5%) were typed as AdV17 and 5 samples (12.5%) as AdV54. For 6 positive samples (15%) the serotype was not determined. Finally, it was concluded that the strain Adenovirus 17 (Ad 17) was the cause of the epidemic between March and June 2012. There are poor epidemiological and molecular data for this particular disease in Greece. This study is one of the very few on conjunctivitis determination in Greece. This research underscores the need for a national surveillance system for conjunctivis outbreaks.

A Laringotraqueíte Infecciosa (LTI) é uma doença respiratória altamente contagiosa, que acomete principalmente galinhas, é causada por um Gallid herpesvirus tipo 1. As aves infectam-se através do trato respiratório e por via ocular, sendo as aves com infecção clínica as principais transmissoras do vírus. Outras fontes de transmissão são as aves com infecções latentes, materiais de cama e fômites contaminados. Os sinais clínicos geralmente aparecem entre 6 a 12 dias da exposição natural e em infecções experimentais entre 4 a 7 dias pós infecção (p.i). Na forma subclínica pode-se observar uma leve traqueíte mucoide, sinusite, conjuntivite, com morbidade variável e baixa mortalidade. Na forma severa, as aves apresentam depressão, dispneia, espirros, corrimento nasal, conjuntivite, expectoração de secreção sanguinolenta. A taxa de morbidade é alta, comprometendo 100% do lote e a mortalidade pode ocorrer em até 70% do plantel, embora taxas de 10 a 20% sejam as mais frequentes. O agente causador desta doença pode ser propagado na membrana corio-alantóide (MCA) de embriões de frango em desenvolvimento e replicado em células de rim de frangos adultos, como também, em uma variedade de células epiteliais de embrião como do rim, do fígado e do pulmão. Existem vários procedimentos para o diagnóstico da LTI como: a observação de sinais clínicos, a observação de lesões macroscópicas e lesões histopatológicas e o uso de técnicas moleculares como: RFLP, PCR e PCR em tempo real. Como a PCR em tempo real apresenta uma maior sensibilidade quando comparada com outros métodos de diagnóstico, permite quantificar o número de cópias amplificadas do genoma viral, assim como, a diferenciação da doença na fase aguda ou crônica, reduzindo o número de possíveis falsos positivos, esta foi usada para a detecção deste vírus. O objetivo deste trabalho foi isolar o VLTI em ovos embrionados, descrever as lesões macroscópicas causadas pelo vírus, detectar o vírus pela reação de PCR em tempo real, usando como alvo da reação o gene ICP4, padronizar uma reação de PCR em Tempo Real usando a glicoproteína E como alvo da reação e propor o seu uso no diagnóstico de rotina. / The Infectious Laryngotracheitis (ILT) is a highly contagious respiratory disease that affects mainly chickens; caused by a Gallid herpesvirus type 1. Infect birds, by the respiratory tract and by the ocular route, the birds with clinical infection the main transmission of the virus. Other transmission sources are birds with latent infections, bedding materials and contaminated fomites. Clinical signs generally appear between 6 to 12 days exposure of the natural and in experimental infections between 4 and 7 days post infection (p.i). In subclinical form can observe a slight mucoid tracheitis, sinusitis, conjunctivitis, variable morbidity and low mortality. In the severe form, the birds present depression, dyspnea, sneezing, nasal discharge, conjunctivitis, expectoration of bloody discharge. The morbidity rate is high, impairing the lot 100% and mortality may occur in up to 70% of the flock, although 10 to 20% rates are the most frequent. The causative agent of this disease can be propagated in chorioallantoic membrane (CAM) of chicken embryos develop and replicated in adult chicken kidney cells as well as in a variety of epithelial-cell embryo as kidney, liver and lung. There are several procedures for the diagnosis of LTI as observation of clinical signs, observation of gross lesions and histopathological lesions, and the use of molecular techniques as RFLP, PCR and real time PCR. As the real-time PCR has greater sensitivity compared to other diagnostic methods to quantify the number of amplified copies of the viral genome, as well as the differentiation of the disease in the acute or chronic phase, reducing the number of potential false positives, this was used for detection of virus. The objective of this study was to isolate the VLTI in embryonated eggs, describe macroscopic lesions caused by the virus, detect the virus by PCR reaction in real time, using as a target of reaction the ICP4 gene, standardize a PCR reaction in real time using the glycoprotein E as a target of reaction and propose their use in routine diagnosis.