Evaluation of colon cancer histomorphology: a comparison between formalin and PAXgene tissue fixation by an international ring trial.

The aim of our study was to evaluate the quality of histo- and cytomorphological features of PAXgene-fixed specimens and their suitability for histomorphological classification in comparison to standard formalin fixation. Fifteen colon cancer tissues were collected, divided into two mirrored samples and either formalin fixed (FFPE) or PAXgene fixed (PFPE) before paraffin embedding. HE- and PAS-stained sections were scanned and evaluated in a blinded, randomised ring trial by 20 pathologists from Europe and the USA using virtual microscopy. The pathologists evaluated histological grading, histological subtype, presence of adenoma, presence of lymphovascular invasion, quality of histomorphology and quality of nuclear features. Statistical analysis revealed that the reproducibility with regard to grading between both fixation methods was rather satisfactory (weighted kappa statistic (k w) = 0.73 (95 % confidence interval (CI), 0.41-0.94)), with a higher agreement between the reference evaluation and the PFPE samples (k w = 0.86 (95 % CI, 0.67-1.00)). Independent from preservation method, inter-observer reproducibility was not completely satisfactory (k w = 0.60). Histomorphological quality parameters were scored equal or better for PFPE than for FFPE samples. For example, overall quality and nuclear features, especially the detection of mitosis, were judged significantly better for PFPE cases. By contrast, significant retraction artefacts were observed more frequently in PFPE samples. In conclusion, our findings suggest that the PAXgene Tissue System leads to excellent preservation of histomorphology and nuclear features of colon cancer tissue and allows routine morphological diagnosis.

Identification of Acremonium isolates from grapevines and evaluation of their antagonism towards Plasmopara viticola

Some endophytic fungal genera in Vitis vinifera, including Acremonium, have been reported as antagonists of Plasmopara viticola. Endophytic Acremonium isolates from an asymptomatic grapevine cultivar Inzolia from Italy were identified by morphological features and multigene phylogenies of ITS, 18S and 28S genes, and their intra-specific genomic diversity was analyzed by RAPD analysis. Culture filtrates (CFs) obtained from Acremonium isolates were tested in vitro for their inhibitory activity against the P. viticola sporangia germination. Among 94 isolates, 68 belonged to the Acremonium persicinum and 26 to the Acremonium sclerotigenum. RAPD analysis grouped the A. persicinum isolates into 15 clusters and defined 31 different strains. The A. sclerotigenum isolates, instead, were clustered into 22 groups and represented 25 strains. All A. persicinum CFs inhibited sporangia germination of P. viticola, while not all those of A. sclerotigenum had inhibitory effect. A different degree of inhibition was observed between strains of the same species, while some strains of different species showed identical inhibitory effect. No correlation was found between RAPD groups and inhibitory activity in both Acremonium species.

Italian wild hop (Humulus lupulus L.): from biodiversity evaluation to varietal selection

Hop (Humulus lupulus L.) is a dioecius perennial plant. The cultivation is specific for female plants, used mainly for brewing and pharmacology. Female inflorescence, known as cone or strobili, contains bitter acids, essential oil and polyphenols. Commercial hop cultivation provides better results in regions between 45 and 55 degrees north or south in latitude, an area that also includes the northern part of Italy, where hop is endemic. Despite several studies have been conducted on the characterization of wild hops biodiversity in the U.S.A. and Europe, a lack in literature concerning the description of Italian wild hops genetic variability is still present. The increasing request of hop varieties improved in important traits, like diseases, resistance and valuable aroma profile, is bringing the hop industry. Moreover, Italian agricultural sector needs new impulse to be competitive in the market. In this view, Italian wild hop biodiversity is a resource, useful for the obtaining of Italian hop varieties, characterized by peculiar aromatic traits and more adaptable to Mediterranean climate, making their cultivation more sustainable. Based on this consideration, the present Ph.D. thesis deals with the evaluation of the Italian hop biodiversity, through the characterization of the wild samples under different point of view. The project started with the recovery of wild hop samples in different areas of north of Italy to consitue a collection field, where 11 commercial cultivars of US and European origin were grown, to have a complete vision of the hop panorama. Ph.D. project followed different research lines, the results of each one contributed to completly characterize the northern Italian hop wild biodiversity: • the morphological description showed a high phenological variability (Study 1); • the genetic characterization confirmed the rich biodiversity of the Italian population and showed a significant genetic distance between Italian genotypes and the commercial cultivars, taken in consideration (Study 2); • the need of an early sex discrimination method leads to an improvement of a genetic marker, developing a more efficient marker (Study 3); • a complete morphologic, genetic and chemical analysis of plants gave results to select the most promising genotypes (Study 4); • the comparison between the performance of wild hops and commercial cultivars in the same collection field indicated that some wild genotypes had a higher environment adaptability (Study 5); • the evaluation of the terroir, obtained comparing commercial cultivars in the collection field and the same genotypes purchased in the market, showed the influence of the northern Italian environment on the aromatic profile (Study 5); • a new analytical method for the revelation of bioactive metabolites and a simple extraction procedure were developed (Study 6). In conclusion, the Ph.D. thesis, contains the first characterization of Italian wild hop, made under field condition. The present study: i) permits to obtain a complete and significative description of the genotypes; ii) allows the identification of the most promising wild Italian genotypes; iii) allows the identification of commercial cultivars more adaptable the northern Italian climate.

Analysis of Acanthamoeba polyphaga surface carbohydrate exposure by FITC-lectin binding and flourescence evaluation

Aims: Characterization of the representative protozoan Acanthamoeba polyphaga surface carbohydrate exposure by a novel combination of flow cytometry and ligand-receptor analysis. Methods and Results: Trophozoite and cyst morphological forms were exposed to a panel of FITC-lectins. Population fluorescence associated with FITC-lectin binding to acanthamoebal surface moieties was ascertained by flow cytometry. Increasing concentrations of representative FITC-lectins, saturation binding and determination of K d and relative Bmax values were employed to characterize carbohydrate residue exposure. FITC-lectins specific for N-acetylglucosamine, N-acetylgalactosamine and mannose/glucose were readily bound by trophozoite and cyst surfaces. Minor incremental increases in FITC-lectin concentration resulted in significant differences in surface fluorescence intensity and supported the calculation of ligand-binding determinants, Kd and relative B max, which gave a trophozoite and cyst rank order of lectin affinity and surface receptor presence. Conclusions: Trophozoites and cysts expose similar surface carbohydrate residues, foremost amongst which is N-acetylglucosamine, in varying orientation and availability. Significance and Impact of the Study: The outlined versatile combination of flow cytometry and ligand-receptor analysis allowed the characterization of surface carbohydrate exposure by protozoan morphological forms and in turn will support a valid comparison of carbohydrate exposure by other single-cell protozoa and eucaryotic microbes analysed in the same manner.

Lexical database enrichment through semi-automated morphological analysis

Derivational morphology proposes meaningful connections between words and is largely unrepresented in lexical databases. This thesis presents a project to enrich a lexical database with morphological links and to evaluate their contribution to disambiguation. A lexical database with sense distinctions was required. WordNet was chosen because of its free availability and widespread use. Its suitability was assessed through critical evaluation with respect to specifications and criticisms, using a transparent, extensible model. The identification of serious shortcomings suggested a portable enrichment methodology, applicable to alternative resources. Although 40% of the most frequent words are prepositions, they have been largely ignored by computational linguists, so addition of prepositions was also required. The preferred approach to morphological enrichment was to infer relations from phenomena discovered algorithmically. Both existing databases and existing algorithms can capture regular morphological relations, but cannot capture exceptions correctly; neither of them provide any semantic information. Some morphological analysis algorithms are subject to the fallacy that morphological analysis can be performed simply by segmentation. Morphological rules, grounded in observation and etymology, govern associations between and attachment of suffixes and contribute to defining the meaning of morphological relationships. Specifying character substitutions circumvents the segmentation fallacy. Morphological rules are prone to undergeneration, minimised through a variable lexical validity requirement, and overgeneration, minimised by rule reformulation and restricting monosyllabic output. Rules take into account the morphology of ancestor languages through co-occurrences of morphological patterns. Multiple rules applicable to an input suffix need their precedence established. The resistance of prefixations to segmentation has been addressed by identifying linking vowel exceptions and irregular prefixes. The automatic affix discovery algorithm applies heuristics to identify meaningful affixes and is combined with morphological rules into a hybrid model, fed only with empirical data, collected without supervision. Further algorithms apply the rules optimally to automatically pre-identified suffixes and break words into their component morphemes. To handle exceptions, stoplists were created in response to initial errors and fed back into the model through iterative development, leading to 100% precision, contestable only on lexicographic criteria. Stoplist length is minimised by special treatment of monosyllables and reformulation of rules. 96% of words and phrases are analysed. 218,802 directed derivational links have been encoded in the lexicon rather than the wordnet component of the model because the lexicon provides the optimal clustering of word senses. Both links and analyser are portable to an alternative lexicon. The evaluation uses the extended gloss overlaps disambiguation algorithm. The enriched model outperformed WordNet in terms of recall without loss of precision. Failure of all experiments to outperform disambiguation by frequency reflects on WordNet sense distinctions.

IN VIVO analysis of ocular morphological changes during phakic accommodation

The principal theme of this thesis is the in vivo examination of ocular morphological changes during phakic accommodation, with particular attention paid to the ciliary muscle and crystalline lens. The investigations detailed involved the application of high-resolution imaging techniques to facilitate the acquisition of new data to assist in the clarification of aspects of the accommodative system that were poorly understood. A clinical evaluation of the newly available Grand Seiko Auto Ref/ Keratometer WAM-5500 optometer was undertaken to assess its value in the field of accommodation research. The device was found to be accurate and repeatable compared to subjective refraction, and has the added advantage of allowing dynamic data collection at a frequency of around 5 Hz. All of the subsequent investigations applied the WAM-5500 for determination of refractive error and objective accommodative responses. Anterior segment optical coherence tomography (AS-OCT) based studies examined the morphology and contractile response of youthful and ageing ciliary muscle. Nasal versus temporal asymmetry was identified, with the temporal aspect being both thicker and demonstrating a greater contractile response. The ciliary muscle was longer in terms of both its anterior (r = 0.49, P <0.001) and overall length (r = 0.45, P = 0.02) characteristics, in myopes. The myopic ciliary muscle does not appear to be merely stretched during axial elongation, as no significant relationship between thickness and refractive error was identified. The main contractile responses observed were a thickening of the anterior region and a shortening of the muscle, particularly anteriorly. Similar patterns of response were observed in subjects aged up to 70 years, supporting a lensocentric theory of presbyopia development. Following the discovery of nasal/ temporal asymmetry in ciliary muscle morphology and response, an investigation was conducted to explore whether the regional variations in muscle contractility impacted on lens stability during accommodation. A bespoke programme was developed to analyse AS-OCT images and determine whether lens tilt and decentration varied between the relaxed and accommodated states. No significant accommodative difference in these parameters was identified, implying that any changes in lens stability with accommodation are very slight, as a possible consequence of vitreous support. Novel three-dimensional magnetic resonance imaging (MRI) and analysis techniques were used to investigate changes in lens morphology and ocular conformation during accommodation. An accommodative reduction in lens equatorial diameter provides further evidence to support the Helmholtzian mechanism of accommodation, whilst the observed increase in lens volume challenges the widespread assertion that this structure is incompressible due to its high water content. Wholeeye MRI indicated that the volume of the vitreous chamber remains constant during accommodation. No significant changes in ocular conformation were detected using MRI. The investigations detailed provide further insight into the mechanisms of accommodation and presbyopia, and represent a platform for future work in this field.

Aerobic performance and morphological modifications after a 4 month physical activity training program in elderly women

Research clearly shows that physical activity (PA) is an important factor to develop and maintain good health and adequate body functions in older people. In this context, the purpose of this study was to determine aerobic performance and morphological modifications after a 4 month physical activity program (PAP) in elderly. METHODS Forty subjects divided in two groups (control, n=20; and experimental, n=20) were evaluated twice, at the beginning and after a 4-month-activity program period. This program called “+ age + health” consists of 3 week sessions of one hour each, based on walking and aerobic exercises. The control group had, at its first evaluation, the followings characteristics: average body mass 68kg±15, 28±5 BMI, 37%±5 body fat, 2.2kg±0.4 bone mass, 42%±9 lean body mass and did 129 repetitions ± 46 on a 2-Minute Step Test (2MST). The assessment of anthropometric and morphological variables was measured through an electrical bioimpedance scale (TANITA - BC 545). Aerobic endurance was evaluated from a 2MST.RESULTS In the control group only the percentage of body fat changed significantly, and increased over time. In the experimental group we found a positive relationship between PAP and the majority of morphological variables. The percentage of variation changed in: body fat (-4.3%±7.6, p=0.014), bone mass (2.4%±3.1, p=0.004) and 2MST (33.6%±63.1, p=0.023). In the remaining variables there were no significant modifications. The significant modification in 2MST after the activity period means that the aerobic performance can be improved in elderly, and attenuates the negative effects of age. Moreover, the benefits of PAP can be seen by positive alterations registered in lean body mass and in the percentage of body fat.

Evaluation of the reaction of watermelon parent and F1 plants to Meloidogyne enterolobii.

The aim of this study was to evaluate the performance of progenies from Citrullus lanatus var. lanatus (cultivated watermelons) when crossed with progenies from C. lanatus var. citroides (fodder watermelon with a historic of resistance to the nematode Meloidogyne enterolobii). The parents and their F1s were evaluated for resistance to this nematode. In the initial stages of eleven treatments, watermelon seedlings plantlets were transplanted to plastic bags of six kilograms once the first leaves developed. Ten inoculated plants with 5,200 eggs in the soil near the stem of the plant and four non-inoculated ones were used in each treatment, in a complete block design. Sixty-two days after sowing, the following characteristics were evaluated: the length of the aerial part of the plant (LAP, in m), fresh mass of the aerial part (FMAP, in g), root fresh mass (RFM, in g), egg number (EN) and reproduction factor (RF). A comparison between the averages of inoculated and non-inoculated plants was performed using Scott-Knott test at 5% and the diallelic analysis was performed using the GENES program. The morphological characteristics did not allow for the identification of the parent plants or the F1s with respect to nematode resistance, but the variables EN and RF were useful for such identification. The analyses of the general and specific combining abilities indicate highly significant effects with respect to this resistance, showing additive gene effects as well as dominance and epistatic gene effects, allowing for identification of parents and F1s that can be used in watermelon breeding programs to improve resistance to the M. enterolobii.

Analytical evaluation of paper degradation

The aim of this work was in first place to define a methodology for the use of Py-GC/MS as a characterization technique for the organic compounds present in paper samples containing foxing stains, paper have a complex structure and mostly consist with cellulose fibers. Additionally, it was intent to characterize paper samples containing foxing stains with a batch of non-destructive analytical techniques. The work intent to deepen our knowledge on foxing stains, its chemical nature and morphological aspects. For characterization of the morphology of paper samples and foxing stains was used photography under different illuminations and optical microscopy. The presence of fibers disruption was observed with scanning electron microscopy coupled with energy dispersive spectroscopy (SEM-EDS), and also the nature of the fillers that is present in different areas. Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) was used for identification of the sizing agents, determination of the chemical composition of additives that were used for production of paper, and comparison between foxing stains and unfoxed areas was allowed. Micro X-ray diffraction was used to evaluate the crystalline fillers in the sample. Pyrolysis-Gas Chromatography/Mass Spectrometry (Py-GC/MS was used for chemical analysis to identify the organic components and different classes of organic compounds; Resumo: O objetivo deste trabalho foi definir, em primeiro lugar, uma metodologia para o uso de Py-GC / MS como técnica de caracterização dos compostos orgânicos presentes em amostras de papel contendo manchas de foxing, o papel tem uma estrutura complexa e consiste principalmente com fibras de celulose. Além disso, pretendia caracterizar amostras de papel contendo manchas de raposas com técnicas analíticas não destrutivas. Para a caracterização da morfologia das amostras de papel e das manchas de foxing foi usada fotografia sob diferentes iluminações e microscopia óptica. A presença de fibras de ruptura foi observada por microscopia electrónica de varrimento juntamente com espectroscopia dispersiva de energia (EDS-SEM), assim como a natureza dos materiais de enchimento que está presente em diferentes áreas. Espectroscopia de infravermelho com transformada de Fourier em modo de reflexão total atenuada (ATR-FTIR) foi utilizada na identificação dos agentes de colagem, e na determinação da composição química de aditivos usados na produção de papel, e a comparação entre foxing manchas e áreas unfoxed foi deixada. Micro difracção de raios X foi usada para avaliar o enchimentos cristalinos na amostra. Cromatografia pirólise-gasosa / espectrometria de massa (Py-GC / MS) foi utilizada para análise química para identificar os componentes orgânicos e diferentes classes de compostos orgânicos.

Acquired and inherited cardiomyopathies: evaluation of cardiotoxic effects in preclinical models

Cardiomyopathies are a heterogeneous group of myocardial disorders defined by structural and functional alterations of the heart. These cardiac diseases can have both non-genetic and genetic origin. Nevertheless, a different etiology can trigger the same phenotype, as in the case of anthracycline-induced cardiotoxicity and desmin-related cardiomyopathy (DRM). Therefore, the aim of this study was to investigate the cellular mechanisms driving the development of these cardiotoxic conditions in in vitro models. Doxorubicin (DOX) is a commonly used antineoplastic drug for the treatment of a wide range of tumors. Besides, its clinical use is restricted because of dose-dependent cardiotoxicity. Our findings provided evidence that phospholipase C Beta 2 (PLCβ2) may have a critical role in DOX-induced cardiotoxicity in undifferentiated and differentiated H9c2 cell line. Interestingly, the results obtained revealed that cardiomyocytes are less sensitive to DOX, following the evaluation of cellular mechanisms such as: oxidative stress, apoptosis and cell proliferation. Nonetheless, the treatment induced a significant upregulation of PLCβ2 associated to morphological changes in both models, demonstrating the implication in a hypertrophic response. On the other hand, a hereditary DRM was associated to a missense mutation of aB crystallin (CRYAB), a chaperone protein involved in the regulation of the intermediate filament network. Since research has only been conducted on transgenic (TG) mice and neonatal rat cardiomyocytes, this study aimed at investigating cellular mechanisms triggered by CRYABR120G mutation in a hiPSC-derived DRM model. Our model confirmed the impairment of the cytoskeletal organization resulting in the formation of desmin and CRYAB aggregates and myofibril misalignment. Moreover, the missense mutation confirmed a hypertrophic cardiomyopathy phenotype, a feature of DRM patients, on cardiac engineered tissues. Lastly, these data obtained suggest that further research on PLCβ2 and CRYAB are needed to comprehend the molecular mechanisms behind the development of these 2 cardiac diseases.

Evaluation Of The Radiopacity Of Esthetic Root Canal Posts.

The radiopacity of esthetic root canal posts may impair the assessment of their fit to the root canal when using radiographic images. This study determined in vitro the radiographic density of esthetic root canal posts using digital images. Thirty-six roots of human maxillary canines were assigned to six groups (N=6 per group): Reforpost (RP); Aestheti-Plus (AP); Reforpost MIX (RPM); D.T. Light Post (LP); Reforpost Radiopaque (RPR); and White Post DC (WP). Standardized digital images of the posts were obtained in different conditions: outside the root canal, inside the canal before and after cementation using luting material, and with a tissue simulator. Analysis of variance was used to compare the radiopacity mean values among the posts outside the root canal and among the posts under the other conditions, and the t unpaired test to compare the radiopacity between the posts and the dentin, and between the posts and the root canal space. There was no statistically significant difference in radiopacity between RP and RPM, and LP and WP. AP posts showed radiopacity values significantly lower than those for dentin. No statistically significant difference was found between posts (RP and AP) and the root canal space. A statistically significant difference was observed between the luted and non-luted posts; additionally, luted posts with and without tissue simulator showed no significant differences. Most of the cement-luted posts analyzed in this study were distinguishable from the density of adjacent dentin surfaces, allowing radiographic confirmation of the fit of the post in the canal. The success of using esthetic root canal posts depends mainly on the fit of the post within the canal.[1] The radiopacity of a post allows for radiographic imaging to be used to determine the fit, an important factor in a clinical perspective.

Otoneurological Evaluation: Current Good Practice.

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Endoscopic Endonasal Transsphenoidal Resection Of Pituitary Adenomas: Preliminary Evaluation Of Consecutive Cases.

Endoscopic endonasal transsphenoidal surgery has gained increasing acceptance by otolaryngologists and neurosurgeons. In many centers throughout the world, this technique is now routinely used for the same indications as conventional microsurgical technique for pituitary tumors. To present a surgical experience of consecutive endoscopic endonasal trans-sphenoidal resections of pituitary adenomas. In this study, consecutive patients with pituitary adenomas submitted to endoscopic endonasal pituitary surgery were evaluated regarding the rate of residual tumor, functional remission, symptoms relief, complications, and tumor size. Forty-seven consecutive patients were evaluated; 17 had functioning adenomas, seven had GH producing tumors, five had Cushing's disease, and five had prolactinomas. Of the functioning adenomas, 12 were macroadenomas and five were microadenomas; 30 cases were non-functioning macroadenomas. Of the patients with functioning adenomas, 87% improved. 85% of the patients with visual deficits related to optic nerve compression progressed over time. Most of the patients with complaints of headaches improved (76%). Surgical complications occurred in 10% of patients, which included with two carotid lesions, two cerebrospinal fluid leaks, and one death of a patient with a previous history of complications. Endoscopic endonasal pituitary surgery is a feasible technique, yielding good surgical and functional outcomes, and low morbidity.

Microbial Evaluation Of Traumatized Teeth Treated With Triple Antibiotic Paste Or Calcium Hydroxide With 2% Chlorhexidine Gel In Pulp Revascularization.

Revascularization outcome depends on microbial elimination because apical repair will not happen in the presence of infected tissues. This study evaluated the microbial composition of traumatized immature teeth and assessed their reduction during different stages of the revascularization procedures performed with 2 intracanal medicaments. Fifteen patients (7-17 years old) with immature teeth were submitted to the revascularization procedures; they were divided into 2 groups according to the intracanal medicament used: TAP group (n = 7), medicated with a triple antibiotic paste, and CHP group (n = 8), dressed with calcium hydroxide + 2% chlorhexidine gel. Samples were taken before any treatment (S1), after irrigation with 6% NaOCl (S2), after irrigation with 2% chlorhexidine (S3), after intracanal dressing (S4), and after 17% EDTA irrigation (S5). Cultivable bacteria recovered from the 5 stages were counted and identified by means of polymerase chain reaction assay (16S rRNA). Both groups had colony-forming unit counts significantly reduced after S2 (P < .05); however, no significant difference was found between the irrigants (S2 and S3, P = .99). No difference in bacteria counts was found between the intracanal medicaments used (P = .95). The most prevalent bacteria detected were Actinomyces naeslundii (66.67%), followed by Porphyromonas endodontalis, Parvimonas micra, and Fusobacterium nucleatum, which were detected in 33.34% of the root canals. An average of 2.13 species per canal was found, and no statistical correlation was observed between bacterial species and clinical/radiographic features. The microbial profile of infected immature teeth is similar to that of primarily infected permanent teeth. The greatest bacterial reduction was promoted by the irrigation solutions. The revascularization protocols that used the tested intracanal medicaments were efficient in reducing viable bacteria in necrotic immature teeth.

Evaluation Of Different Treatment Methods Against Denture Stomatitis: A Randomized Clinical Study.

The aim of this clinical study was to determine the efficacy of Uncaria tomentosa (cat's claw) against denture stomatitis (DS). Fifty patients with DS were randomly assigned into 3 groups to receive 2% miconazole, placebo, or 2% U tomentosa gel. DS level was recorded immediately, after 1 week of treatment, and 1 week after treatment. The clinical effectiveness of each treatment was measured using Newton's criteria. Mycologic samples from palatal mucosa and prosthesis were obtained to determinate colony forming units per milliliter (CFU/mL) and fungal identification at each evaluation period. Candida species were identified with HiCrome Candida and API 20C AUX biochemical test. DS severity decreased in all groups (P < .05). A significant reduction in number of CFU/mL after 1 week (P < .05) was observed for all groups and remained after 14 days (P > .05). C albicans was the most prevalent microorganism before treatment, followed by C tropicalis, C glabrata, and C krusei, regardless of the group and time evaluated. U tomentosa gel had the same effect as 2% miconazole gel. U tomentosa gel is an effective topical adjuvant treatment for denture stomatitis.