Dissertations / Theses on the topic 'Protocol characterization'

2011/2012
Le principali attività di ricerca svolte durante il dottorato hanno riguardano la validazione e caratterizzazione dell’applicabilità di un protocollo per l’analisi della cinematica di spalla in ambito clinico (ISEO - INAIL Shoulder and Elbow Outpatient protocol). Lo scopo principale era quello di creare uno strumento che fornisse al personale sanitario informazioni sulla performance motoria dei pazienti, supportando, con informazioni di tipo quantitativo, la valutazione ambulatoriale delle patologie della spalla. E’ possibile suddividere l’attività di ricerca in tre temi principali: caratterizzazione e validazione di ISEO; applicazione di ISEO per valutazioni di tipo clinico; applicazione di ISEO per valutazione di performance motoria in ambito sportivo. Grazie ai processi di validazione e caratterizzazione svolti e alle applicazioni di ISEO, ad oggi il protocollo può essere utilizzato in studi clinici e sportivi riguardanti la cinematica di spalla (coordinazione scapolo-omerale), per i quali la sensibilità dello strumento può essere considerata adatta alle esigenze valutative.
The main research activities carried out during the PhD were related to the validation and characterization of the applicability of a protocol for the analysis of the kinematics of the shoulder in a clinical setting (ISEO - Shoulder and Elbow INAIL Outpatient protocol). The main purpose was to create a tool that provides quantitative information about the motor performance of patients, supporting clinicians for the assessment of ambulatory shoulder disorders. The research activity can be split in three main themes: characterization and validation od ISEO; application of ISEO for clinical assessments; application of ISEO for sport performance assessments. Thanks to the validation and characterization of the protocol and its application in several contests, it can be concluded that ISEO can be used to evaluate the kinematics of the shoulder (in particular the scapulohumeral coordination) in clinical and sport performance studies, for which the sensitivity of the protocol can be considered appropriate.
XXV Ciclo
1983

Background & rationale: Multiparametric MRI (mpMRI) is the imaging modality of choice for the diagnostic workup of men with clinically suspected prostate cancer. However, mpMRI has several limitations, including: limited accessibility in routine clinical practice; high interreader variability; low specificity and positive predictive value. Aim of the work: The aim of the work is to address current limitations of mpMRI with focus on MRI protocol simplification, interreader agreement assessment and development of a novel MRI technique called Luminal Index MRI (LI-MRI). Material & Methods: I) The interreader variability of mpMRI was investigated in a multireader study reflecting the real-world clinical practice in 200 patients. Seven radiologists reviewed and scored the mpMRI scans using PI-RADS v2.1. Agreement on index lesion detection was evaluated with Conger’s k coefficient, agreement coefficient 1 (AC1), percentage of agreement (PA) and indexes of specific agreement. II) The feasibility of MRI protocol simplification was tested on 151 men who underwent mpMRI and transperineal template prostate mapping biopsies. Three experienced radiologists scored MRI scans using three different protocols (mpMRI, bpMRI and abbreviated bp-MRI [a-bpMRI]). The diagnostic performance and interreader agreement of the different protocols was compared. III) LI-MRI diagnostic performance was tested on a total of 178 men who underwent mpMRI for clinically suspected PCa. A validation cohort was then used to validate the results and to test LI-MRI reproducibility. IV) Retrospective analysis on men who underwent LI-MRI (+/-biopsy) was performed to develop a dedicated scoring system for LI-MRI. Results: I) Agreement on index lesion detection among was substantial (AC1 0.738; 95% CI 0.695–0.782); dedicated radiologists had higher agreement compared with non-dedicated readers. II) The sensitivity and specificity of a-bpMRI were 92% and 48%, respectively. There was no significant difference in sensitivity compared to bpMRI and mpMRI. Interreader agreement of a-bpMRI was moderate (AC1 0.58). III) LI-MRI performed using a simplified 8-echo protocol was feasible, repeatable and yielded high specificity for the detection of clinically significant prostate cancer (65-78% specificity for 89-90% sensitivity). IV) A dedicated scoring system was developed for LI-MRI based on both multiecho-T2W and LI-maps. Conclusion: The variability of mpMRI is lower than previously thought. Short MRI protocols may perform as well as mpMRI in expert hands and should be considered to address the limited accessibility of mpMRI. LI-MRI is a promising tool that could improve PCa characterization with MRI.
Background e razionale: La risonanza magnetica mutiparametrica (mpMRI) è la modalità diagnostica di scelta per la diagnosi del tumore prostatico. Tuttavia, la mpMRI ha diverse limitazioni, tra cui la limitata accessibilità, l'alta variabilità inter-osservatore, la bassa specificità e il basso valore predittivo positivo. Scopo dello studio: Con particolare riferimento alla possibilità di fare fronte alle correnti limitazioni della mpMRI, lo scopo dello studio è quello di valutare la reale variabilità inter-osservatore della mpMRI, di valutare la fattibilità di una semplificazione dei protocolli di RM prostatica, e di sviluppare una nuova tecnica RM chiamata Luminal Index MRI (LI-MRI). Materiali e Metodi: I) La reale variabilità inter-osservatore è stata valutata in uno studio multi-osservatore progettato per rispecchiare il più possibile la normale pratica quotidiana, su un campione di 200 pazienti. Sette radiologi hanno rivalutato le risonanze secondo i criteri PI-RADS v2.1. Si è valutata la concordanza sulla identificazione della lesione principale (Index Lesion) mediante il coefficiente K di Conger, il coefficiente di agreement 1 (AC1), le percentuali di concordanza e gli indici di concordanza specifici. II) La fattibilità di un protocollo semplificato di RM prostatica è stata testata su 151 pazienti che avessero effettuato mpMRI e mappaggio prostatico mediante biopsie transperineali (TTPM). Tre radiologi esperti hanno rivalutato le immagini utilizzando tre diversi protocolli (risonanza multiparametrica (mpMRI), biparametrica (bpMRI) e biparametrica abbreviata (a-bpMRI)). È stata valutata la performance diagnostica per ogni protocollo e la concordanza inter-osservatore. III) La performance diagnostica della LI-MRI è stata testata su 178 pazienti che avessero fatto una mpMRI e LI-MRI per sospetto tumore prostatico. I risultati ottenuti sono stati validati su una coorte di valutazione, oltre alla riproducibilità della metodica su scansioni ripetute. IV) Gli esami di LI-MRI sono stati rivalutati retrospettivamente in confronto alla mpMRI e a i risultati delle biopsie per sviluppare un sistema di interpretazione ad hoc per LI-MRI. Risultati: I) La concordanza sulla identificazione della Index Lesion è stata sostanziale (AC1 0.738; 95% CI 0.695–0.782), maggiore per i radiologi esperti nei confronti dei meno esperti. II) La sensibilità e la specificità della a-bpMRI è stata di 92% e 48%, rispettivamente, senza significativa differenza nei valori di sensibilità se confrontata con la bpMRI e mpMRI. La concordanza interosservatore per a-bpMRI è però inferiore (AC1 0.58). III) La tecnica LI-MRI che utilizza un protocollo semplificato è fattibile, riproducibile e ottiene ottimi valori di sensibilità e specificità per la identificazione del tumore prostatico (65-78% specificità per 89-90% sensibilità). IV) Uno scoring system dedicato è stato sviluppato per la tecnica LI-MRI e basato sull'interpretazione delle sequenze T2-multieco delle mappe di luminal index. Conclusioni: La variabilità inter-osservatore della risonanza magnetica è inferiore a quanto precedentemente descritto dalla letteratura. I protocolli abbreviati di RM prostatica sono efficaci quanto quelli standard (se interpretati da lettori esperti) e dovrebbero essere presi in considerazione per aumentare l'accessibilità alla RM prostatica. La tecnica LI-MRI ha ottenuto risultati promettenti e potrebbe migliorare la caratterizzazione del tumore prostatico utilizzando la risonanza magnetica.

Current brachytherapy dosimetry protocols assume that a physical source may be approximated by a point source. A new brachytherapy dosimetry protocol, recently proposed by the American Association of Physicists in Medicine Task Group 43, has the advantage of using functions derived solely from measurements performed in the medium and uses a more realistic source geometry than the point source approximation. The aim of this work is to obtain the dosimetric functions required by this new protocol for both a low and a high dose-rate Iridium-192 brachytherapy source through dose measurements in a water-equivalent phantom.
Dose measurements have been performed using lithium fluoride thermoluminescent detectors positioned in a polystyrene phantom at distances from the source that vary from 1 cm to 10 cm, with 1-cm intervals, and at angles that vary from 0$ sp circ$ to 170$ sp circ$ with 10$ sp circ$ intervals.
Our experimental results have clearly shown that the point-source approximation model can overestimate the dose to water, especially for the high dose-rate source, where we have found that differences between point-source estimates and exact measured values can differ by almost 30% for points along the longitudinal axis of the source.

A digital thematic map of the shallow marine habitats surrounding the Tobago Cays and the Horseshoe Reef was created using a low-cost remote sensing methodology. Colour aerial photographs were selected because of their high spatial resolution and availability. The aerial photographs were scanned, georeferenced, rectified (ground control points and a second order polynomial) and mosaicked to cover the entire study arm. Benthic classes were derived and described objectively using agglomerative hierarchical classification of field data. Supervised classification of the Tobago Cays was obtained using this field derived classification. The final thematic map comprises 8 classes (mixed live coral community, dead coral substratum with mixed algae, seagrass dominated, macro algae dominated, sand dominated, rubble dominated, deep water and beach sands) with an overall accuracy of 87% and a Kappa and Tau coefficients of 85%. Producer and user accuracies of individual classes range between 53% and 100%. (Abstract shortened by UMI.)

Thesis (Ph. D.)--Worcester Polytechnic Institute.
Keywords: characterization; test protocol; finite element method; performance curve; thermocouple; rutting; strain gauge; MMLS3; fatigue; instrumentation. Includes bibliographical references (p. 117-119).

The thesis focuses on the characterization of the alteration of the mineral and organic phases, investigated with different approaches, of human bone tissue from different burial contexts, with ages spanning from the Late Roman period to our time. This topic is very important in paleontological, archaeo-anthropological and forensic contexts in order to understand the taphonomic agents and then to provide biological data as possibly to discern human behavior in ancient funerary as well as in recent forensic contexts. It is well-known that peri and post mortem events may leave marks that have to be interpreted in the light of the state of the conservation or degradation of the skeletal remains. In fact, physical anthropologists are frequently required to date human bone remains, in order to recognize if osteological samples have an archaeological, historic or forensic interest. The determination of post mortem interval (PMI), the time elapsed between the death and the discovery of the corpse or skeletal remains, is extremely difficult to evaluate in absence of direct chronometric dating (e.g. C14), since bones might undergo several alterations, both structural and chemical, depending on the environment in which they deposited in. Because of bone tissue is an intimate association of mineral (carbonate-hydroxyapatite) and organic components (collagen) arranged in an ordinary structure, different levels of degradation are possible. Over time post mortem degradation is dominated by loss of structural collagen by collagenolytic enzymes, which caused a rapid swelling and hydrolysis of the protein fibers. Collagen dissolution is generally accompanied by the alteration of mineral crystals, which are vulnerable to diagenetic changes due to their small size. During diagenesis, the protein can be totally or partially removed and can replaced by inorganic precipitates, the most common beign hydroxyapatite, which in the process is subjected to recrystallization, ion exchange and substitution. As consequence, when depositional conditions are favorable for bone preservation, the mineral crystallinity increase, the porosity and chemical composition change. The quality and the assessement of organic and inorganic phase, can act positively or negatively both on bone mechanical properties in live, both on decomposition process after death, reducing or accelerating it. Several studies were performed to better understand the taphonomy of bone material during burial time. It appears that bone degradation depends on a wide range of environmental interactions, including biological, chemical and physical factors. These include: average temperature and humidity, microbilological composition and activity, soil chemistry (mineralogy and pH) and permeability, mechanical pressure and other numerous factors. Different type of bone degradation are observable at different scale of observation; particularly, in this study, bone preservation was investigated at macroscopic, biomolecular, microscopic, ultramicroscopic and chemical scale. The aim of this research is thus to further describe the impact of environmental conditions on bone preservation, and the effect of time, by applying and comparing the results from different analitical techniques. For this study 40 human skeletons of adult individuals from four different dated burial location in the Milan area were analyzed. The first one is a necropolis dated to the Late Roman age (3th-4th century AD), the second one is a 17th century AD mass grave, the third one is an ossuary contanining bones dated between 15th and 18th century AD, and the last one is a modern cemetery. The macroscopic analysis evaluated the general appearance of the remains and their state of preservation, through the observation of specific macroscopic parameters and morphological characteristics. The Luminol test, a fast and inexpensive method developed to detect blood traces, was performed to investigate the presence of haemoglobin preserved in bone. The histological analysis, conducted on calcified thin sections, considered the presence or absence of tunneling and bioerosion, in accordance to the Oxford Histological Index (OHI). Also, to evaluate the state of preservation of the organic component, primarily collagen, the samples were decalcified and stained with Hematoxylin and Eosin. Because of the lack of literature in this field, we created a new Decalcified Histological Index (DHI). Both calcified and decalcified bone thin sections were observed in transmitted and polarized light microscopy, in order to test the optical properties of structural components. Scanning electron microscope coupled with energy-dispersive X-ray spectroscopy (SEM-EDS) was used to evaluate exogenous chemical elements and minerals, adsorbed from burial environment, and histological changes, as well as recrystallization, tunnelling and fractures, due to fungal or bacteria action. X-ray micro-computed tomography of bone sections was performed at the SYRMEP beamline of the third-generation Synchrotron Light Laboratory (ELETTRA) located in Trieste (Italy), with the purpose to evaluate and quantify the preservation of bone structure, such as canals and lacunae, and the porosity changes due to diagenetic process. Fourier transform infrared spectrometry (FT-IR) and micro-spectrometry (mFTIR) were performed at Simon Fraser University (Burnaby) in Canada to investigate the preservation of both mineral and organic phases. Finally, 23 skeletons from the archaeological site of Travo (PC), dating from 7th-8th century AD, and their burial ground sediments were sampled and analyzed. Macroscopic, microscopic and chemical analyses were performed on bones to evaluate the tissue preservation state at different scales; the soil samples collected from the graves were characterized for color, particle size distribution, pH, organic carbon and calcium carbonate concentration. This study shows that macroscopic, biomolecular, microscopic, ultramicroscopic and chemical alterations follow independent paths that affect the bone preservation at different scales of observation. Therefore, the estimation of the diagenetic process cannot be limited to the macroscopic aspect of the bone tissue but must take into account biomolecular, microscopic and chemical alterations, since these may have affected the bone tissue differently at different scale. Bone degradation can be employed to estimate the post mortem interval, or to reconstruct the burial environment of human remains. As long as the evaluation of taphonomic alterations is performed at different scales with different ad hoc methodologies. In fact, age and environment can play an equal role on the degradation of organic and mineral phases, producing different effects on bone conservation at different levels.

Dissertation toobtaina Master of Science degree in Bioorganics
There has been a worldwide acknowledgement that nature derived saccharides can provide the raw materials needed for the production of numerous industrial consumer goods. As such, sucrose is a low molecular weight renewable carbohydrate feedstock from which it is possible to elaborate new materials, like water-soluble and/or amphiphilic and biocompatible polymers. In this thesis we will describe some synthetic procedures (both conventional synthesis protocols (CSP) and microwave assisted protocols (MAPs)) by introducing and altering sugar hydroxyl groups, with the intent to produce functionalized polymers for use as biodegradable/biocompatible polymers with sugar linked side chains. The most widely used method for the synthesis of poly(vinyl saccharide)s has been based on free radical polymerizations of vinyl sugars. In this work, eleven compounds based on sucrose derivatization were synthesized using anhydrides, bromide halides, silyl chlorides, non-selective esterification and Mitsunobu reaction. Optimization and scale-up studies were made on monomer synthesis. Four of these compounds were used as monomers for radical copolymerization with styrene using as catalysts 2,2’-Azobis(2-methylpropionitrile) and sodium persulfate whether organic solvents or water was used as reaction media. From this copolymerization’s, four polymers were obtained and polystyrene was also synthesized to be used as a standard for comparison. The polymers, poly(1’,2,3,3’,4,4’,6-hepta-O-benzyl-6’-O-methacryloyl sucrose)-co-polystyrene, poly(1’,2,3,3’,4,4’,6’-hepta-O-acetyl-6-O-methacryloyl sucrose)-co-polystyrene, poly(6-O-methacryloyl sucrose)-co-polystyrene and poly(O-methacryloyl sucrose)-co-polystyrene, were characterized by Proton nuclear magnetic resonance (to assess sucrose vinyl ester/styrene ratio), Fourier transform infrared spectroscopy, Differential scanning calorimetry, Powder X-ray diffraction, Atomic force microscopy (topology studies as thin films and aggregates), Viscometry and polarimetry.

Apicomplexa parasites are single-celled, obligate intracellular cyst-forming protozoa, infecting humans and animals, that pose major threats to world health and global economy. Among the most relevant species for farm animals, Besnoitia besnoiti, Neospora caninum and Toxoplasma gondii are parasites of medical (T. gondii) and veterinary (T. gondii, N. caninum, B. besnoiti) importance in domestic ruminants. Bovine besnoitiosis, caused by Besnoitia besnoiti, is a chronic and debilitating parasitic disease of cattle, characterized by both cutaneous and systemic manifestations, compromising animal welfare and responsible for economic losses on affected farms. In Europe, including Italy, bovine besnoitiosis is an emerging or re-emerging disease, with an increasing geographical distribution and the number of cases of infection. Neospora caninum, a coccidian protozoan, represents an important cause of bovine abortion. It was suggested that the parasite may have adverse effects on fertility and milk production, but only few and contrasting data are available to date. Besides, while only a single genotype of N. caninum exists worldwide, available parasite strains show considerable variation in vitro and in vivo, including different virulence in cattle. Microsatellite markers allow to fingerprint N. caninum isolates or DNAs and undertake population studies. Toxoplasmosis represents an important public health issue, with the consumption of raw or undercooked meat being a major way of human infection. The role of beef in the transmission of the parasite to humans is questioned due to lower quantity of tissue cysts compared with other meat-producing species. However, the habit of consuming raw beef is regionally diffused, and the risk posed by Toxoplasma gondii infection in cattle should not be overlooked. The aim of my doctoral thesis project was to investigate on the epidemiology and molecular characterization of selected protozoa parasites of medical and veterinary importance in domestic ruminants, i.e., B. besnoiti, N. caninum and T. gondii in cattle. A multidisciplinary approach based on clinical features, laboratory tests including serological and molecular techniques, was applied throughout the research project, to achieve a multi-level comprehension of the epidemiology of these parasite infections. Three main research lines were developed: Research Line 1: Exploring bovine besnoitiosis: a multi method approach. A case of bovine besnoitiosis in a dairy farm housing 217 cattle in Italy was reported. A serological screening was performed on the whole herd using the recommended approach of ELISA and confirmatory Western Blot. Seropositive animals were clinically examined to reveal symptoms and lesions of besnoitiosis. Risk factors and the effects of the parasite infection on reproductive and productive performances were evaluated. Histopathology and molecular analyses on tissues from a slaughtered cow affected by the chronic phase of the disease were carried out. An overall seroprevalence of 23.5%, which increased up to 43.5% considering only cows, was recorded. Clinical examination of 33 of the seropositive cows evidenced the presence of tissue cysts in at least one of the typical localizations (sclera, vulva, or skin) in 25 animals. Statistical analysis did not evidence any significative impact of the parasite infection on herd efficiency; however, a decrease of productive parameters was recorded in cows showing cutaneous cysts. Concerning the chronically affected cow, histopathology revealed B. besnoiti tissue cysts in the skin of the neck, rump, hind legs, eyelid and vulva, in the muzzle, in mucosal membranes of the upper respiratory tract, and in the lungs. Parasite DNA was detected also in masseter muscles, tonsils, mediastinal lymph nodes, liver, cardiac muscle, aorta wall, ovaries, uterus, and vulva. Moreover, alterations of laboratory parameters, i.e., hematological and biochemical parameters, enzyme activities and serum cortisol levels in Besnoitia besnoiti naturally infected cows were deeply investigated. Laboratory parameters of 107 cows, 61 seronegative and 46 seropositive to B. besnoiti, including 27 with clinical signs of bovine besnoitiosis, were compared. Generalized Linear Models were used to evaluate the effect of Besnoitia infection on the considered laboratory parameters. Hematological analyses revealed that B. besnoiti infection determined a significant alteration of the leukocyte differential with a higher percentage of neutrophil granulocytes and a lower percentage of lymphocytes in seropositive animals; Erythrocyte and Platelet counts did not show any difference between the considered groups of cows. Biochemistry evidenced that the parasite infection influenced serum protein values in seropositive animals and GLDH in clinically affected cows. No or only slight differences were revealed for all the other biochemical and enzyme activity parameters in B. besnoiti infected animals. Seropositive and clinically affected cows evidenced mild higher concentrations of serum cortisol values if compared to seronegative animals, indicating that bovine besnoitiosis could be related to stress in infected animals and that the parasite could compromise animal welfare and also influence disease onset and progression. Furthermore, a form of generalized demodectic mange in two dairy cows infected with Besnoitia besnoiti was described. Two out of the cows seropositive to B. besnoiti, at clinical examination presented skin nodules, widespread all over the body, and in particular in anterior regions. Skin biopsies from the region of the neck were collected and the nodules were microscopically examined through compression method. B. besnoiti tissue cysts were not revealed but a semi-solid yellowish content was evidenced with the presence of several mites, morphologically identified as Demodex bovis. Histological examination of skin biopsies evidenced slight acanthosis and hyperkeratosis of the epidermis and superficial dermatitis with oedema and macrophagic and eosinophilic infiltration. Cystic formations located in the deep dermis were lined by metaplastic squamous epithelium and severe cellular infiltration. A treatment with eprinomectin was attempted and clinical improvement of both cows was observed, particularly at the fifteenth day after treatment, with nodules reduced in size and mites in there degenerated. Moreover, an additional sub research line from this one was implemented to investigate on these Apicomplexa protozoa, particularly Besnoitia spp. infection, in Italian equids: Research Line 1 bis: Exposure of Italian equids to selected protozoa infections and investigation on clinical besnoitiosis in donkeys. A serosurvey was planned to estimate the prevalence of Sarcocystidae species (Besnoitia spp., Toxoplasma gondii and Neospora spp.) in Italian equids. Serum samples from 268 horses and 18 donkeys raised in Italy were collected and serologically analyzed to detect anti-Besnoitia spp., anti-T. gondii and anti-Neospora spp. antibodies: an approach based on an initial screening by in-house ELISA followed by a confirmatory Western Blot was used. Two horses (0.7%) and four donkeys (22.2%), showed antibodies anti-Besnoitia spp. Ten horses (3.7%) resulted positive to T. gondii and one of these (0.4%) was seropositive also to Neospora spp. This is the first detection of anti-Besnoitia spp. specific antibodies in Italian horses and donkeys. Low prevalence of T. gondii and Neospora spp. in horses raised in Italy was reported. A case of clinical besnoitiosis in two donkeys was reported. Two donkeys, one male and one female, reared in northern Italy, showed suspected skin lesions and poor body condition. The animals were clinically examined, and endoscopy of upper respiratory tract and of the vagina for the female donkey was performed. Blood samples and skin biopsies were collected. Serum samples were analyzed by Western Blot to detect anti-Besnoitia spp. antibodies; a PCR targeting ITS-1 region and sequencing were carried out on DNA extracted from skin biopsies. Moreover, blood samples were examined for hematology, biochemistry, and enzyme activity. Clinical examination revealed numerous scleral pearls in the eyes of both animals; besides, alopecia and hyperkeratosis with skin nodules in the region of the neck, hind leg and on the pinnae were detected. No cysts were evidenced in the nares, in the upper respiratory tract and in the vagina and vulva in the female animal. Both animals resulted seropositive according to Western Blot results. Skin biopsies collected from the donkeys resulted positive for the presence of parasitic DNA. Sequencing demonstrated a homology of 100% with Besnoitia spp. sequences deposited in GenBank. Hematology evidenced light anemia, leukocytosis with eosinophilia, and lymphocytosis, whereas biochemistry and enzyme activity revealed hypoalbuminemia with decreased albumin/globulin ratio and elevated alkaline phosphatase values. This first clinical case of besnoitiosis in donkeys in Italy confirmed the circulation of Besnoitia spp. in Italian and European equids. Research Line 2: Genetic characterization of Neospora caninum isolates in cattle and impact of neosporosis on herd performances. With the aim of evaluating the spread of neosporosis and its effects on herd efficiency, an epidemiological study was designed in two dairy farms recruited as case-study. Both selected farms, located in Lombardy region, performed genetic improvement of Holstein Friesian, and reported cases of abortions ascribable to N. caninum. Blood samples were collected from 540 animals, including cows and heifers above 24 months, and analyzed by indirect immunofluorescent antibody test. Epidemiological data (individual, reproductive and productive data) were noted. Overall, 94 animals (17.4%) resulted positive to N. caninum (15.5% and 18.5% in Farm 1 and Farm 2), with differences between the farms concerning the antibody titres (Chi-square, p-value = 0.04), particularly in cows (Chi-square, p-value = 0.018). Regarding the episodes of abortions, a different pattern was depicted in the two investigated farms. Data on fertility and production were considered. The number of insemination necessary to get an animal pregnant resulted higher in seropositive animals (2.4 and 2.9) than in seronegative animals (2.1 and 2.4 in Farm 1 and 2, respectively). Similarly, particularly in Farm 1, the number of days in lactation of not-pregnant cows resulted higher in seropositive (167.7) than seronegative animals (133.4). Moreover, although the association between N. caninum infection and milk production is still unclear, both the daily production and the mature equivalent milk yield were lower in seropositive (31.02 and 11838.94) than seronegative cows (33.59 and 12274.88) in Farms 1. To genotype N. caninum in aborted bovine foetuses from Lombardy, the proportion of N. caninum PCR-positive aborted foetuses in this area was determined and the available isolates were characterized by multi-locus microsatellite genotyping. Aborted bovine foetuses were collected between 2015 and early 2019 from Italian Holstein Friesian dairy herds suffering from reproductive problems. A total of 198 samples were collected from 165 intensive farms located in Lombardy, northern Italy. N. caninum positive samples were then subjected to multilocus-microsatellite genotyping (MLMG) using ten previously established microsatellite markers. In addition to own data, those from a recent study providing data on five markers were included and analyzed. 55 aborted foetuses were positive for N. caninum by RT-PCR, yielding a prevalence of 27.8%; 43 farms recorded at least one positive fetus (26.1%). Of the 55 samples finally subjected to MLMG, 35 were typed at all or 9 out of 10 loci. Linear regression revealed a statistically significant association between the spatial distance of the sampling sites with the genetic distance of N. caninum MLMGs (P< 0.001). Including data from a previous north Italian study (eBURST analysis) revealed that part of N. caninum MLMGs from northern Italy separate into four groups; most of the samples from Lombardy clustered in one of these groups. Principle component analysis revealed similar clusters and confirmed MLMG groups identified by eBURST. These findings confirm the concept of local N. caninum subpopulations. The geographic distance of sampling was associated with the genetic distance as determined by MLMGs. Research Line 3: Evaluation of Toxoplasma gondii infection in beef cattle raised in Italy. To update information on T. gondii in cattle reared in Italy, a multicentric seroepidemiological survey was designed and implemented in four northern regions (Liguria, Lombardy, Piedmont, and Trentino Alto Adige) and Sardinia. A convenience sampling was performed, collecting 1444 serum samples from 57 beef cattle herds. Thirteen beef breeds were sampled, besides crossbreed; bovines age varied from 3 months to over 12 years. Sera were tested with a commercial ELISA for the detection of anti-T. gondii antibodies. Individual and herd data were analyzed by binary logistic regression analysis. A T. gondii seroprevalence of 10.2% was recorded, with differences among regions and values ranging from 5.3% in Liguria to 18.6% in the Piedmont region (p value = 0.0001). Both young and adult animals and males and females tested positive, without any significant difference (age and gender: p value >0.05). Lower seroprevalence values were recorded in cattle born in Italy (8.7%) if compared with animals imported from abroad (13.4%) (p value = 0.046). To obtain epidemiological and molecular data on T. gondii infection in cattle slaughtered in Italy, 80 animals were sampled from one of the biggest Italian slaughterhouses. Dairy (Holstein Friesian) or dual purpose (Alpine Brown and Pezzata Rossa Italiana) breeds or crossbreeds from 15 farms located in northern Italy were sampled; age of animals varied from six months to three years. Approximately 50 g of diaphragm was collected to obtain meat juice and muscle homogenate samples. Individual data were noted. Meat juice samples were analyzed with a commercial ELISA to detect anti-T. gondii antibodies. DNA extracted from muscle homogenate samples were subjected to B1 real-time PCR. Anti-T. gondii antibodies were found in 10 (12.5%) out of 80 examined animals, whereas parasitic DNA was detected in 26 diaphragm muscle samples (32.5%). Only seven samples scored positive in both test: a fair agreement between ELISA and B1 real-time PCR results was achieved (κ value = 0.254). Nevertheless, higher ELISA S/P% values were recorded in diaphragm samples scoring positive to PCR. Higher number of positive samples were found in younger than older animals considering both ELISA and B1 real-time PCR results. Similarly, considering the provenience, animals that have been acquired from other holdings scored more frequently positive to both ELISA and B1 real-time PCR compared to animals that have never left the holding of origin until slaughter. Statistical analysis showed an effect of ELISA S/P% values on B1 real-time PCR results, increasing the risk of parasitic DNA detection when increasing the S/P% values. The other considered variables (age and provenience) did not show any effect on neither ELISA nor B1 real-time PCR. In conclusion, obtained results from the studies of my PhD project allowed to update information on protozoa of medical and veterinary importance both in domestic ruminants and in equids. It was demonstrated that bovine besnoitiosis continues to spread in Italy: both clinical and laboratory tests are needed for an accurate diagnosis, and thus to implement plans for the control of the disease. Moreover, Besnoitia spp. infection circulates in Italian equids and besnoitiosis may be considered an emerging disease of donkeys in Italy and also in Europe. Serological screening of cows and molecular analysis of aborted foetuses confirmed the role of N. caninum in abortion and reproductive failure in dairy herds in northern Italy; besides, multilocus microsatellite genotyping confirmed the concept of local N. caninum subpopulations. The zoonotic importance of T. gondii should not be underestimated in animal species destined to human consumption, including cattle and horses. Serological data are useful to give an indication of the population exposure to the parasite, whereas molecular methods allow to detect tissue cysts in the edible parts reflecting the risk for human infection.

alpha-actinin is a ubiquitous protein found in most eukaryotic organisms. The ability to form dimers allows alpha-actinin to cross-link actin in different structures. In muscle cells alpha-actinin is found at the Z-disk of sarcomeres. In non-muscle cells alpha-actinin is found in zonula adherens or focal adhesion sites where it can bind actin to the plasma membrane.

alpha-actinin is the shortest member of the spectrin superfamily of proteins which also includes spectrin, dystrophin and utrophin. Several hypotheses suggest that alpha-actinin is the ancestor of this superfamily.

The structure of alpha-actinin in higher organisms has been well characterized consisting of three main domains: an N-terminal actin-binding domain with two calponin homology domains, a central rod domain with four spectrin repeats and a C-terminal calcium-binding domain. Data mining of genomes from diverse organisms has made possible the discovery of new and atypical alpha-actinin isoforms that have not been characterized yet.

Invertebrates contain a single alpha-actinin isoform, whereas most of the vertebrates contain four. These four isoforms can be broadly classified in two groups, muscle isoforms and non-muscle isoforms. Muscle isoforms bind actin in a calcium independent manner whereas non-muscle isoforms bind actin in a calcium-dependent manner.

Some of the protozoa and fungi isoforms are atypical in that they contain fewer spectrin repeats in the rod domain. We have purified and characterized two ancestral alpha-actinins from the parasite Entamoeba histolytica. Our results show that despite the shorter rod domain they conserve the most important functions of modern alpha-actinin such as actin-bundling formation and calcium-binding regulation. Therefore it is suggested that they are genuine alpha-actinins.

The phylogenetic tree of alpha-actinin shows that the four different alpha-actinin isoforms appeared after the vertebrate-invertebrate split as a result of two rounds of genome duplication. The atypical alpha-actinin isoforms are placed as the most divergent isoforms suggesting that they are ancestral isoforms. We also propose that the most ancestral alpha-actinin contained a single repeat in its rod domain. After a first intragene duplication alpha-actinin with two spectrin repeats were created and a second intragene duplication gave rise to modern alpha-actinins with four spectrin repeats.

This dissertation contains the results of two different projects. The first one is a study of the transport of protozoa pathogens Cryptosporidium parvum and Encephalitozoon intestinalis in soils. The aim of this project was to investigate the movement and retention mechanisms of these microorganisms in natural porous media. The work determined that in the case of C. parvum, the retention was primarily produced by straining and in the case of E. intestinalis the main retention mechanism was attachment. The results of C. parvum lysimeter experiment compared to the results from the 7 cm column experiments suggest that retention is proportional to the length of the column. The second study evaluated the Polymerase Chain Reaction (PCR) as a tool to identify dechlorinating bacteria in groundwater contaminated with chloroethenes. The target DNA regions to identify these microorganism were the 16s rDNA specific for dehalococcoides sp. and Desulfuromonas and DNA sequences coding for the reductive dehalogenase enzymes pceA, tceA, bvcA and vcrA. Bacteria able to transform PCE into DCE were detected in all groundwater samples. Bacteria able to transform VC into ethene were found only in one of the samples. This study shows that PCR analysis of 16s rDNA and reductive dehalogenase gene sequences together with microcosm results are useful tools to analyze the populations of reductive dechlorinators and their activity in a given site.

The protozoan parasite Giardia intestinalis (syn. G. lamblia, G. duodenalis) is one of the most common causes of diarrheal disease throughout the world, where an estimated 500 million people are infected annually. Despite efforts in trying to elucidate factors associated with virulence in G. intestinalis little is currently known. The disease outcome is highly variable in Giardia infected individuals, ranging from asymptomatic carriers to severe disease. The reasons behind the differences in disease outcome are vaguely understood and studies trying to link infectivity to different Giardia assemblages or sub-assemblages have rendered conflicting results. Prior to this study, little was known about the prevalence and genetic diversity of different G. intestinalis assemblages across the world. In this thesis, molecular characterization of clinical G. intestinalis samples from Eastern Africa and Central America, has been performed, enabling a better understanding of the prevalence of different Giardia genotypes in endemic areas (Papers I and II). A correlation between Giardia colonization and the presence of Helicobacter pylori in the human host was established. We found that the currently available genotyping tools provide low resolution when used to characterize assemblage A Giardia. Also, genotyping of assemblage B isolates at these loci is troublesome due to the polymorphic substitutions frequently found in the sequencing chromatograms. This ambiguity was investigated by using micromanipulation to isolate single assemblage B Giardia cells (Paper III). Both cultured trophozoites and cysts from giardiasis patients were analyzed. The data showed that allelic sequence heterozygosity (ASH) does occur at the single cell level, but also that multiple sub-assemblage infections appear to be common in human giardiasis patients. Furthermore, genome-wide sequencing followed by comparative genomics was performed in order to better characterize differences between and within different Giardia assemblages. The genome of a non-human infecting, assemblage E isolate (Paper IV) was sequenced.  The genomes of two freshly isolated human infecting assemblage AII isolates were also sequenced (Paper V). Subsequent, comparative analyses were performed and included the genomes of two human infecting isolates, WB (AI) and GS/M (B). Several important differences were found between assemblages A, B and E, but also within assemblage A; including unique gene repertoires for each isolate, observed differences in the variable gene families and an overall difference in ASH between the different isolates. Also, a new multi-locus genotyping (MLG) strategy for genotyping of assemblage A Giardia has been established and evaluated on clinical samples from human giardiasis patients.

Desde la aparición de las primeras bases de datos distribuidas hasta los actuales sistemas de replicación modernos, la comunidad de investigación ha propuesto múltiples protocolos para administrar la distribución y replicación de datos, junto con algoritmos de control de concurrencia para manejar las transacciones en ejecución en todos los nodos del sistema. Muchos protocolos están disponibles, por tanto, cada uno con diferentes características y rendimiento, y garantizando diferentes niveles de coherencia. Para saber qué protocolo de replicación es el más adecuado, dos aspectos deben ser considerados: el nivel necesario de coherencia y aislamiento (es decir, el criterio de corrección), y las propiedades del sistema (es decir, el escenario), que determinará el rendimiento alcanzable. Con relación a los criterios de corrección, la serialización de una copia es ampliamente aceptada como el más alto nivel de corrección. Sin embargo, su definición permite diferentes interpretaciones en cuanto a la coherencia de réplicas. En esta tesis se establece una correspondencia entre los modelos de coherencia de memoria, tal como se definen en el ámbito de la memoria compartida distribuida, y los posibles niveles de coherencia de réplicas, definiendo así nuevos criterios de corrección que corresponden a las diferentes interpretaciones identificadas sobre la serialización de una copia. Una vez seleccionado el criterio de corrección, el rendimiento alcanzable por un sistema depende en gran medida del escenario, es decir, de la suma del entorno del sistema y de las aplicaciones que se ejecutan en él. Para que el administrador pueda seleccionar un protocolo de replicación apropiado, los protocolos disponibles deben conocerse plena y profundamente. Una buena descripción de cada candidato es fundamental, pero un marco común es imperativo para comparar las diferentes opciones y estimar su rendimiento en un escenario dado. Los resultados presentados en esta tesis cumplen los objetivos establecidos y constituyen una contribución al estado del arte de la replicación de bases de datos en el momento en que se iniciaron los trabajos respectivos. Estos resultados son relevantes, además, porque abren la puerta a posibles contribuciones futuras.
Ruiz Fuertes, MI. (2011). On the Consistency, Characterization, Adaptability and Integrity of Database Replication Systems [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/11800
Palancia

Tissue characterization is a major step in tissue mechanobiological studies. By characterization methods, tissue quality i.e. the combination of tissue structural, compositional and mechanical properties, is determined. This research focuses on mechanical characterization methods. Among all mechanical characterization methods, we propose those ones which are: 1) Non-destructive, (i.e. that reserves the capability of doing other characterization tests at the end of mechanical test; and, 2) In-line, (that enables tissue progression observation during experiment, and without transferring the specimen from one apparatus to another). However, in-line characterization raises the question of whether conducting tissue observation methods during experimentation modifies tissue progression over time. Therefore, the purpose of this study was to deepen our knowledge about the parameters which could affect tissue quality during mechanical testing. This requires a better understanding of viscoelasticity and viscoplasticity, two key behaviors of tissue, affecting the impact of these parameters (e.g. tissue quality, stimulation parameters) on the response of live tissue to biophysical stimuli. Thus, the objectives of this study were: 1. To review the literature to find information about two mechanical behaviors of tissue i.e. viscoelasticity and viscoplasticity, and the way they affect tissue properties 2. To investigate whether diagnostic tests, as mechanical characterization tests to observe tissue properties, affect tissue progression We explain that viscoelasticity and viscoplasticity of tissue originate from structure and components of the extracellular matrix. We also describe the way they affect tissue dynamic competition between repair, enzymatic degradation and mechanical degradation of the extracellular matrix. Moreover, we specify some tissue stimulation parameters, such as stimulation control type or stimulus history, which could affect tissue progression in response to biophysical stimuli because of viscoelasticity and viscoplasticity. Moreover, by conducting a series of 3-day experiments on frshly extracted tendons, we investigated whether applying "stress relaxation" tests at physiological amplitudes affects tissue response. We divided the tendons into two groups based on the characterization protocol (24 and 0 stress relaxation tests each day), and compared the progression of these groups over time. The stress relaxation tests at physiological amplitude modified tissue response to mechanical stimuli in vitro . In general, the modulus increased for 0 stress relaxation tests, while it first decreased and then increased slightly for 24 stress relaxation tests each day. The difference of mechanical properties between the two groups was significant. Therefore, applying stress relaxation tests at physiological amplitude during the rest periods between mechanical stimuli can affect live tissue progression over time. Therefore, it is essential to take into account the viscoelasticity and viscoplasticity of tissue while developing a stimulation protocol for bioreactor studies or clinical applications.

Cryptosporidiosis is an infection caused by Cryptosporidium; a protozoan parasite that infects the gastrointestinal tract. The infection is of major public health concern in both developed and developing countries. Faecal samples were collected from 160 in-patient adults, with complaint of diarrhoea, admitted at Victoria hospital in Alice, Nkonkobe Municipality. Twenty apparently healthy subjects were included as controls. All diarrhoea positive patients were interviewed to record socio-demographic information, water supply and animal contact. Initial screening was carried out by microscopy and ELISA to detect positive Cryptosporidium. Genomic DNA was extracted from microscopically positive samples and a PCR reaction was perform to amplify the (18S) SSUrRNA gene for further identification and epidemiology of Cryptosporidium. Data were analysed using Pearson‘s χ2 and Fisher‘s exact test to assess the univariate association between Cryptosporidium infection and the possible risk factors. Of the 180 subjects screened for cryptosporidial infection, Cryptosporidium antigen was detected in 122 giving an overall prevalence of 67.8 percent. In HIV-positive diarrhoea patients, prevalence increased with ages; between 31-43 (mean age 36.5 yr) and 70-82 (mean age 75.8 yr) had a higher prevalence (100 percent) of the antigen than 18-30 (mean age 23.2 yr) and 83-95 (mean age 88.8 yr) (50.0 percent) in HIV-positive diarrhoea patients (P > 0.05). In HIV-negative diarrhoea patients, prevalence was highest in the 18-30 (mean age 23.2 yr) (87.5 percent) and least (35.7 percent) in those aged 83-95 (mean age 88.8 yr) (P > 0.05). Cryptosporidium antigen was higher in females than in males. Of 115 females (mean age 46.7yr) who participated in the study, antigen was detected in 90 (78.2 percent) against 32 (71.1 percent) of 45 males (mean age 42.6yr). None of the 20 apparently healthy control subjects was found to be infected with Cryptosporidium. Cryptosporidium was detected in 27 HIV-positive and 97 HIV-negative diarrhoea patients by any one of the techniques. Antigen detection by ELISA 14 showed the highest positivity 96 (76.8 percent) in HIV- negative and 26 (74.3 percent) in HIV- positive diarrhoea patients. PCR detected eighty-nine (71.2 percent) cases in HIV-negative and 23 (65.7 percent) in HIV-positive patients with diarrhoea. Only 13 (37.1 percent) HIV-positive and 34 (27.2 percent) HIV-negative diarrhoea patients were found positive for Cryptosporidium by modified ZN. No significant difference was observed in sensitivity of antigen detection by ELISA and PCR (96.9 percent) in HIV-negative diarrhoea patients, respectively. Specificity of the staining technique was 88.9 percent in HIV-positive and 96.6 percent in HIV-negative diarrhoea patients. No significant difference was found in specificity of antigen detection by ELISA and PCR in HIV-positive and HIV-negative diarrhoea patients, respectively. Positive predictive value of ZN staining in both HIV-positive and HIV-negative diarrhoea patients (92.3 and 96.9 percent) was statistically higher than ELISA and PCR. No significant difference was observed in negative predictive value of ZN technique for detection of Cryptosporidium between HIV-positive and HIV- negative diarrhoea patients. Differences found in prevalence rates due to water source, suggest that the high infection rates of specific groups are associated with their exposure to the contaminated water supply. The results indicate that Cryptosporidium infection is highly prevalent in adult faecal specimens in the Nkonkobe Municipality, an indication of active infection that is likely to emerge as major human pathogen in this location due to socioeconomic changes which favour transmission. However, sequencing analysis is required to differentiate between Cryptosporidium genotypes in the various outbreaks

Ligand-gated and ligand-modulated ion channel (IC) sensors have received increased attention for their ability to transduce ligand-binding events into a readily measurable electrical signal. Ligand-binding to an IC modulates the ion flux properties of the channel in label-free manner, often with single-molecule sensitivity and selectivity. As a result, ICs are attractive sensing elements in biosensoring platforms, especially for ligands lacking optical (e.g. fluorescent) or electrochemical properties. Despite the growing number of available ligand-gated and ligand-modulated ICs and artificial lipid bilayer platforms for IC reconstitution, significant work remains in defining the analytical performance capabilities of IC sensors. Particularly, few studies have described platforms for making measurements with rapid temporal resolution and high sensitivity. In this work, we describe an artificial lipid bilayer platform which enables rapid measurement of ion channel activity, a key parameter for developing IC sensors suitable for studying biological events, e.g. single cell exocytosis (Chapter 2 and 3). Additionally, we developed expression, purification, and reconstitution protocols for Kir6.2, a model ligand-gated ion channel, for use in sensor development (Chapter 4). The final goal is to reconstitute ion channel-coupled receptors (ICCRs), G protein-coupled receptor-Kir6.2 fusion proteins, into artificial lipid bilayers to detect small molecules and hormones targeting GPCRs. Towards this goal, we characterized the expression and function of two ICCRs, M2-Kir and D2-Kir, in HEK293 cells (Chapter 5).

The livestock sector is facing different challenges, and the demand for higher sustainability seems to be one of the most urgent. This PhD project debated, in particular, the environmental impacts related to ruminant nutrition, focusing on dairy cows, since nutrition is bound tightly to two of the most important sources of impact: enteric CH4 emission and land use change (LUC). Enteric CH4 emission from ruminants represents 29-38% of the total (anthropic + natural) emission of this powerful (21 CO2 equivalent) greenhouse gas. The production of CH4 is a physiological process used by ruminants to discharge the [H] resulting from rumen fermentation. Different strategies can be implemented to mitigate this impact, and they can be roughly grouped into three main categories: animal and feed management, diet formulation, and rumen manipulation. The second issue investigated in the project is the high reliance of European livestock on soybean meal as a protein source for diet formulation. A total of 30 million tonnes of this feedstuff was imported into Europe in 2020. The main countries of origin are in South America (65% of total import), where 20% of soybean meal production was linked with deforestation (and consequently LUC) in the last decades. Clearing these areas means loss of carbon sink and emission of CO2 in the atmosphere. Other feedstuffs, like grain legumes, oilseed meals alternative to soybean, and high quality forages could be considered to provide protein feed with a lower environmental cost. In this context, the PhD project was developed as follows:  To address the problem of CH4 emission, plant essential oils, as modulators of rumen fermentation, were evaluated (Experiment 1). Furthermore, the effect on CH4 emission of different forages in the diet of dairy cows was investigated (Experiment 2). For validation of mitigation strategies and inventory computation of emissions at a national scale, country-specific equations to quantify CH4 emission were evaluated (Experiment 3).  To address the problem of soybean meal environmental impact, soybean silage and responsible soybean meal (not connected with land use change) were evaluated as protein source alternatives to soybean meal in the diet of lactating cows (Experiments 4 and 5). Enteric methane direct emission In the first experiment, Achille moschata essential oil and its main pure components, namely bornyl acetate, camphor, and eucalyptol, were evaluated in an in vitro experiment. The trial comprehended a short-term in vitro incubation (48 h), with 200 mg of compound per L of inoculum, and a long-term one by continuous fermenter (9 d), with 100 mg/L for each compound. In the first incubation, no differences due to the treatments were found for in vitro gas production (on average, 30.4 mL/200 mg DM, P = 0.772 at 24 h and 45.2 mL/200 mg DM, P = 0.545 at 48 h). Camphor and eucalyptol reduced CH4 production when expressed as % of gas production at 48 h (P < 0.05): -7.4% and -7% compared to control. In the second incubation, CH4 was reduced by eucalyptol (-18%, P < 0.05). Regarding volatile fatty acids, the main effects were a decrease of total production for camphor (-19.5%, P < 0.05) and an increase in acetate production at 9 d with bornyl acetate and camphor (+13% and 7.6%, respectively, P < 0.05) compared to control. Total protozoa count was increased compared to the control (on average: +37%, P = 0.006, at 48 h and +48%, P < 0.001, at 9 d) with all the pure compounds tested. In the short-term incubation, all the treatments reduced Bacteroidetes (30.3%, on average, vs. 37.1% of control, P = 0.014) and Firmicutes (26.3%, on average, vs. 30.7% of control, P = 0.031) abundances but increased Proteobacteria (36.0%, on average, vs. 22.5% of control, P = 0.014). In the long-term incubation, eucalyptol increased the genus Ruminococcus abundance (2.60% vs. 1.18% of control, P = 0.011). An adaptation at long time incubation was observed. In particular, considering eucalyptol addition at 9 d incubation, VFA production was reduced (26.8 vs. 33.3 mmol of control, P < 0.05) contrary to the 48 h incubation (P = 0.189). Furthermore, the treatments affected protozoa genera relative abundances at 24 h (increased abundance for Entodinium with all the treatments, P < 0.001, and reduced for Diplodinium, P = 0.001); at 9 d, instead, protozoa genera relative abundances were not affected by the treatment. The additives tested showed potential in reducing CH4 production without compromising the overall fermentation efficiency. A meta-analysis (Experiment 2) investigated the effects on lactation performance and enteric CH4 of the main forage included in the diet. In the dataset, composed of in vivo experiments, four main forage bases were evaluated: corn silage, alfalfa silage, grass silage, and green forage. Cows fed corn, and alfalfa silages had the highest DMI (21.9 and 22.0 kg/d, P < 0.05) and milk yield (29.7 and 30.4 kg/d, P < 0.05). On the opposite, NDF digestibility was highest for grass silage and green forage (67.6% and 73.1%, P < 0.05) than corn and alfalfa silages (51.8% on average). CH4 production was lower (P < 0.05) for green forage (332 g/d) than the silage diets (on average 438 g/d). Instead, corn silage and alfalfa silage gave the lowest CH4 per kg of milk yield (14.2 g/kg and 14.9 g/kg, P < 0.05). Considering CH4 per kg of DMI, the only difference was between corn silage and grass silage (19.7 g/kg vs. 21.3 g/kg respectively for corn and grass silage, P < 0.05). Finally, prediction models for CH4 production were obtained through a step-wise multi regression. In particular, the models for the prediction of: CH4 in g/d (CH4 = - 65.3(±63.7) + 11.6(±1.67) × DMI - 4.47(±1.09) × CP - 0.86(±0.33) × Starch + 2.62(±0.78) × OM digestibility + 30.8(±9.45) × Milk fat) and for CH4 in g/kg of milk yield (CH4/milk yield = - 55.5(±20.1) - 0.37(±0.13) × DMI + 0.18(±0.05) × Total forage inclusion on diet DM - 0.10(±0.04) × Inclusion of the main forage on diet DM + 0.48(±0.21) × OM + 0.14(±0.06) × NDF + 1.98(±0.86) × Milk fat +4.34(±1.66) × Milk protein) showed high precision (R2 = 95.4% and 88.6%, respectively), but the best AIC value (320) was found for the model predicting CH4 in g/kg DMI: CH4/kg DMI = 6.16(±3.89) - 0.36(±0.03) × CP + 0.12(±0.05) ×OM digestibility + 3.77(±0.56) × Milk fat - 3.94(±1.07) × Milk fat yield. A dataset (66 observations in total) of three in vivo experiments conducted in Italy on lactating cows in respiration chambers was built to evaluate IPCC Tier 2 equations to estimate enteric CH4 production (Experiment 3). In the dataset, the CH4 conversion factor (conversion of gross energy intake into enteric CH4 energy) was lowest for a diet based on grass and alfalfa silages (5.05%, P < 0.05), while the others values ranged between 5.41 and 5.92%. On average, energy digestibility was 69.0% across the dataset, but the diet based on hays had a lower value (64.8%, P < 0.05). The IPCC (2019) Tier 2 (conversion factor = 5.7% or 6.1% for diet with NDF concentration < 35% or >35%, respectively; digestible energy = 70%) gave, on average, a value of CH4 production not statistically different from the ones measured in vivo (382 vs. 388 g/d in vivo, P > 0.05). The IPCC (2006) Tier 2 (conversion factor = 6.5%, digestible energy = 70%) over-predicted CH4 emission (428 vs. 388 g/d in vivo, P < 0.05; μ = -1.05). The most precise models were the two considering digestible energy equal to 70% and average values of conversion factor for IPCC (2006) and IPCC (2019) (R = 0.630); the most accurate models was the one considering a conversion factor equal to 5.7% and energy digestibility measured in vivo (Cb = 0.995). Overall, the best performance among the predicting models tested was for the one based on a conversion factor equal to 5.7% and energy digestibility of 70% (CCC = 0.579 and RMPSE = 9.10%). Use of alternative protein source to conventional soybean meal The dietary inclusion of soybean silage in partial replacement of soybean meal for dairy cows was evaluated in vivo in lactating cow diets (Experiment 4). Cows were fed two diets, one with 12.4% of DM from soybean silage in substitution of 35% of the soybean meal of the control diet. The treatment did not affect DMI and milk yield (on average, 23.7 kg/d, P = 0.659, and 33.0 kg/d, P = 0.377, respectively). Cows fed the soybean silage diet had lower milk protein concentration (3.43% vs. 3.55% of the control, P < 0.001) and higher milk urea (30.5 vs. 28.7 mg/dL, P = 0.002). The soybean silage had lower nutrient digestibility than the control: DMD 65.2% vs. 68.6%, OMD 66.4% vs. 69.8%, NDFD 31.5% vs. 38.8% (respectively for soybean silage and control diet; P < 0.001 for all of them). Regarding N balance, cows fed soybean silage excreted more nitrogen in the urines (32.3 % of N intake vs. 28.9%, P = 0.005) and less in the milk (31.3% vs. 32.7%, P =0.003) than the control. When used as a protein source alternative to soybean meal, soybean silage sustained comparable milk production, but NDF digestibility and N use efficiency should be improved. The environmental impact of the use of soybean silage in comparison to a control diet with soybean meal as the main protein source was evaluated through an LCA approach (Experiment 5). In addition, two scenarios were included in the study, considering the two diets mentioned before, but with soybean meal not connected to LUC (responsible soybean meal). Regarding the single forages, soybean silage had higher global warming potential than alfalfa hay (477 vs. 201 kg CO2eq/ton DM), also when this was expressed per tonnes of protein production (2439 and 1034 kg CO2eq/ton CP, respectively), probably due to the lower contribution of the cultivation phase for alfalfa, being a multi-year crop. The scenario with soybean silage reduced the global warming potential per kg of fat and protein corrected milk (1.17 kg CO2eq) compared to the control (1.38 kg CO2eq). Responsible soybean meal reduced the global warming potential per kg of fat and protein corrected milk (1.13 kg CO2eq/kg vs. 1.38 of the scenario with the control diet). Overall, the best result per kg of fat and protein corrected milk was obtained when responsible soybean meal and soybean silage were used in combination (1.01 kg CO2eq). Also, when global warming potential was evaluated per daily fed TMR, the impact was lowest for the scenario with responsible soybean meal (13.4 kg CO2eq/d) due to the lower contribution of soybean meal to the total impact (11% vs. 43% of the control). Therefore, the two alternative protein sources tested should be preferred when considering environmental impact compared to conventional soybean meals.

Orientador: Regina Maria Puppin Rontani
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: Este estudo teve por objetivos: produzir lesões artificiais de cárie em esmalte bovino, com características clínicas similares àquelas identificadas pelo protocolo ICDAS II 2009 (International Caries Detection and Assessment System), códigos 1, 2, e 3, através da inspeção visual; identificar o tempo de desmineralização necessário para a produção de lesões artificiais de cárie em esmalte bovino; caracterizar segundo a área de secção transversal, profundidade (LD) e volume da lesão, topografia da superfície, a razão entre a porcentagem de perda mineral e a profundidade da lesão (ratio) e perda mineral das lesões, por meio de diferentes metodologias de análise: Análise visual, Microtomografia (?-CT) Microscopia Eletrônica de Varredura (MEV), Microrradiografia Transversal (TMR) e Microscopia óptica em Luz Polarizada (MLP). Foram utilizados 45 blocos de esmalte bovino distribuídos em 3 grupos (n=15), de acordo com o Código ICDAS 1, 2 e 3, e submetidos ao desafio ácido pelo método biológico, com S. mutans. Os blocos foram imersos em meio BHI suplementado por sacarose a 1% com inóculo de S. mutans, sendo o meio trocado a cada 24h, e para identificação do tempo de produção das lesões (estudo piloto). Em seguida, os espécimes foram removidos do meio de cultura de acordo com o período de tempo identificado para cada grupo (24h, 4 dias e 14 dias), fotografados (análise visual), submetidos à análise por ?-CT, MEV e posteriormente, seccionados longitudinalmente em relação à lesão de cárie e avaliados em TMR e MLP. Os dados obtidos da MEV foram avaliados descritivamente; aqueles provenientes da ?-CT, TMR e MLP foram submetidos aos testes ANOVA e Tukey (p<0,05). O tempo necessário para produção de lesões clinicamente semelhantes àquelas para cada código ICDAS II foi identificado visualmente. Para a produção de lesões in vitro similares ao código 1, o tempo necessário foi de 24 horas; para o código 2, 4 dias; e para o código 3, 14 dias. Para as lesões similares àquelas Código 1, a MLP e TMR identificaram a presença de lesões subsuperficiais de cárie, porém o ?-CT não permitiu visualizá-las. O MEV mostrou superfície mais regular, na maioria das amostras, sem sinais de erosão e com pequenos orifícios provocados pelo S. mutans. Para as lesões Código 2 a análise por ?-CT identificou áreas e volume de lesão menores do que para o Código 3. A profundidade da lesão não pode ser identificada pelo ?-CT para ambos os Códigos. MLP e TMR identificaram maiores profundidades de lesão para o Código 3, comparado ao 2, porém, evidenciaram a presença de desmineralização e cavitação, também maiores no grupo ICDAS 3. A profundidade média das lesões dos três grupos foi mensurada pelos métodos TMR e MLP, e ambas as análises evidenciaram que, quanto maior o código ICDAS, maior a profundidade das lesões; porém, não foi observada correlação significativa entre estes dois métodos. Baseando-se nos resultados obtidos pode-se concluir que o método biológico de produção de cárie por S. mutans possibilita a produção de lesões similares às observadas clinicamente pelo protocolo ICDAS II. Diferentes metodologias empregadas podem ser recomendadas de acordo com o tipo de lesão produzida artificialmente
Abstract: This study was developed in order to identify the effect of demineralization process to provide bovine enamel caries-like lesions, using Streptococcus mutans biofilms with clinical features similar to those found in ICDAS II (2009) criteria, codes 1, 2 and 3 by visual inspection; to identify the time required for providing the demineralization; to characterize the lesions obtained from different analysis methodologies: X-ray microtomography (?-CT), Scanning Electron Microscopy (SEM), Transversal Microradiography (TMR) and light polarized optical microscopy (MLP). It was used 45 enamel block divided into 3 groups (n =15) submitted to caries-like lesions by biological method, with Streptococcus mutans biofilm. The specimens were immersed in BHI broth supplemented with 1% sucrose inoculum of S. mutans and the medium was changed every 24 hours. Then, the specimens were removed from the culture media in each period of time (24h, 4 and 14 days) and pictures were taken from their demineralized surface (visual analysis). Next, the specimens were submitted to ?-CT and SEM evaluations and longitudinally sectioned by the caries lesions and they were evaluated in TMR e MLP. Data obtained from SEM were descriptively evaluated. Data from ?-CT, TMR and MLP were submitted to ANOVA and Tukey's tests (p<0,05). The time required to produce clinical lesions similar to those ICDAS II for each protocol code was visually identified. For the production of lesions in vitro similar to code 1, the time required was 24 hours, code 2 for 4 days and code 3 for 14. For the similar lesion to those from code 1, the MLP and TMR identified the presence of subsurface carious lesions, but ??CT did not allow viewing it. The SEM analysis showed smoother surface, mostly without signs of erosion and pinholes caused by S mutans. For code 2 lesions the ??CT analysis identified lesions areas and volume smaller than Code 3. The lesion depth cannot be identified by ??CT for both codes. MLP and TMR identified higher depths to code 3 as compared to 2, however, showed the presence of demineralization and cavitation also higher than code 3 ICDAS. The lesion depth average from the three groups was measured by TMR and MLP methods, and both analyses showed that the higher the ICDAS II code, the deeper the lesions were. Different methods may be employed in accordance with the recommended type of lesion produced artificially
Mestrado
Odontopediatria
Mestra em Odontologia

Cette thèse porte sur le développement d’un moyen d’essai mécanique permettant de caractériser l’effet de la pression partielle d’oxygène sur le comportement mécanique des oxydes sous-stoechiométriques de type pérovskite utilisé pour leur propriété de semi-perméation. Pour étudier ces matériaux, un nouveau protocole expérimental a été développé, ainsi qu’une méthode numérique de dépouillement ad hoc. Les essais de compression diamétrale cyclique et fluage par compression diamétrale mis en place sont réalisés dans un four d’essais mécaniques à étanchéité renforcée permettant de maitriser l’atmosphère gazeuse dans la zone utile pendant l’essai. Le four, conçu pour ces essais, permet la réalisation de mesures optiques en temps réels. Pour l’identification des paramètres matériaux, une nouvelle routine nommée DigImCo.v2 a été développée. Celle-ci couple les techniques de corrélation d’images numérique (CIN) et la méthode d’identification par analyse inverse à travers la simulation numérique des essais et les méthodes d’optimisation. L’algorithme d’optimisation proposée découle de celui de Levenberg-Marquardt modifié et la loi de fluage implémentée dans le logiciel de simulation éléments finis est une loi de type Norton. Les résultats des essais montrent une faible influence de la pression partielle d’oxygène sur les matériaux étudiés, qui présentent également des déformations de fluage négligeables comparativement aux matériaux de la littérature
This thesis deals with the development of mechanical set up that allows characterizing the oxygen partial pressure effect on the mechanical behavior of sub-stoichiometric perovskites oxides, used due to their potential oxygen semi-permeation properties. In order to study these materials, a new experimental set up and a new post-process routine are developed. Diametric compression tests and creep by diametric compression are conducted in an original mechanical test furnace with reinforced sealing and controlled atmospheres. The tests are instrumented by optical measurements. In order to control the oxygen partial pressure in the test zone, and to study its real influence on mechanical properties, an oxygen sensor is monitored to the device. To analyze experiences, a new post-process routine called DigImCo.v2 is developed that permits to identify material parameters. This approach combine Digital Image Correlation method and an inverse identification method. The optimization technic is based on Levenberg-Marquardt module and the numeric model simulation of the tests are performed on Abaqus software. For the creep parameters identification, Norton creep model is simulated in Abaqus. Tests results reflect the relatively weak influence of oxygen partial pressure on studies materials, which exhibit negligible creep strains compared to literature

Plusieurs évolutions sont constatées dans la recherche en biologie. Tout d’abord, les études menées reposent souvent sur des approches expérimentales quantitatives. L’analyse et l’interprétation des résultats requièrent l’utilisation de l’informatique et des statistiques. Également, en complément des études centrées sur des objets biologiques isolés, les technologies expérimentales haut débit permettent l’étude des systèmes (caractérisation des composants du système ainsi que des interactions entre ces composants). De très grandes quantités de données sont disponibles dans les bases de données publiques, librement réutilisables pour de nouvelles problématiques. Enfin, les données utiles pour les recherches en biologie sont très hétérogènes (données numériques, de textes, images, séquences biologiques, etc.) et conservées sur des supports d’information également très hétérogènes (papiers ou numériques). Ainsi « l’analyse de données » s’est petit à petit imposée comme une problématique de recherche à part entière et en seulement une dizaine d’années, le domaine de la « Bioinformatique » s’est en conséquence totalement réinventé. Disposer d’une grande quantité de données pour répondre à un questionnement biologique n’est souvent pas le défi principal. La vraie difficulté est la capacité des chercheurs à convertir les données en information, puis en connaissance. Dans ce contexte, plusieurs problématiques de recherche en biologie ont été abordées lors de cette thèse. La première concerne l’étude de l’homéostasie du fer chez la levure pathogène Candida glabrata. La seconde concerne l’étude systématique des modifications post-traductionnelles des protéines chez la levure pathogène Candida albicans. Pour ces deux projets, des données « omiques » ont été exploitées : transcriptomiques et protéomiques. Des outils bioinformatiques et des outils d’analyses ont été implémentés en parallèle conduisant à l’émergence de nouvelles hypothèses de recherche en biologie. Une attention particulière et constante a aussi été portée sur les problématiques de reproductibilité et de partage des résultats avec la communauté scientifique
Biological research is changing. First, studies are often based on quantitative experimental approaches. The analysis and the interpretation of the obtained results thus need computer science and statistics. Also, together with studies focused on isolated biological objects, high throughput experimental technologies allow to capture the functioning of biological systems (identification of components as well as the interactions between them). Very large amounts of data are also available in public databases, freely reusable to solve new open questions. Finally, the data in biological research are heterogeneous (digital data, texts, images, biological sequences, etc.) and stored on multiple supports (paper or digital). Thus, "data analysis" has gradually emerged as a key research issue, and in only ten years, the field of "Bioinformatics" has been significantly changed. Having a large amount of data to answer a biological question is often not the main challenge. The real challenge is the ability of researchers to convert the data into information and then into knowledge. In this context, several biological research projects were addressed in this thesis. The first concerns the study of iron homeostasis in the pathogenic yeast Candida glabrata. The second concerns the systematic investigation of post-translational modifications of proteins in the pathogenic yeast Candida albicans. In these two projects, omics data were used: transcriptomics and proteomics. Appropriate bioinformatics and analysis tools were developed, leading to the emergence of new research hypotheses. Particular and constant attention has also been paid to the question of data reproducibility and sharing of results with the scientific community

The accelerated ash loading of diesel particulate filters (DPFs) by lube-oil derived products is investigated in the present study. A 517-cc single-cylinder, naturally aspirated direct-injection diesel engine is used to accelerate ash formation by artificially increasing the rate of lube-oil consumption to approximately 40 times that observed during normal engine operation. Lube-oil consumption (LOC) is accelerated by blending diesel fuel with 5% by volume of standard 15-w40 lube oil and is subsequently injected through the fuel injector into the combustion chamber. The ash loading protocol is a backpressure-based method of determining the amount of soot present within the DPF and initiating active regeneration upon achieving the target soot loading of 3 grams per liter. The final protocol employed a backpressure threshold that is defined for each individual loading by adding 0.20 psi to the baseline backpressure observed for that cycle, and consistently achieved the target soot loading. The active regeneration strategy was also refined to gradually increasing DPF temperatures to approximately 700ºC. A total of five full experiments are carried out in the present investigation. Two cordierite substrates, one silicon carbide substrate, and two mullite substrates are utilized to evaluate the performance of the accelerated ash loading protocol and make necessary refinements. The rate of backpressure increase with respect to ash accumulation varies substantially between substrates. Soot lightoff temperatures for all substrates are observed to be approximately 600ºC, with ash having a minimal effect on this value except in the highly-catalyzed substrates, where lightoff temperatures are initially lower but increase as ash accumulation limits exposure of the PGM to the soot layer. Characterization techniques such as Electron Probe Microanalysis (EPMA), Scanning Electron Microscopy with Energy Dispersive Spectroscopy (SEM-EDS), X-ray Diffraction (XRD), and Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) are used to analyze the ash layer for comparison to previously published results. All characterization results depict an ash layer that increases in thickness along the direction of flow within the DPF. The relative thickness of each ash layer is observed to be a strong function of the channel wall topography as well as the presence of catalyst and washcoat material.

博士
國立臺灣師範大學
生命科學系
105
The aim of the current study was to develop an iron oxide nanoparticle (ION) labelling and magnetic resonance imaging (MRI)-based protocol to allow visualization of the differentiation process of mesenchymal stem cells (MSCs) into neural-like cells (NCs) in vitro. Ferucarbotran, a clinically available ION, which can be visualized under MRI, is used for tracking cells implanted in vivo. The NCs were verified morphologically and histologically by light microscopy, and their functions were verified by measuring their action potentials. Conformational conversion of axon-like structures was observed under light microscopy. These NCs exhibited frequent, active action potentials compared with cells that did not undergo neural differentiation. The labelling of ION had no influence on the morphological and functional differentiation capacity of the MSCs. We conclude that the MSCs that were differentiated into NCs exhibited in vitro activity potential firing and may be used to replace damaged neurons.

Panic disorder (PD) is a common psychiatric illness with highly stereotyped symptoms including a sense of shortness of breath or feelings of suffocation. PD is characterized by spontaneous and recurrent panic attacks (PAs) that consist of incapacitating periods of acute-onset respiratory, cardiovascular, gastrointestinal, autonomic and cognitive symptoms. According to the DSM-5, recurrent panic attacks in PD are categorized as being either spontaneous (unexpected) or cued (expected). Accumulating evidence suggests that spontaneous PAs may be provoked by interoceptive sensory triggers caused by fluctuations in the internal homeostatic milieu. An important internal homeostatic trigger for the genesis of PAs, supported by an emerging body of work, is acid–base imbalance and associated pH chemosensory mechanisms. Largely founded on panic provocation studies with agents promoting homeostatic pH imbalance (e.g. sodium lactate or CO2) and related to the false suffocation alarm theory the role of acid–base and chemosensory systems in panic provides strong scientific insights on the genesis of PAs. Heightened sensitivity to carbon dioxide (CO2) is an established biological correlate of PD. Indeed inhaled CO2 triggers PAs in most individuals with PD but only a minority of unaffected controls. More recently the acid sensing ion channel-1 (ASIC1) has been proposed as a candidate gene responsible for these phenomena. Indeed this channel is expressed in central nervous system and in particular in amygdala, brainstem, structures involved in chemosensory detection and breathing. According to twin studies, shared genetic determinants appear to be the major underlying cause of the developmental of adult PD and of altered sensitivity to CO2. Moreover, in addition to genetic determinants, environmental risk factors affect the liability to these traits, and several life events that influence the susceptibility to PD also predict heightened CO2 reactivity. There is evidence that genetic and environmental determinants interact to influence human responses to CO2 suggesting also epigenetic mechanisms to underlie the development of PD. Valuable animal models for PD are lacking due to the difficulty to measure “panic” in animals. Indeed models of PD used in pre-clinical research measure the defensive behaviors showed by the animals in response to a real aversive stimulus and not spontaneously and/or in the absence of real dangerous situation, as in PD. Not being able to interview the animal, about its symptoms, such as fear of dying or going crazy as in human PD patients, CO2 hypersensitivity, observed in patients with panic and their unaffected relatives, represents a valid endophenotype to model this disorder in animals. PD is a chronic disorder with variable course and treatments available until now are not specific and are often only modestly efficacious. Typical pharmacologic treatments are antidepressants (SSRI) or anxiolytics (benzodiazepines). An alternative strategy is psychotherapy (cognitive behavioral therapy) and often patients are treated with a combination of psycho- and pharmaco-therapies. In some cases therapies (e.g., benzodiazepines) may be associated with treatment-specific side effects or risks such as sedation or the risk of dependence or tolerance. For these reasons is important to find a therapy specific for PD, possibly with the fewest side effects. The aims of my PhD Thesis were: 1) to validate the RCF protocol in mice as a useful manipulation procedure affecting individual emotionality, different from the classical maternal separation (Handling), usually applied in rodents, to evaluate the effects of an early adverse environment. I evaluated the short and long-term effects of these early manipulations on several behavioral, molecular and physiological parameters (mother-pups interaction; stress response; emotionality; CO2 panic-related response; gluco- and mineral-corticoid receptors mRNA expression; etc.) 2) to analyze possible molecular mechanisms underlying the panic-related CO2 hypersensitivity showed by RCF animals and evaluate different pharmacological treatments (chloridiazepoxide, chlorogenic acid and amiloride) able to recover the normal respiratory response to hypercapnia, on the basis of molecular suggestions 3) to verify the cognitive capability of RCF animals trough learning tests (such as active avoidance test and novel recognition test) and investigate the capability of exposure to 6% CO2 on animals’ behavioral conditioning, in RCF and Control subjects. Indeed, humans with PD show behavioral conditioning to panic attacks and develop PA also in absence of unconditioned stimulus. 4) to investigate whether the CO2 hypersensitivity showed by RCF animals was a transgenerational transmissible trait. First of all, results reported in this study suggest that the behavioral and physiological phenotypes observed during development and adulthood depend on characteristics and timings of early adversities capable of activate different biological processes. Reasonably, the response of the animal to the early manipulations is different and aimed at maximizing individual fitness: the early environment could exert its programming role during this developmental plastic period, through specific epigenetic modifications. Short, even if repeated, separations from the mother (Handling protocol) induce habituation to a relatively low stressing environment, enhancing the capability of the subject to face new stressful situations. By contrast, the disruption of the infant attachment bond (RCF protocol) is associated to a modification in the respiratory response to high CO2 in breathing air, an endophenotype these animals share with PD patients. The disruption of infant-mother bond in RCF animals suggested by the enhanced separation anxiety at 8 days age supports the relation between SAD and PD already suggested in literature. In addition the CO2 reactivity showed by these animals represents a useful tool to study PD in pre-clinical research. Molecular alterations found in RCF animals (experiment 2a) supported the involvement of acid-base balance dysregulation in development of CO2 hypersensitivity. Indeed RCF animals showed a higher expression in ASIC1 gene that codifies for acid sensing ion channels. These channels are sensitive to lower levels of pH being able to detect changes in CO2 concentration in the body and adjust the respiratory function to receive enough O2 not to compromise biological processes. Molecular investigations in addition revealed alterations in GABAergic transmission in RCF animals supporting the idea of an involvement of this neurotransmitter in the development of PD. RCF animals showed an increased expression of Dbi which is an inhibitor of GABAergic transmission. These molecular findings have provided indications suggesting that a possible rescue treatment for PD patients should consist in reducing CO2 hypersensitivity. Lowering of this increased respiratory response to modest increase in CO2 could reduce the negative feeling associated to condition, reducing the conditioning potentiality that favor the development of panic disorder, after repeated panic attacks. The use of benzodiazepine such as chlordiazepoxide was able to restore the normal respiratory response to CO2 as well, giving pharmacological validation to RCF model. However, benzodiazepines have several contraindications, especially for chronic treatments and their sedative effect should also be taken into consideration. Even if I only present few data on the effects of chlorogenic acid and amiloride on RCF animals, I think these results are very interesting and need further and deeper evaluation. Both these compounds interacted with the pH sensitive channels (asics) and their administration was able to restore the respiratory response observed in control animals. It is well known, that panic attacks are able to condition behaviors of PD patients. They indeed tend to avoid situations and places similar to those where a panic attack previously occurred. Similarly RCF animals showed, in experiment 3, behavioral conditioning to the situation previously paired with CO2 (tone exposure). It should be now explored whether RCF animals generalize the conditioned fear, suggesting how an initial panic attack can evolve into panic disorder in humans. Finally RCF model demonstrated a transgenerational transmission of the respiratory endophenotype (experiment 4) supporting the hypothesis of gene-enviroment interplay role to predisposition to panic disorder (Spatola et al., 2011). The epigenetic mechanisms responsible for this trans-generational transmission are under investigation as well as possible strategies to prevent this phenomenon. In conclusion, the Repeated Cross-Fostering protocol seems a valid mouse model of Panic Disorder in humans: RCF mice show typical features of this disorder such as separation anxiety during childhood, CO2 hypersensitivity and CO2 conditioned and avoidance behaviors. Acid sensing ion channels are interesting molecular markers which can be used as new targets for pharmacological treatments and can help to explain hyper-responsiveness to CO2 in PD patients as well.

[ES] La fabricación de nanopartículas con tamaños por debajo de los 100 nm ha permitido el desarrollo de innovadores nanodispositivos capaces de interactuar de forma directa con sistemas vivos a nivel celular y molecular, convirtiéndose en una parte fundamental dentro del campo de la nanomedicina. Uno de los principales retos a los que se enfrenta la ingeniería de nanopartículas es el desarrollo de nanodispositivos con propiedades físico-químicas bien definidas, ya que de ellas depende el comportamiento y biodistribución de dichos sistemas una vez introducidos en el organismo. No menos importante es el desarrollo de protocolos de síntesis reproducibles y optimizados, indispensables para la fabricación y escalado de nanodispositivos que puedan ser trasladados a futuras aplicaciones biomédicas. El principal objetivo de este proyecto de doctorado es el estudio y fabricación de nanopartículas magnéticas mesoporosas de sílice con estructura "core-shell" para su aplicación como agentes teranósticos en el campo de la nanomedicina. En este estudio se analiza en profundidad la síntesis y caracterización de dichos nanomateriales con el objetivo de producir nanopartículas con unas propiedades físico-químicas bien definidas de forma controlada y reproducible. La obtención de dichas nanopartículas supondría un gran avance de cara al desarrollo de nanodispositivos más complejos y sofisticados. El contenido de la tesis se ha estructurado en distintos capítulos que se detallan brevemente a continuación: ¿El capítulo 1 es una introducción a la nanomedicina, destacando el papel fundamental que tienen las nanopartículas en el desarrollo de nuevas aplicaciones biomédicas. A continuación se presentan las nanopartículas de sílice mesoporosa, mostrando la gran versatilidad de dichos nanomateriales para el desarrollo de dispositivos teranósticos así como sistemas para la liberación controlada de fármacos. Por último, se destaca la importancia de fabricar nanodispositivos con unas propiedades físico-químicas bien definidas como requisito indispensable para la traslación de los resultados experimentales hacia el campo clínico. ¿El capítulo 2 incluye los objetivos principales de la tesis. ¿El capítulo 3 se centra en la síntesis y caracterización de nanopartículas superparamagnéticas de óxido de hierro (USPIONs), siendo estas utilizadas en capítulos posteriores para la síntesis de las nanopartículas mesoporosas tipo "core-shell". Las USPIONs son preparadas a través de un método sencillo de coprecipitación en el que se emplean condiciones de reacción moderadas. Las nanopartículas obtenidas son caracterizadas en profundidad, analizando sus propiedades magnéticas para su aplicación en hipertermia magnética y como agentes de contraste dual en imagen por resonancia magnética (MRI). ¿El capítulo 4 está dedicado a la preparación de nanopartículas magnéticas mesoporosas de sílice con estructura "core-shell". Los conceptos fundamentales relacionados con los mecanismos de formación de este tipo de nanomateriales son ampliamente analizados, así como los parámetros de reacción involucrados en la síntesis. Como punto de partida, se propone un protocolo de síntesis general para la obtención de las nanopartículas tipo "core-shell". A continuación, se analiza en profundidad el efecto que los distintos parámetros de reacción tienen en las propiedades físico-químicas de dichas nanopartículas. Para la fase de optimización se utiliza un modelo semi-empírico como referencia, racionalizando los resultados experimentales observados en base a un posible mecanismo de formación. ¿El capítulo 5 se centra en el análisis y caracterización de la estructura mesoporosa de las nanopartículas tipo "core-shell". Además, se analiza el efecto que los distintos parámetros de reacción tienen sobre la estructura final de las nanopartículas, aportando información adicional sobre su posible mecanismo
[CAT] La fabricació de nanopartícules amb grandàries per davall dels 100 nm ha permés el desenvolupament d'innovadors nanodispositius capaços d'interactuar de forma directa amb sistemes vius a nivell cel¿lular i molecular, convertint-se en una part fonamental dins del camp de la nanomedicina. Un dels principals reptes als quals s'enfronta l'enginyeria de nanopartícules és el desenvolupament de nanodispositius amb propietats físic-químiques ben definides, ja que d'elles depén el comportament i biodistribució d'aquests sistemes una vegada introduïts en l'organisme. No menys important és el desenvolupament de protocols de síntesis reproduïbles i optimitzats, indispensables per a la fabricació a gran escala de nanodispositius que puguen ser utilitzats en futures aplicacions biomèdiques. El principal objectiu d'aquest projecte de doctorat és l'estudi i fabricació de nanopartícules magnètiques mesoporoses de sílice amb estructura "core-shell" per a la seua aplicació com a agents teranòstics en el camp de la nanomedicina. En aquest estudi s'analitza en profunditat la síntesi i caracterització d'aquests nanomaterials amb l'objectiu de produir nanopartícules amb unes propietats físic-químiques ben definides de forma controlada i reproduïble. L'obtenció d'aquestes nanopartícules suposaria un gran avanç de cara al desenvolupament de nanodispositius més complexos i sofisticats. El contingut de la tesi s'ha estructurat en diferents capítols que es detallen breument a continuació: ¿El capítol 1 és una introducció a la nanomedicina, destacant el paper fonamental que tenen les nanopartícules en el desenvolupament de noves aplicacions biomèdiques. A continuació es presenten les nanopartícules de sílice mesoporosa, mostrant la gran versatilitat d'aquests nanomaterials per al desenvolupament de dispositius teranòstics així com sistemes per a l'alliberament controlat de fàrmacs. Finalment, es destaca la importància de fabricar nanodispositius amb unes propietats físic-químiques ben definides com a requisit indispensable per a la translació dels resultats experimentals al camp clínic. ¿El capítol 2 inclou els objectius principals de la tesi així com els objectius específics proposats per a cada capítol de la tesi. ¿El capítol 3 està dedicat a la síntesi i caracterització de nanopartícules superparamagnétiques d'òxid de ferro (USPIONs), sent aquestes utilitzades en capítols posteriors per a la síntesi de les nanopartícules mesoporoses tipus "core-shell". Les USPIONs són preparades a través d'un mètode senzill de coprecipitació en el qual s'empren condicions de reacció moderades. Les nanopartícules obtingudes són caracteritzades en profunditat, analitzant les seues propietats magnètiques per a la seua aplicació en hipertèrmia magnètica i com a agents de contrast dual en imatge per ressonància magnètica (MRI). ¿El capítol 4 està dedicat a la preparació de nanopartícules magnètiques mesoporoses de sílice amb estructura "core-shell". Els conceptes fonamentals relacionats amb els mecanismes de formació d'aquest tipus de nanomaterials són àmpliament analitzats, així com els paràmetres de reacció involucrats en la síntesi. Com a punt de partida, es proposa un protocol de síntesi general per a l'obtenció de les nanopartícules tipus "core-shell". A continuació, s'analitza en profunditat l'efecte que els diferents paràmetres de reacció tenen en les propietats físic-químiques d'aquestes nanopartícules. Per a la fase d'optimització s'utilitza un model semi-empíric com a referència, racionalitzant els resultats experimentals observats sobre la base d'un possible mecanisme de formació. ¿El capítol 5 està dedicat a l'anàlisi i caracterització de l'estructura mesoporosa de les nanopartícules tipus "core-shell". A més, s'analitza l'efecte que els diferents paràmetres de reacció tenen sobre l'estructura final de les nanopartícules, aportant informació
[EN] The fabrication of nanoparticles with sizes below 100 nm has opened the door to the development of innovative nanodevices that directly interact with living systems at the cellular and molecular level, becoming an essential part of nanomedicine. One of the main challenges that nanoparticle engineering is currently facing is the design of nanodevices with well-defined physico-chemical properties, which ultimately determine the fate and function of these systems inside the organism. Similarly, the development of reproducible and versatile synthetic protocols is of great importance for manufacture purposes, a fundamental requirement for an efficient translation of this technology into the clinic. The main objective of this PhD thesis is the study and fabrication of core-shell-type magnetic mesoporous silica nanoparticles (M-MSNs) for their application as theranostic nanodevices in the field of nanomedicine. A comprehensive study about the synthesis and characterization of this type of nanomaterials is presented with the aim of obtaining core-shell M-MSNs with well-defined physico-chemical properties in a robust and reproducible way. The fabrication of such particles would provide a versatile and reliable platform for the development of more complex nanodevices with advanced functionalities. The thesis has been structured into several chapters that are briefly summarized as follows: ¿Chapter 1 is an introduction to the topic of nanomedicine, highlighting the importance of nanoparticles in the development of new biomedical applications. Mesoporous silica nanoparticles are then introduced, showing the great versatility that this nanomaterials offer for the development of theranostic nanodevices and smart drug delivery systems. Finally, the development of nanodevices with well-defined physico-chemical properties is identified as a crucial requirement for overcoming biological barriers and facilitate the translation of nanomedicines from the bench to bedside. ¿Chapter 2 presents the aims of this thesis and the specific objectives that are addressed in the following chapters. ¿Chapter 3 is devoted to the synthesis and characterization of ultrasmall superparamagnetic iron oxide nanoparticles (USPIONs), which are later used as magnetic seeds for the synthesis of core-shell M-MSNs. USPIONs are prepared through a simple coprecipitation method using mild reaction conditions. The obtained nanoparticles are fully characterized and their magnetic properties are analyzed focusing on magnetic hyperthermia and dual MR imaging applications. ¿Chapter 4 is a comprehensive study about the preparation of monodisperse core-shell M-MSNs. The main concepts related to the synthesis and formation mechanisms of this type of nanomaterials are revised, together with the reaction parameters that are expected to have a major contribution on the reaction. As a starting point, a general synthetic protocol for the synthesis of core-shell M-MSNs is presented. Then, specific reaction parameters are investigated in order to understand their effect on the physico-chemical properties of the obtained nanoparticles. The application of a semi-empirical model to the optimization stage is presented in an attempt to provide an adequate reference framework to understand the formation of this complex nanodevices. ¿Chapter 5 presents a detailed analysis about the characterization of mesoporous silica materials and, in particular, the assessment of the mesoporous structure of MSNs with a radial distribution of wormhole-like channels. The effects that specific reaction parameters have on the mesoporous silica structure of core-shell M-MSNs are also analysed, providing additional information about the formation of this type of nanoparticles. ¿Chapter 6 gathers the main conclusions of this thesis.
Sánchez Cabezas, S. (2019). Development of a reproducible and optimized synthetic protocol for the preparation of monodisperse core-shell-type magnetic mesoporous silica nanoparticles [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/129878
TESIS

Protein-protein interactions (PPI) mediate numerous biological processes, but inhibiting these interactions with small molecules has been difficult to achieve in drug discovery. A small number of successes have shown that some PPIs are amenable to inhibition. Computational algorithms designed to measure the druggability of PPIs have been developed based on these successes. These algorithms have identified the interaction between the NF-κB essential modulator (NEMO) and I𝜅B kinase β (IKKβ) as a candidate for inhibition. Furthermore, in vivo peptide-based inhibition of the NEMO-IKKβ interface has shown benefits in attenuating the NF-𝜅B response in cellular and animal models. In addition to its intrinsic interest as a drug target, developing inhibitors against the NEMO/IKKβ interaction may help in the development of improved methods for PPI inhibition. In this thesis, the production of full-length, recombinant forms of soluble NEMO is described. This protein was used in a variety of biochemical assays to advance our understanding of NEMO structure and function. Furthermore, a fluorescence anisotropy (FA) assay was developed to screen for compounds inhibiting the NEMO/IKKβ PPI. Hits from the FA assay were tested by several methods to confirm true inhibition. Additionally, the FA assay was used to accurately measure the affinity of NEMO for IKKβ and to assess the degree of cooperativity in IKKβ binding. The oligomeric state of NEMO has been characterized through the development of a panel of NEMO cysteine to alanine mutants, using polyacrylamide gel electrophoresis analysis, analytical ultracentrifugation, and fluorescence anisotropy. These data represent the first comprehensive characterization of full-length human NEMO, and may provide a path toward development of drug-like inhibitors of the NEMO/IKKβ interaction.

博士
國立臺灣大學
電機工程學研究所
100
Time synchronization protocols play an important role in numerous applications, such as instrumentation, power systems and high-speed communications. With the rapid advances in technology, the demands placed on the accuracy of time synchronization have increased. The precision time synchronization protocol (PTP) was the first protocol developed to successfully synchronize distributed devices in the sub-microsecond (10-6 seconds) range. This dissertation firstly introduces the background knowledge regarding time synchronization protocols as well as the measurement of time and frequency. Then the ideas of PTP are investigated. New applications of PTP are proposed then. For example, PTP can be used as the case of the time measurement system to replace the Time Interval Counter (TIC). The benefits of PTP based time frequency measurement include low cost and high efficiency. The Gray Proportional Integral (GPI) controller and the Threshold Proportional Integral (TPI) controller are proposed to reduce the transient time interval during the time synchronization processes. Since PTP can provide time synchronization precision in the sub-microsecond (10-6 seconds) level, the time stamps are used as the encryption parameters. The pass words are revised every second. Such hind of Time Varying Encryption System (TVES) is quite safe since it is difficult to be cracked.

Thesis (Ph. D.)--University of Georgia, 2002.
Directed by Ron Orlando. Includes articles submitted to Rapid communications in mass spectrometry, and Biochemistry. Includes bibliographical references.

Thesis (M.S.)--University of Wisconsin--Madison, 2000.
Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 114-116).

This thesis investigates the use of density centrifugation with Percoll to separate and enrich the organisms involved in the first step of toluene degradation within a methanogenic toluene degrading consortium. Protocol development resulted in the enrichment of bacteria and archaea in separate layers. However the separation of Eub-1 (an organism suspected to be responsible for the first step in toluene degradation), and bssA (a gene encoding the benzylsuccinate synthase enzyme) using previously developed qPCR primers could not be established. Cloning and sequencing of the toluene degrading consortia were conducted and phylogenetic analysis showed a change in community composition from what had previously been observed, suggesting why previously established primers were not effective. In parallel with these studies, microcosms using soil obtained from a petroleum hydrocarbon-contaminated area in North Carolina were constructed. These microcosms showed benzene degradation in all but one sample over the 444 day period.

MSc (Biochemistry)
Department of Biochemistry
Malaria is caused by protozoa of the genus Plasmodium. Malaria accounts for approximately more than half a million deaths yearly. Of the five species of Plasmodium, P. falciparum accounts for the most deadly form of the disease. P. falciparum survives under various physiological conditions during its life cycle. The parasite employs its molecular chaperones machinery particularly heat shock proteins (Hsps) to protect its protein constituents during physiological stress. Hsps are conserved molecules that constitute a major part of the cell’s molecular chaperone system. P. falciparum Hsps play an important cyto-protective role guaranteeing that the malarial parasite survives under the severe conditions that prevail in the host environment. PFF1010c is a type IV P. falciparum heat shock protein 40. PFF1010c is predicted to be expressed only at the gametocyte stage of the malarial parasite’s life cycle. The aim of the current study was to investigate the expression PFF1010c by parasites and the gametocyte stage as well as characterize the structure-function features of the protein. PFF1010c was successfully expressed in E. coli cells. Despite successful expression of the protein, its purification proved problematic. The attempt to purify PFF1010c was carried out under both native and denaturing conditions. Far Western blot analysis to investigate direct interaction between PFF1010c and PfHsp70-1 was conducted and no interaction was observed. Malarial parasites were harvested at different stages and total protein was isolated. The expression of PFF1010c was confirmed to occur at the gametocyte stage of the parasite’s development using Western blot analysis. This study confirmed that PFF1010c is only expressed at the gametocyte stage of the malarial parasite. Furthermore, PFF1010c was not expressed at the asexual stage. Possible interactors of PFF1010c were predicted by STRING, a bioinformatics based tool. The expression of PfHsp90, PfHop and PfHsp70-1 at the gametocyte stage was investigated and confirmed by Western blot analyses.

Dissertação de Mestrado em Biologia Celular e Molecular apresentada à Faculdade de Ciências e Tecnologia da Universidade de Coimbra.
Multiple myeloma (MM) is a plasma cell malignancy characterized by uncontrolled production of monoclonal immunoglobulins within the bone marrow. Despite the recent major treatment advances, obtained with the introduction of new therapeutic agents such as bortezomib and lenalidomide, drug resistance is a major obstacle to therapeutic success. Therefore, it is urgent to identify biomarkers of MM drug resistance. Extracellular vesicles (EVs) (including microvesicles and exosomes) are released by various cell types, contributing to intercellular communication. Tumor cells such as MM cells also release EVs, whose cargo may be transferred into recipient cells. Importantly, the presence of markers in the circulating EVs shed by tumor drug resistant cells, including particular microRNAs such as miR-21, may allow the detection of biomarkers of drug resistance. Therefore, this study aimed at selecting a method and optimizing a protocol for the isolation of EVs with different sizes (possibly exosomes and microvesicles) from plasma samples of MM patients. Thus, a comparison of methods was carried out, including ultracentrifugation, a commercial kit (ExoQuick) and qEV size exclusion chromatography (SEC) columns. The isolated EVs were characterized in terms of size and purity, by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. In addition, the analysis of EV markers such as Hsp70, CD63 and CD9 was also carried out (by Western Blot), together with the detection of possible cellular contaminants and protein contaminants from the plasma. Finally, the expression levels of miR-21 and miR-16 was attempted (by qRT-PCR). All the three methods allowed the isolation of EVs, but with distinct sizes. The ultracentrifugation allowed the isolation of EVs together with contaminants (possibly lipoproteins). In addition, this protocol had the disadvantage of requiring an expensive equipment (ultracentrifuge) and taking several hours. Moreover, the amount of total protein recovered from the ultracentrifugation protocol was very low. The ExoQuick kit allowed higher yields of total protein to be recovered from the isolated EVs and it was a much quicker protocol than the ultracentrifugation one; however, it only isolated the smaller vesicles (possibly exosomes). The qEV SEC columns were the preferred approach, since they enabled a rapid isolation of EVs with various sizes (possiblyexosomes and microvesicles). In addition, by pooling the various fractions collected, it is possible to have enough protein to be analysed by Western blot. Finally, preliminary results indicate that RNAse treatment is necessary when analysing miRs from EV samples. In addition, when pre-treating samples with RNase, the three protocols seem to allow detecting similar levels of miRs. However, these preliminary results need to be confirmed before conclusions can be taken.
O mieloma múltiplo é uma neoplasia maligna caracterizada pela produção descontrolada de imunoglobulinas monoclonais na medula óssea. Apesar dos recentes avanços nos tratamentos, obtidos sobretudo com a introdução de novos agentes terapêuticos como o bortezomib e a lenalidomida, a resistência aos fármacos continua a ser um dos maiores obstáculos para o sucesso terapêutico. Assim, é urgente identificar biomarcadores de resistência em mieloma múltiplo. Vesículas extracelulares (incluindo microvesículas e exossomas) são libertadas por vários tipos celulares, contribuindo para a comunicação intercelular. Células tumorais de mieloma múltiplo também libertam essas vesículas extracelulares, que transportam no seu interior componentes que podem ser transferidos parar outras células. A presença de marcadores nas vesículas extracelulares circulantes no sangue, libertadas por células tumorais resistentes a fármacos, nomeadamente microRNAs como o miR-21, podem permitir a detecção de biomarcadores de resistência. Assim, este projecto teve o objectivo de selecionar um método e optimizar um protocolo de extração de vesículas extracelulares com diferentes tamanhos (possivelmente exossomas e microvesículas) existentes no plasma de doentes com mieloma múltiplo. Para isso, foi realizada uma comparação entre métodos, incluindo a ultracentrifugação, um kit comercial (ExoQuick) e colunas de cromatografia de exclusão por tamanho (qEV). As vesículas extracelulares isoladas foram caracterizadas em termos de perfil de tamanho e pureza, respectivamente, por “Dynamic light scattering” (DLS) e por microscopia electrónica de transmissão (TEM). Além disso, a presença de marcadores como o Hsp70, CD63 e CD9 foi também analisada (por Western blot), bem como a presença de possíveis contaminantes celulares e proteicos com origem no plasma. Por fim, a expressão de miR-21 e miR-16 foi igualmente realizada (por qRT-PCR). Os três métodos permitiram a extração de vesículas extracelulares, mas com diferentes tamanhos. A ultracentrifugação permitiu isolar vesículas extracelulares mas contendo contaminantes (possivelmente lipoproteínas). Este protocolo tem também as desvantagens de requerer equipamento dispendioso (ultracentrífuga) e de ser muito moroso. Além disso, a quantidade total de proteína conseguida com este método foi muito reduzida. Por sua vez, ExoQuick permitiu precipitar grandes quantidades de proteína total a partir de amostras de vesículas extracelulares, tendo sido ainda um protocolo muito maisrápido em comparação com a ultracentrífugação; contudo, apenas permitiu isolar vesículas de pequenos tamanhos (possivelmente exossomas). As colunas de cromatografia qEV mostraram ser o protocolo preferido, uma vez que permitiram o rápido isolamento de vesículas extracelulares com diferentes tamanhos (possivelmente exossomas e microvesículas). Além disso, juntando as várias frações recolhidas das colunas qEV, é possível obter quantidade de proteína suficiente para ser analisada por Western blot. Por fim, resultados preliminares indicaram que o tratamento com RNase é necessário para a análise de miRs a partir de amostras de vesículas extracelulares. Além disso, com o pré-tratamento das amostras com RNase, os três protocolos parecem permitir detectar quantidades semelhantes de miRs. Contudo, estes resultados preliminares precisam ser confirmados para que se possam retirar conclusões.
cancro, mieloma múltiplo, resistência aos fármacos, vesículas extracelulares, miRs