Practical Management of the Endangered African Wild Dog (Lycaon Pictus): Implementation of Research Methodologies, Management Tools and Validation of Non-Invasive Endocrinology Applications

The porpoise of this study was to implement research methodologies and assess the effectiveness and impact of management tools to promote best practices for the long term conservation of the endangered African wild dog (Lycaon pictus). Different methods were included in the project framework to investigate and expand the applicability of these methodologies to free-ranging African wild dogs in the southern African region: ethology, behavioural endocrinology and ecology field methodologies were tested and implemented. Additionally, research was performed to test the effectiveness and implication of a contraceptive implant (Suprenolin) as a management tool for the species of a subpopulation hosted in fenced areas. Attention was especially given to social structure and survival of treated packs. This research provides useful tools and advances the applicability of these methods for field studies, standardizing and improving research instruments in the field of conservation biology and behavioural endocrinology. Results reported here provide effective methodologies to expand the applicability of non-invasive endocrine assessment to previously prohibited fields, and validation of sampling methods for faecal hormone analysis. The final aim was to fill a knowledge gap on behaviours of the species and provide a common ground for future researchers to apply non-invasive methods to this species research and to test the effectiveness of the contraception on a managed metapopulation.

Non invasive assessment of right ventricle in patients with operated tetralogy of Fallot

The objective of this study was to evaluate right ventricular function in patients with right ventricular volume overload in patients with (tetralogy of Fallot, and pulmonary atresia + VSD ) underwent corrective surgery; with echocardiography measure that can be easily applied; and to study the relationship between ProBNP and the contractile function of the right ventricle, dilated right atrium, and the consequences of pulmonary insufficiency . Methods: The study included 50 patients (50% males, mean age 30.64 ± 13.30 years) with prior cardiac surgical intervention of TDF (90%) or pulmonary atresia + VSD (10%). (49 pz) have performed a cardiac MRI and clinical evaluation, (47 pz) echocardiogram, (48 pz) ECG, (34 pz) a cardiopulmonary exercise testing, (29 pz) a dosage of ProBNP. Results: The S-wave velocity (p <0.0001), the TAPSE (p <0.0001) correlated significantly with RVEF estimated by cardiac MRI. The VO2 max was 27.93 ± 12.91 ml / kg / min, 15% of patients had VE/VCO2 The peak> 35. ProBNP correlated positively and significantly with the area of the right atrium (p = 0.0001), and negative and significant with VO2 max (p = 0.04). Those who have increased pulmonary insufficiency (PVR fraction> 30%) have a significantly increased RVED volume (p = 0.01), reduced VO2 max (p = 0.04), and lower ejection fraction of LV (p = 0.02) than the group of patients with PVR ≤ 30. Conclusion: The TAPSE and S-wave velocity are fundamental and may become the technique of choice for routine assessment of RV systolic function in adult patients with TOF. The monitoring of the Pro BNP is probably a choice, given the simplicity and their information that correlate with the test cardiopulmonary. In view of the ventricular-ventricular interaction, so measures to maintain or restore the functioning of the pulmonary valve could preserve biventricular function.

Non Invasive Tools for Early Detection of Autism Spectrum Disorders

Autism Spectrum Disorders (ASDs) describe a set of neurodevelopmental disorders. ASD represents a significant public health problem. Currently, ASDs are not diagnosed before the 2nd year of life but an early identification of ASDs would be crucial as interventions are much more effective than specific therapies starting in later childhood. To this aim, cheap an contact-less automatic approaches recently aroused great clinical interest. Among them, the cry and the movements of the newborn, both involving the central nervous system, are proposed as possible indicators of neurological disorders. This PhD work is a first step towards solving this challenging problem. An integrated system is presented enabling the recording of audio (crying) and video (movements) data of the newborn, their automatic analysis with innovative techniques for the extraction of clinically relevant parameters and their classification with data mining techniques. New robust algorithms were developed for the selection of the voiced parts of the cry signal, the estimation of acoustic parameters based on the wavelet transform and the analysis of the infant’s general movements (GMs) through a new body model for segmentation and 2D reconstruction. In addition to a thorough literature review this thesis presents the state of the art on these topics that shows that no studies exist concerning normative ranges for newborn infant cry in the first 6 months of life nor the correlation between cry and movements. Through the new automatic methods a population of control infants (“low-risk”, LR) was compared to a group of “high-risk” (HR) infants, i.e. siblings of children already diagnosed with ASD. A subset of LR infants clinically diagnosed as newborns with Typical Development (TD) and one affected by ASD were compared. The results show that the selected acoustic parameters allow good differentiation between the two groups. This result provides new perspectives both diagnostic and therapeutic.

Induction of Hebbian associative plasticity through paired non-invasive brain stimulation of premotor-motor areas to elucidate the network's functional role

The ventral premotor cortex (PMv) is believed to play a pivotal role in a multitude of visuomotor behaviors, such as sensory-guided goal-directed visuomotor transformations, arbitrary visuomotor mapping, and hyper-learnt visuomotor associations underlying automatic imitative tendencies. All these functions are likely carried out through the copious projections connecting PMv to the primary motor cortex (M1). Yet, causal evidence investigating the functional relevance of the PMv-M1 network remains elusive and scarce. In the studies reported in this thesis we addressed this issue using a transcranial magnetic stimulation (TMS) protocol called cortico-cortical paired associative stimulation (ccPAS), which relies on multisite stimulation to induce Hebbian spike-timing dependent plasticity (STDP) by repeatedly stimulating the pathway connecting two target areas to manipulate their connectivity. Firstly, we show that ccPAS protocols informed by both short- and long-latency PMv-M1 interactions effectively modulate connectivity between the two nodes. Then, by pre-activating the network to apply ccPAS in a state-dependent manner, we were able to selectively target specific functional visuo-motor pathways, demonstrating the relevance of PMv-M1 connectivity to arbitrary visuomotor mapping. Subsequently, we addressed the PMv-to-M1 role in automatic imitation, and demonstrated that its connectivity manipulation has a corresponding impact on automatic imitative tendencies. Finally, by combining dual-coil TMS connectivity assessments and ccPAS in young and elderly individuals, we traced effective connectivity of premotor-motor networks and tested their plasticity and relevance to manual dexterity and force in healthy ageing. Our findings provide unprecedent causal evidence of the functional role of the PMv-to-M1 network in young and elderly individuals. The studies presented in this thesis suggest that ccPAS can effectively modulate the strength of connectivity between targeted areas, and coherently manipulate a networks’ behavioral output. Results open new research prospects into the causal role of cortico-cortical connectivity, and provide necessary information to the development of clinical interventions based on connectivity manipulation.

Plasma-activated liquids as promising tools for non-invasive selective treatment of epithelial ovarian cancer

Plasma medicine is a branch of plasma-promising biomedical applications that uses cold atmospheric plasma (CAP) as a therapeutic agent in treating a wide range of medical conditions including cancer. Epithelial ovarian cancer (EOC) is a highly malignant and aggressive form of ovarian cancer, and most patients are diagnosed at advanced stages which significantly reduces the chances of successful treatment. Treatment resistance is also common, highlighting the need for novel therapies to be developed to treat EOC. Research in Plasma Medicine has revealed that plasma has unique properties suitable for biomedical applications and medical therapies, including responses to hormetic stimuli. However, the exact mechanisms by which CAP works at the molecular level are not yet fully understood. In this regard, the main goal of this thesis is to identify a possible adjuvant therapy for cancer, which could exert a cytotoxic effect, without damaging the surrounding healthy cells. An examination of different plasma-activated liquids (PALs) revealed their potential as effective tools for significantly inhibiting the growth of EOC. The dose-response profile between PALs and their targeted cytotoxic effects on EOC cells without affecting healthy cells was established. Additionally, it was validated that PALs exert distinct effects on different subtypes of EOC, possibly linked to the cells' metabolism. This suggests the potential for developing new, personalized anticancer strategies. Furthermore, it was observed that CAP treatment can alter the chemistry of a biomolecule present in PAL, impacting its cytotoxic activity. The effectiveness of the treatment was also preliminarily evaluated in 3D cultures, opening the door for further investigation of a possible correlation between the tumor microenvironment and PALs' resistance. These findings shed light on the intricate interplay between CAP and the liquid substrate and cell behaviour, providing valuable insights for the development of a novel and promising CAP-based cancer treatment for clinical application.

Non-invasive detection of acute cell-mediated graft rejection in paediatric heart transplant recipients: the role of cardiovascular magnetic resonance

Background and Aim: Acute cardiac rejection is currently diagnosed by endomyocardial biopsy (EMB), but multiparametric cardiac magnetic resonance (CMR) may be a non-invasive alternative by its capacity for myocardial structure and function characterization. Our primary aim was to determine the utility of multiparametric CMR in identifying acute graft rejection in paediatric heart transplant recipients. The second aim was to compare textural features of parametric maps in cases of rejection versus those without rejection. Methods: Fifteen patients were prospectively enrolled for contrast-enhanced CMR followed by EMB and right heart catheterization. Images were acquired on a 1,5 Tesla scanner including T1 mapping (modified Look-Locker inversion recovery sequence – MOLLI) and T2 mapping (modified GraSE sequence). The extracellular volume (ECV) was calculated using pre- and post-gadolinium T1 times of blood and myocardium and the patient’s hematocrit. Markers of graft dysfunction including hemodynamic measurements from echocardiography, catheterization and CMR were collated. Patients were divided into two groups based on degree of rejection at EMB: no rejection with no change in treatment (Group A) and acute rejection requiring new therapy (Group B). Statistical analysis included student’t t test and Pearson correlation. Results: Acute rejection was diagnosed in five patients. Mean T1 values were significantly associated with acute rejection. A monotonic, increasing trend was noted in both mean and peak T1 values, with increasing degree of rejection. ECV was significantly higher in Group B. There was no difference in T2 signal between two groups. Conclusion: Multiparametric CMR serves as a noninvasive screening tool during surveillance encounters and may be used to identify those patients that may be at higher risk of rejection and therefore require further evaluation. Future and multicenter studies are necessary to confirm these results and explore whether multiparametric CMR can decrease the number of surveillance EMBs in paediatric heart transplant recipients.

Study of variability and genetic structure of European populations of Myotis emarginatus and Myotis capaccinii (Chiroptera, Vespertilionidae)

The Geoffroy’s bat Myotis emarginatus is mainly present in southern, south-eastern and central Europe (Červerný, 1999) and is often recorded from northern Spain (Quetglas, 2002; Flaquer et al., 2004). It has demonstrated the species’ preference for forest. Myotis capaccinii, confined to the Mediterranean (Guille´n, 1999), is classified as ‘vulnerable’ on a global scale (Hutson, Mickleburgh & Racey, 2001). In general, the species preferred calm waters bordered by well-developed riparian vegetation and large (> 5 m) inter-bank distances (Biscardi et al. 2007). In this study we present the first results about population genetic structure of these two species of genus Myotis. We used two methods of sampling: invasive and non-invasive techniques. A total of 323 invasive samples and a total of 107 non-invasive samples were collected and analyzed. For Myotis emarginatus we have individuated for the first time a set of 7 microsatellites, which can work on this species, started from a set developed on Myotis myotis (Castella et al. 2000). We developed also a method for analysis of non-invasive samples, that given a good percentage of positive analyzed samples. The results have highlighted for the species Myotis emarginatus the presence on the European territory of two big groups, discovered by using the microsatellites tracers. On this species, 33 haplotypes of Dloop have been identified, some of them are presented only in some colonies. We identified respectively 33 haplotypes of Dloop and 10 of cytB for Myotis emarginatus and 25 of dloop and 15 of cytB for Myotis capaccinii. Myotis emarginatus’ results, both microsatellites and mtDNA, show that there is a strong genetic flow between different colonies across Europe. The results achieved on Myotis capaccinii are very interesting, in this case either for the microsatellites or the mitochondrial DNA sequences, and it has been highlighted a big difference between different colonies.

Hormonal parameters in foal hair: from birth to post weaning

The aim of this study was to investigate cortisol and progesterone (P4) trends in hair from birth up to postweaning in Italian trotter foals. Hair sampling is non-invasive and hair concentrations provide retrospective information of integrated hormone secretion over periods of several months. Samples were collected at birth and at a distance of 30 days, collecting only regrowth hair, up to post weaning. From birth to 3 months, foals cortisol falls from 47.64±5.6 to 4.9±0.68 pg/mg (mean±standard error), due to the interruption of foetal-placental connection and progressive adaptation to extrauterine life. From the third month of life to post weaning concentrations don’t vary significantly, underlining a non-chronic activation of the HPA axis. Hair P4 significantly decreases in the first two samples (from 469.68±72,54 to 184.65±35.42 pg/mg). At 2 (111.78±37.13 pg/mg) and 3 months (35.96±6.33 pg/mg) hair concentrations don’t show significant differences. These concentrations are not due to interactions of the utero-placental tissues with foals, animals are still prepuberal and P4 isn’t produced by adrenals as a result of high stress. We could therefore hypothesize that the source of foal hair P4 could be milk, suckled from mares. The high individual variability in hair at 2 and 3 months is due to a gradual and subjective change in foal diet, from milk to solid food, and to the fact that mares do not allow to suckle. From fourth month to post weaning P4 concentration in hair remains around 37.56±6.45 pg/mg. In conclusion, hair collected at birth, giving information about last period of gestation, could be used along with traditional matrices, to evaluate foals maturity. Hair cortisol could give indications about foals capacity to adapt to extra-uterine life. Finally milk, configuring as a bringer of nutrients and energy and assuming the characteristic of a nutraceutical, could give fundamental information about parental care.

Landscape ecology and genetics of the wolf in Italy

This PhD Thesis includes five main parts on diverse topics. The first two parts deal with the trophic ecology of wolves in Italy consequently to a recent increase of wild ungulates abundance. Data on wolf diet across time highlighted how wild ungulates are important food resource for wolves in Italy. Increasing wolf population, increasing numbers of wild ungulates and decreasing livestock consume are mitigating wolf-man conflicts in Italy in the near future. In the third part, non-invasive genetic sampling techniques were used to obtain genotypes and genders of about 400 wolves. Thus, wolf packs were genetically reconstructed using diverse population genetic and parentage software. Combining the results on pack structure and genetic relatedness with sampling locations, home ranges of wolf packs and dispersal patterns were identified. These results, particularly important for the conservation management of wolves in Italy, illustrated detailed information that can be retrieved from genetic identification of individuals. In the fourth part, wolf locations were combined with environmental information obtained as GIS-layers. Modern species distribution models (niche models) were applied to infer potential wolf distribution and predation risk. From the resulting distribution maps, information pastures with the highest risk of depredation were derived. This is particularly relevant as it allows identifying those areas under danger of carnivore attack on livestock. Finally, in the fifth part, habitat suitability models were combined with landscape genetic analysis. On one side landscape genetic analyses on the Italian wolves provided new information on the dynamics and connectivity of the population and, on the other side, a profound analysis of the effects that habitat suitability methods had on the parameterization of landscape genetic analyses was carried out to contributed significantly to landscape genetic theory.

Valutazione non invasiva dell'epatopatia Fontan indotta

Introduzione: L’intervento di Fontan comporta un aumento istantaneo della pressione venosa centrale che causa, nel medio-lungo termine, una forma di epatopatia specifica detta FALD. Il monitoraggio della FALD è complesso ma potrebbe consentire di bloccarne o rallentarne l’insorgenza. Lo studio ha valutato l’efficacia delle modalità di monitoraggio non invasivo. Materiale e metodi: Sei pazienti (età media 24 anni) operati presso l’IRCCS Azienda Ospedaliero Universitaria di Bologna sono stati sottoposti a RMN 4D-Flow e ad Ecodoppler epatico. Sono stati raccolti i dati anagrafici, morfologici, anamnestici e i markers sierologici per il calcolo degli scores MELD-XI, APRI, FIB4, i valori di Shear Stress assiale e circonferenziale e gli indici di pulsatilità e resistenza delle arterie epatica e renale. Risultati: Il tempo trascorso tra la Fontan e lo studio è stato di 17,8 anni. Età media alla Fontan 6,8 anni. Tutti i pazienti avevano un quadro compatibile con epatopatia. I markers sierologici e gli scores MELD-XI,APRI e FIB4 si sono dimostrati di scarsa utilità. All’ecografia tutti i pazienti avevano ecostruttura irregolare, splenomegalia e valori elevati di pulsatilità e resistenza dell’arteria epatica e splenica. La rigidità epatica media è stata di 12,4 Kpa. Alla RMN 4DF lo Shear stress assiale è stato massimo a livello del condotto (0,16 Pa) e minimo a livello delle vene sovra epatiche (0,05 Pa). Lo Shear Stress si è mostrato massimo nei pazienti con emodinamica sfavorevole e peggior quadro ecografico addominale, evidenziando aree di inefficienza energetica. Conclusioni: La combinazione delle diagnostiche di imaging non invasive potrebbe rivelarsi adeguata per il monitoraggio della FALD. In particolare, la RMN 4D Flow potrebbe rivelare aree di inefficienza energetica predisponenti alla FALD. Questo potrebbe indirizzare in modo specifico la terapia dei pazienti operati o addirittura indurre la modifica del disegno della Fontan verso forme più efficienti.

Ruolo dell'elastometria pulsata monodimensionale ad ultrasuoni (fibroscan) nella stadiazione della fibrosi epatica in pazienti sottoposti a trapianto ortotopico di fegato con recidiva di epatite C

PREMESSA: La progressione della recidiva d’epatite C è accelerata nei pazienti sottoposti a trapianto di fegato e ciò ha portato alla necessità di sviluppare nuove e validate metodiche non invasive per la quantificazione e la misura della fibrosi epatica. SCOPI: Stabilire l’efficacia dell’elastometria epatica (Fibroscan®) e dei parametri sierici di fibrosi, attualmente disponibili nella pratica clinica, per predire il grado di fibrosi nei pazienti sottoposti a trapianto epatico. METODI: La correlazione fra fibrosi epatica, determinata mediante biopsia epatica ed esame istologico, e Fibroscan® o indici clinico-sierologici di fibrosi (Benlloch, Apri, Forns, Fibrotest and Doppler resistance index), è stata studiata in pazienti che avevano ricevuto un trapianto ortotopico di fegato con evidenza di recidiva d’epatite da HCV. Un totale di 36 pazienti, con la seguente classificazione istologica: fibrosi secondom METAVIR F1=24, F2=8, F3=3, F4=1, sono stati arruolati nella popolazione oggetto di studio. Un totale di 29 individui volontari sani sono serviti come controllo. Le differenze fra gli stadi di fibrosi sono state calcolate mediante analisi statistica non parametrica. Il miglior cut-off per la differenziazione di fibrosi significativa (F2-F4) è stato identificato mediante l’analisi delle curve ROC. RISULTATI: La rigidità epatica ha presentato valori di 4.4 KPa (2.7-6.9) nei controlli (mediane e ranges), con valori in tutti i soggeti <7.0 KPa; 7.75 KPa (4.2-28.0) negli F1; 16.95 KPa (10.2-31.6) negli F2; 21.10 KPa nell’unico paziente F4 cirrotico. Le differenze sono state statisticamente significative per i soggetti controllo versus F1 e F2 (p<0.0001) e per F1 versus F2 (p<0.0001). Un cut-off elastografico di 11.2 KPagarantisce 88% di Sensibilità, 90% di Specificità, 79% di PPV e 95% di NPV nel differenziare i soggetti F1 dagli F2-F4. Le AUROC, relativamente alla capacità di discriminare fra i differenti gradi di fibrosi, evidenziavano un netto vantaggio per il Fibroscan® rispetto ad ognuno degli indici non invasivi di fibrosi. CONCLUSIONI: L’elastometria epatica presenta una buona accuratezza diagnostica nell’identificare pazienti con fibrosi epatica di grado significativo, superiore a quella di tutti gli altri test non invasivi al momento disponibili nella clinica, nei pazienti portatori di trapianto epatico ortotopico da cadavere con recidiva di HCV.