An investigation of the possible health-promoting modes of action of regular- and super- doses of phytase in the broiler chicken

The overall objective of this thesis was to study the effects of regular and high (super-) doses of phytase in the gut of broilers, with the aim of documenting the mechanism of their action leading to improvements in animal health. Phytase is often supplemented to commercial broiler diets to facilitate the hydrolysis of plant phytate and release of phosphorus for utilisation. Although not the original intention of its addition, phytase supplementation leads to improvements in growth performance parameters and enhanced nutrient utilisation. Further benefits have also been observed following the addition of super-doses of phytase which are not explained by an increase in phosphorus release, and thus have been termed ‘extra-phosphoric effects’. Using diets formulated to be adequate or marginally deficient in available phosphorus (aP; forming the negative control, NC), phytase was supplemented at 1,500 and 3,000 FTU/kg phytase in the first study (both super-doses) and the partitioning of nutrients within the body was investigated. It appeared that there were some metabolic changes between 1,500 and 3,000 FTU/kg, switching between protein and fat accretion, potentially as a consequence of nutrient availability, although these changes were not reflected by changes in growth performance parameters. However, the loss of the NC treatment without phytase on day 12 limits the comparison of the phytase within the NC treatment, but does allow for comparison of each dose at adequate or low dietary aP levels. As expected, a greater degree of phytate hydrolysis was achieved with 3,000 than with 1,500 FTU/kg phytase, but changes in carcass accretion characteristics were greater with 1,500 than 3,000 FTU/kg. Using these findings and the observation that there were no further changes in the parameters measured by increasing phytase from 1,500 to 3,000 FTU/kg (aside from phytate hydrolysis), 1,500 FTU/kg phytase was selected as the super-dose to be used in subsequent studies. The next study considered the influence of regular (500 FTU/kg) and super doses (1,500 FTU/kg) of phytase from within the gut. Overall, it was observed that changes were occurring to the gut environment, which ultimately would influence the absorptive capacity and conditions for further phytate hydrolysis. Dietary treatment influenced gut conditions such as pH, intestinal morphology and bacterial populations which can subsequently influence nutrient utilisation and potential for growth. The subsequent study was designed to investigate the effects within the gut in more detail. The release of nutrients from phytate hydrolysis and their bioavailability within the digesta can influence conditions within intestine, facilitating enhanced absorption. One of the parameters investigated was the expression of genes involved in the transport of nutrients in the intestine. Overall, there were few significant dietary treatment influences on gene expression in the intestine, however there was a dose-dependent response of phytase on the expression of the jejunual divalent mineral transporter. This indicates a change in divalent mineral bioavailability in the intestine, with correlations with inositol phosphate esters (IPs) being identified. This is likely explained by the IPs produced by phytase hydrolysis and accumulating in the digesta, differing between regular and high doses of phytase. It became apparent that interactions between the products of phytate hydrolysis (IP3, IP4) and minerals in the digesta had the potential to influence the gut environment and subsequent nutrient bioavailability and overall phytase action. The final study was designed to increase the content of the IPs, and investigate the influence of phytase under these conditions. As the complete hydrolysis of phytate to myo-inositol has been reported to be beneficial due to its proposed insulin mimetic effects, myo-inositol was also supplemented to one of the diets to see if any further benefits would be observed when supplemented alongside super-doses of phytase. Neither increased concentrations of the higher IP esters (IP6, IP5 and IP4) nor myo-inositol (myo-) had any effect on broiler growth performance, however there were still apparent beneficial influences of phytase supplementation. The results suggest considerable and important interactions between minerals and IP esters within the digesta, which ultimately have the potential to influence gut conditions and thus nutrient utilisation and growth performance. Reduced concentrations of blood glucose in the high IP ester diet with additional phytase supplementation suggest some insulin-like effects of myo- production. Additionally, the lack of effect of myo- supplementation on blood glucose and insulin concentrations suggests a difference between the structure of phytase-produced myo- and supplemented myo-. Although there were no improvements in growth performance by increasing phytase from 500 to 1,500 FTU/kg, there were changes occurring at the level of the gut and expression of genes in the intestine, influencing nutrient utilisation and the partitioning of nutrients within the body. There are many factors to be considered when supplementing phytase, with dietary nutrient content and nutrient release and IP production during phytate hydrolysis having an influence on phytase action, nutrient absorption and conditions within the gut. Super-doses of phytase may be beneficial for maintaining optimal gut conditions, clearing IP esters from the digesta, reducing their potential to form complexes with minerals and other nutrients, ultimately influencing the efficiency of production.

Heterogeneous Catalysts: Design, Applications and Research Insights

The cyclization of pseudoionone yields a mixture of alpha-ionone, beta-ionone and gamma-ionone. By careful control of reagent and reaction conditions, either the alpha- and beta- isomer can be favoured. The alpha-ionone has violet odour and is widely used in perfumery and flavours. beta-Ionone is the main precursor of Vitamin A and beta-carotene. Traditionally, strong homogeneous catalysts, like sulphuric acid and phosphoric acid have been used. These problems can be overcome by the use of solid acid catalysts. This work reports the cyclization of pseudoionone over USY zeolites, at 80ºC. USY It is observed that the initial activity increases with the Si/Al ratio of zeolite until a maximum, which is obtained with USY3. With higher Si/Al ratio, a decrease in the catalytic activity is observed. Selectivity to ionone isomers is around 42 %, at 75% of pseudoionone conversion, after 24 h of reaction. USY3 zeolite was reused four times with the same catalyst sample in the same condicions. It was observed a stabilization of the catalytic activity, after the second use.

Gold Catalyzed Functionalization Of Alkynes And Allenamides

The gold(I)-catalyzed chemoselective dearomatization of β-naphthols is reported through a straightforward approach via [3,3]-sigmatropic rearrangement /allene-cyclyzation cascade processes. Easily accessed naphthyl-propargyl ethers and derivatives in this work are employed as starting materials. Delightfully, an array of deoramatized dyhydrofuryl -naphthalen-2(1H)-ones featured densely functional groups are obtained in high yields (up to 98%) in 10 min reaction time under extremely mild reaction conditions like reagent grade solvent and exposure to air. The potential of accessing to high enantioselectivety on the dearomatized dyhydrofuryl- naphthalen-2(1H)-ones is also approved by the good ee (65%) relying on (R)-xylyl- BINAP(AuCl)2. In addition, complete theoretical elucidation of the reaction pathway is also proposed which addresses a rationale for essential motivation such as regio- and chemoselectivity. Moreover, an efficient gold catalyzed intermolecular dearomatization of substituted β-naphthols with allenamides is presented here. PPh3AuTFA (5 mol %) approves the efficient dearomatively allylation protocol under mild conditions and exhibits high tolerance on substrates scope (24 examples) in good to excellent yield accompanied with high regioselectivity and stereoselectivity. Moreover, the synergistic catalytic system also highlight the synergistic function between the [PPh3Au]+ (π-acid) and TFA− (Lewis base). At last, a new chiral BINOL phosphoric acid silver salt is successfully synthesized and used as the chiral counter anion, which strongly promotes the enantioselectivity (up to 92%). At last but not least, crucially, SmI2 induced enantioselective formal synthesis of strychnine, a complex alkaloid and a classical target used to benchmark new synthetic methods is developed. Enantioselective dearomatising radical cyclisation on to the indole unit and further ET will then give organosamarium that is quenched diastereoselectively by the ester to deliver Strychnine in 7 steps.

Comparative Analysis of Genus Bifidobacterium: Insight into its Host Adaptation

Bifidobacteria is amongst one of the health promoting bacteria. The role of this important probiotic genera can be elucidated by understanding its genome. Comparative analysis of the whole genus of these bacteria can reveal their adaptation to a diverse host range. This study comprises of four research projects. In the first study, a reference library for genus Bifidobacterium was prepared. The core genes in each genus were selected based on a newly proposed statistical definition of core genome. Comparative analysis of Bifidobacterium with another probiotic genus Lactobacillus revealed the metabolic characteristics of genus Bifidobacterium. The second study investigated the immunomodulatory role of a B. bifidum strain TMC3115. The analysis of TMC3115 provided insights into its extracellular structures which might have their role in host interaction and immunomodulation. The study highlighted the variability among these genomes just not on species level but also on strain level in terms of host interaction. The last two studies aim to inspect the relationship between bifidobacteria and its host diet. Bifidobacteria, are both host- and niche-specific. Such adaptation of bifidobacterial species is considered relevant to the intestinal microecosystem and hosts’ oligosaccharides. Many species should have co-evolved with their hosts, but the phylogeny of Bifidobacterium is dissimilar to that of host animals. The discrepancy could be linked to the niche-specific evolution due to hosts’ dietary carbohydrates. The distribution of carbohydrate-active enzymes, in particular glycoside hydrolases (GHs) that metabolize unique oligosaccharides was examined. When bifidobacterial species were classified by their distribution of GH genes, five groups arose according to their hosts’ feeding behaviour. The distribution of GH genes was only weakly associated with the phylogeny of the host animals or with genomic features such as genome size. Thus, the hosts’ dietary pattern is the key determinant of the distribution and evolution of GH genes.

Synthesis of enantiopure cyclopentanones by desymmetrization of allylic cyclobutanols

The interest in five-membered ring molecules derives from their important application in many different fields, such as pharmaceutical and agrochemical areas. A common strategy for their formation is four-membered ring expansion, which also allows to add molecular complexity and functional handles within one single operation starting from readily available starting materials. Organocatalysis can be exploited to promote the reaction and to obtain a good enantio- and diastereoselection. This technique involves the exclusive use of organic molecules as catalysts, without resorting to metals. The aim of this work is to obtain enantiopure cyclopentanones starting from achiral allylic cyclobutanols. The reaction consists in a ring expansion promoted by the addition of a halogen to the double bond of the substrate, with formation of a haliranium ion as intermediate, followed by a semipinacol rearrangement to afford the cyclopentanone. The reaction is catalysed by a chiral phosphoric acid that, besides accelerating the rate of the reaction, transmits a specific chirality thanks to its chiral structure, following the asymmetric catalysis principles. Starting from symmetric trans-allylic cyclobutanols, the whole reaction is a desymmetrization and leads to the formation of two new stereogenic centres: a mixture of diastereoisomers is obtained, each as couple of enantiomers; the ratio between the possible configurations is determined by the relative position that the chiral catalyst and the reagent occupy during the reaction. Since the reaction is already optimized, the original aim was to study the scope: first, the synthesis of a set of allylic cyclobutanols and their relative precursors, in order to have a wider range of substrates; then, the identification of the type of substrate that undergoes the expansion, with the study of enantio- and diastereoselectivity obtained in each case. Due to the Covid-19 emergency, most of the work was developed as a bibliographic study.

Considerations on indirect adhesive restorations at the material and tooth interfaces

Objective: Lithium-silicate (LiSi) ceramic is nowadays widely used in dentistry. However, for the longevity of LiSi indirect restorations, it is important to pretreat the material and the dental substrate adequately. However, is not certain how the simplification of the manufacturing and conditioning procedures influences the bonding performances of LiSi ceramic restorations. Accordingly, the aims of this thesis were to investigate the effect of: 1) different LiSi ceramic surface decontamination procedures on the shear bond strength (SBS) to resin composite; 2) different types of lithium-disilicate (LiDi) (pressed vs CAD-CAM) on SBS to resin composite; 3) an experimental metal salt-based zirconium oxynitrate etchant [ZrO(NO3)2] on bonding performances to dentin. Materials and Methods: SBS test was used to investigate the influence of different cleaning protocols applied, or different processing techniques (CAD or PRESS) on the bond strength to composite resin. The third study tackled the interface between restorative materials and dentin, and investigated the microtensile bond strength test (µTBS), nanoleakage expression analysis (NL), gelatin zymography and in situ zymography of dentin conditioned with an experimental metal salt-based zirconium oxynitrate etchant [ZrO(NO3)2]. Results: MEP showed comparable bond strength to the double HP etching and higher compared to other groups. BS of press LiSi to composite was higher than that of CAD/CAM LiSi. ZON pretreatment increased bond strength to dentin when used with a universal adhesive, and inhibited dentinal endogenous enzymes. Conclusions: While simplification of the LiSi conditioning and cleaning procedures seems to yield bond strength comparable to the traditional procedures, it could be recommended in the clinical practice. However, pressed LiSi still seems to perform better in terms of bond strength compared to the CAD/CAM LiSi. Further, the novel ZON etchant seems to perform better compared to the traditional phosphoric dentin etching.

New methodologies in organocatalysis: from discovery to application and mechanistic

This PhD thesis deals with three different topics: i) sulfoxonium ylides, ii) donor-acceptor cyclopropanes, and iii) desymmetrization reactions. Catalysis, and in more detail organocatalysis, is the fil rouge linking the three subjects of study. The main focus treated during this doctorate period is the reactivity of sulfoxonium ylides, and in particular stabilized sulfoxonium ylides. Special attention has been dedicated to the behavior of these particular substrates under asymmetric and non-asymmetric reaction conditions. Moreover, also similarities and differences with the related, less stable, sulfonium ylides were fully analyzed, both experimentally and from a theoretical point of view. Two different reactions were developed in full. One conducted under acidic reaction conditions and the second one exploiting the asymmetric aminocatalysis. Subsequently, the reactivity of donor-acceptor cyclopropanes was studied. After different attempts in the development of a new catalytic methodology based on these substrates, a non-conventional reactivity conducted under phase transfer catalysis was discovered and optimized. In particular, a chemodivergent reaction depending on the reaction conditions was developed. Finally, during the period spent abroad, a preliminary study of a desymmetrization reaction was carried out. The studied reaction is based on an asymmetric elimination reaction conducted under asymmetric phosphoric acid catalysis. In summary, this PhD thesis shows the versatility of different organocatalytic methodologies when applied to different reactions and substrates.