Mammals usually develop immunity to gastrointestinal nematode parasites. However, during late pregnancy and lactation, this immunity often breaks down, resulting in elevated levels of parasitism. This periparturient relaxation of immunity (PPRI) renders lactating hosts main sources of infection for their parasite-naïve offspring, and may have a nutritional basis. Results of studies on parasitized hosts suggest that both crude protein (CP) and metabolizable energy (ME) supply may be important in regulating the degree of PPRI by affecting the immune response towards parasites. However there is a scarcity of data supporting such a role for ME in periparturient hosts, while there is sufficient evidence to support the view that CP in general, and amino acids are potent immunonutrients in various disease states (Chapter 1). In the first experiment (Chapter 2) I seperated effects of CP and ME on PPRI by feeding parasitized lactating rats at two levels of ME supply and one of three levels of CP supply. The results show that PPRI is sensitive to CP scarcity, and not to moderate ME scarcity. Increasing CP supply improved lactational performance and reduced PPRI, as observed by reduced worm burdens. In the second experiment (Chapter 3) I examined the rate at which improved nutrition can restore immunity by feeding low protein diets to rats nursing high number of pups, and then reduced litter size in a sub-group so that host nutritional status would change from scarce to adequate. The egg production of the parasite population of the latter group reduced within days to similarly low levels as rats that had always reared low number of pups and this was associated with an increased number of musosal mast cells and increased dam weight gain. Since host responses to dietary CP are almost by ii definition responses to essential amino acids, the third experiment assessed the sensitivity of PPRI to methionine and leucine deficiency (Chapter 4). The latter resulted in increased worm burdens and egg production to similar levels when low protein diets are fed and imposed penalties in lactational performance. Finally, in the fourth experiment it was investigated whether similar outcomes can be expected in periparturient ruminant hosts by supplementing sheep with field beans, which are deficient in methionine, instead of soybean meal (Chapter 5). Indeed, feeding high protein diets based on field beans was less effective in reducing the worm egg excretion and improving lactational performance. The data from this thesis (Chapter 6) provide novel information on the nutritional basis of PPRI, showing that the latter can be rapidly reduced through improved protein nutrition. This may be seen as a response to the protein quality of the diet and the supply of amino acids in optimum quantities. These results have implications for parasite control strategies in farm animals.

Thesis advisor: Abhishek Chatterjee / Abstract Unnatural amino acid (UAA) incorporation is a powerful tool used by biochemists to discover the nature of protein structure and function. The evolution of orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs enables site-specific incorporation of UAAs proteins inside of living cells. The goal of this study was to further expand the repertoire of genetically encoded unnatural amino acids in E. coli as well as eukaryotes. We first attempted to engineer an aaRS, previously evolved for p-borono-phenylalanine (pBoF), to specifically charge 3-acetyl-p-borono-phenylalanine (AcpBoF). A randomized library of the pBoF-specific synthetases was generated and it was subjected to established selection schemes in a bacterial host. This report also describes the development of a yeast-based selection system to alter the substrate specificity of bacterial leucyl-tRNA synthetase, for genetic code expansion in eukaryotes. / Thesis (MS) — Boston College, 2017. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Wheat (Triticum aestivum L.) grown in controlled environments with a 24-hour photoperiod was analyzed for phenylpropanoid and amino acid metabolites. Discrete spectral environments, including a metal halide, high-pressure sodium and low-pressure sodium lamps, provided both photosynthetically active radiation and phenylpropanoid inducing fluences of light . A greenhouse spectral environment supplemented with fluorescent lamps was also used to culture wheat . All. four spectral envi ronments were used to culture wheat to maturity separately. The activities of phenylalanine and tyrosine ammonia-lyase were photoinduced in wheat tissue obtained from plants grown in the metal halide, high -pressure sodium and greenhouse spectral environments. These enzyme activities are the committed catalytic step in phenylpropanoid biosynthesis and were induced in wheat tissue by fluences of light in the ultraviolet and blue regions of the spectrum. The low-pressure sodium lamp , which does not provide strong irradiance in these wavelengths , produced significantly lower ammonia-lyase activities than were observed in wheat grown within the metal halide , high-pressure sodium, or greenhouse spectral environments. These effects were not caused by phytochrome, since calculation of PfrfPtotal for the low-pressure sodium lamp was higher than the ratio obtained from metal halide or high-pressure sodium lamps. Lignin was also significantly reduced in wheat grown with low-pressure sodium lamps . Several essentialamino acids were in lower molar concentration in protein from wheat grown under low-pressure sodium lamps. However, phenylalanine and tyrosine were in significantly higher concentration in wheat grain produced in this spectral environment, and amino acid concentrations of wheat cultured with the low-pressure sodium lamp are regarded as a direct result of the spectral properties of this light source.

The study of cancer plays an important role in modern medical science. Over the years, a lot has been learnt about the properties and treatment of cancer cells. Despite the remarkable progress made in understanding the genesis of cancer, the work so far has had very little impact in the clinic especially the design of new and improved drugs. Platinum-based drugs such as cis-diamminedichloroplatinum(II) and its anolgue, carboplatin, are the most effective chemotherapeutic agents used in the treatment of testicular, ovarian, bladder and lung cancers. Nevertheless, the emergence of toxic side-effects compromises its clinical effectiveness. It is generally agreed that most of the toxic effects of platinum-based drugs arise from their lack of selectivity. This thesis reports on the development of new platinum(II) complexes bound to carrier molecules with the hope of obtaining compounds which display the cytotoxic effects only in tumour tissue. In addition, some information is included about what is known about the causes of cancer, how it kills and the current methods of treatment / Doctor of Philosophy (PhD)

The whole genome duplication (WGD) event is an intriguing mechanism from an evolutionary perspective. Such an event may be the source of new genes, functions or species. Traces of WGD event have been detected in the genome of all four eukaryotic kingdoms: plants, animals, fungi and protists. In fungi, an ancestor of Saccharomyces cerevisiae underwent an event of WGD, about 100 million years ago, after diverging from the Kluyveromyces lineage. In S. cerevisiae, only ten percent of the resulting duplicated genes survived as duplicates. In particular, some of these duplicates encodes for transcription factors in several nutrient sensing pathways. The main subject of this thesis’s work is the external amino acid sensing system in S. cerevisiae. The detection of extracellular amino acids in yeast begins with a transporter homologue devoid of any uptake activity, the Ssy1 sensor. The binding of extracellular amino acids to Ssy1 leads to the successive activation of Ptr3 and the Ssy5 endoprotease. This endoporotease catalyses the processing of two transcriptions factors: Stp1 and Stp2. The Stp factors, released from their N-terminal cytoplasmic-anchored domains, are then translocated into the nucleus, where they activate the transcription of several amino acid permease genes (e.g. AGP1 and DIP5). Starting this work, the Stp factors were considered as functionally redundant. We first determined that the STP1 and STP2 genes derivate from the event of WGD. The conservation of these two genes in S. cerevisiae was accompanied by a functional divergence of their products at several levels: processing sensibility, transcriptional activation capacity, target genes, cellular abundance level and stability. The Stp2 factor with its high abundance in the cells and its higher Ssy5-processing sensibility is specialized towards induction of the AGP1 gene when the external amino acid signaling is weakly stimulated. Under strong stimulation conditions, the amino acids induce cleavage-triggered destabilization of Stp2 through the proteasomal pathway and the induction of AGP1 is mediated mainly by the Stp1 transcription factor. Unlike Stp2, the Stp1 factor is characterized by its high transcriptional activation capacity and weaker sensitivity towards Ssy5-processing. The Stp factors differ also by their genetic targets. Indeed, only Stp2 regulates the expression of DIP5. Finally, we determined that the processing sensibility and the transcriptional activation capacity of each Stp factors is directly linked to their N- and C-terminal domains, respectively. The phosphorylation states and the degradation of the Stp2 factor were also examined. The event of degradation concerns only the processed forms of this factor and takes places principally in the nucleus. Some data indicate that such an event might be important to limit the activation capacity of this factor. The role of the Stp2 phosphorylation in the external amino acid signaling pathway is still unknown but this event might be important for the Stp2 degradation or its transcriptional activity. The unique Stp factor from Kluyveromyces lactis (Kl-Stp), a pre-WGD species, was also studied. The Kl-Stp factor shares at least two characteristic with the S. cerevisiae Stp2 factor: high sensibility towards processing and high levels of degradation. This observation leads us to conclude to that the STP genes may have been conserved after WGD though a mechanism called neofunctionalization (one of the duplicate obtained after duplication retains the ancestral function while the other evolves to perform a novel function). Finally, a new model for the external amino acid signaling pathway that brings together all the data obtained during this thesis’s work is proposed.

Hydrogen bonding interactions have been applied to the synthesis of chiral vicinal diamines and the generation of helical chirality. A stereospecific synthesis of vicinal diamines was developed by using the diaza-Cope rearrangement reaction driven by resonance-assisted hydrogen bonds (RAHBs). This process for making a wide variety of chiral diamines requires only a single starting chiral diamine, 1,2-bis(2-hydroxyphenyl)-1,2-diaminoethane (HPEN) and aldehydes. Experimental and computational studies reveal that this process provides one of the simplest and most versatile approaches to preparing chiral vicinal diamines including not only C2 symmetric diaryl and dialkyl diamines but also unsymmetrical alkyl-aryl and aryl-aryl diamines with excellent yields and enantiopurities. Weak forces affecting kinetics and thermodynamics of the diaza-Cope rearrangement were systematically studied by combining experimental and computational approaches. These forces include hydrogen bonding effects, electronic effects, steric effects, and oxyanion effects. As an example of tuning diamine catalysts, a vicinal diamine-catalyzed synthesis of warfarin is described. Detailed mechanistic studies lead to a new mechanism involving diimine intermediates. Decreasing the NCCN dihedral angle by varying the diamine structure results in an increase of the enantioselectivity up to 92% ee. Hydrogen bonds have been used to generate helical chirality in a highly stereospecific manner with a single amino acid and 2,2′-dihydroxybenzophenone. DFT computational and experimental data including circular dichroism (CD), X-ray crystallography and 1H NMR data provide insight into the origin of the stereospecificity. A signalling dizao group can be attached to the receptor for general sensing of amino acid enantiopurity.

The shikimate pathway is a primary metabolic pathway involved in the synthesis of aromatic compounds in plants, fungi, apicomplexan parasites and microbes. The absence of this pathway in animals makes it ideal for the synthesis of antimicrobial compounds and herbicides. Additionally, its branching into indole hormone synthesis and phenylpropanoid secondary metabolism makes this pathway attractive for metabolic engineering. Here, the focus is on the first step of the shikimate pathway catalyzed by DAHP synthase. This step consists of the condensation of phosphoenol pyruvate and erythrose-4-phosphate to make DAHP, which undergoes another six catalytic steps to synthesize chorismate, the precursor of the aromatic amino acids. Arabidopsis thaliana contains three DAHP synthase isozymes, which are known to indirectly regulate downstream pathways in response to wounding and pathogen stress. The model presented here proposes that DAHP synthase isozymes are regulated by the end products tyrosine, tryptophan and phenylalanine.

The shikimate pathway is a primary metabolic pathway involved in the synthesis of aromatic compounds in plants, fungi, apicomplexan parasites and microbes. The absence of this pathway in animals makes it ideal for the synthesis of antimicrobial compounds and herbicides. Additionally, its branching into indole hormone synthesis and phenylpropanoid secondary metabolism makes this pathway attractive for metabolic engineering. Here, the focus is on the first step of the shikimate pathway catalyzed by DAHP synthase. This step consists of the condensation of phosphoenol pyruvate and erythrose-4-phosphate to make DAHP, which undergoes another six catalytic steps to synthesize chorismate, the precursor of the aromatic amino acids. Arabidopsis thaliana contains three DAHP synthase isozymes, which are known to indirectly regulate downstream pathways in response to wounding and pathogen stress. The model presented here proposes that DAHP synthase isozymes are regulated by the end products tyrosine, tryptophan and phenylalanine.

Hydrogen bonding interactions have been applied to the synthesis of chiral vicinal diamines and the generation of helical chirality. A stereospecific synthesis of vicinal diamines was developed by using the diaza-Cope rearrangement reaction driven by resonance-assisted hydrogen bonds (RAHBs). This process for making a wide variety of chiral diamines requires only a single starting chiral diamine, 1,2-bis(2-hydroxyphenyl)-1,2-diaminoethane (HPEN) and aldehydes. Experimental and computational studies reveal that this process provides one of the simplest and most versatile approaches to preparing chiral vicinal diamines including not only C2 symmetric diaryl and dialkyl diamines but also unsymmetrical alkyl-aryl and aryl-aryl diamines with excellent yields and enantiopurities. Weak forces affecting kinetics and thermodynamics of the diaza-Cope rearrangement were systematically studied by combining experimental and computational approaches. These forces include hydrogen bonding effects, electronic effects, steric effects, and oxyanion effects. As an example of tuning diamine catalysts, a vicinal diamine-catalyzed synthesis of warfarin is described. Detailed mechanistic studies lead to a new mechanism involving diimine intermediates. Decreasing the NCCN dihedral angle by varying the diamine structure results in an increase of the enantioselectivity up to 92% ee. Hydrogen bonds have been used to generate helical chirality in a highly stereospecific manner with a single amino acid and 2,2′-dihydroxybenzophenone. DFT computational and experimental data including circular dichroism (CD), X-ray crystallography and 1H NMR data provide insight into the origin of the stereospecificity. A signalling dizao group can be attached to the receptor for general sensing of amino acid enantiopurity.

Previous studies have indicated the level of total sulfur amino acids (TSAA) (methionine + cystine) is most limiting in practical diet formulations for hybrid striped bass (HSB), especially if animal feedstuffs are replaced with plant feedstuffs. Reduction of costly animal feedstuffs such as fish meal while maintaining adequate dietary levels of TSAA may enhance cost effectiveness of production. Therefore, this study, consisting of four separate feeding trials, investigated three different aspects of sulfur amino acid nutrition of HSB including: (1) the efficacy of crystalline methionine hydroxy analog (MHA) and liquid MHA (AlimetTM) relative to L-methionine in meeting the requirement for TSAA; (2) the cystine sparing value for methionine; and, (3) the influence of various sulfur amino acid supplements on ammonia excretion. During the feeding trials, juvenile HSB were fed various diets including a basal diet deficient in TSAA (0.33 or 0.51% of diet), and experimental diets supplemented on an equalsulfur basis with different levels of either L-methionine, AlimetTM or crystalline MHA. Diets containing TSAA at 1% of diet and different ratios of cystine to methionine (60:40, 55:45, 50:50, and 45:55) also were fed to re-evaluate sparing effects of cystine on methionine. During the ammonia excretion trial, HSB were fed diets containing either L-methionine,AlimetTM or crystalline MHA after which total ammonia nitrogen (TAN) excretion was determined 4 h postprandial. In trial 1, AlimetTM was 73% as effective in promoting growth as L-methionine at the same concentration while MHA was 83% as effective. In trial 3, fish fed AlimetTM at 1.25% of diet displayed similar growth performance as those fed TSAA at 1.0% of diet while weight gain of fish fed AlimetTM at 1% was only 58% of that displayed by fish fed TSAA at 1.0%. No significant differences in weight gain, feed utilization or survival were observed among fish fed diets containing various ratios of cystine to methionine although the diet with 60:40 cystine to methionine supported the lowest responses. Inclusion of MHA or AlimetTM did not affect TAN excretion of HSB. These findings will aid in refining diet formulations for HSB to ensure adequate sulfur amino acid nutrition.