The Wingless (Wnt) signaling pathway is one of the critical developmental pathways for control of cell differentiation, proliferation, and cell growth. The DEP domain, located on the C-terminus of Dishevelled (Dvl), plays a role in cytoplasm-membrane association, which branches the canonical and non-canonical Wnt signaling pathway within the cell. It has been suggested that the DEP domain requires the recruitment of ionic lipids, such as phosphatidic acid, to regulate its localization to the plasma membrane and association to the frizzle receptor. However, the physical mechanism for DEP association to the plasma membrane is still unknown. We show that mDvl2-DEP interacts with phosphatidic acid at a distinct patch on the surface formed by a positively charged surface area by NMR spectroscopy. The binding of this interaction was also found at physiologically relevant concentration using fluorescence spectroscopy. We also determined that the interaction is pH-dependent and regulated through a 'histidine switch' mechanism at His464 and His465 where there is increased association of mDvl2-DEP to the plasma membrane at higher pH values (7.5). This association is based on tertiary structure conformational changes with rearrangement of the loop regions by a change in local pH, not its interaction with phosphatidic acid. Overall, our work will contribute to elucidate how cells regulate their developmental pathways through localized molecular interactions. / Master of Science

Survival of Listeria monocytogenes in apple, orange, red grape, and white grape juice was evaluated. A six-strain cocktail of L. monocytogenes was used to inoculate (approx. 7 log cfu/ml) fruit juices, which were stored at 4, 10 and 24°C for up to 61 days. Inoculated red grape juice was stored for up to 5 hours only. Samples were withdrawn at appropriate intervals, neutralized with 1.0 N NaOH, serially diluted in 0.1% peptone water, and surface plated onto Tryptic Soy Agar + 0.6% Yeast Extract (TSAYE) and Modified Oxford Agar (MOX), followed by incubation at 32°C for 48 hours. When L. monocytogenes was no longer detected by direct plating, samples were enriched for L. monocytogenes using Listeria Enrichment Broth (LEB), followed by isolation on MOX. L. monocytogenes remained viable in white grape, apple, and orange juices for up to 12, 24 and 61 days, respectively. Over time, recovery of Listeria on TSAYE versus MOX was not significantly different (P>0.05), indicating that limited acid-injury developed during storage. The results of this study demonstrate the ability of L. monocytogenes to survive in apple, orange, and white grape juices during refrigerated and abusive storage conditions. Therefore, measures to prevent or eliminate L. monocytogenes in the fruit juice-processing environment are necessary to ensure the safety of juice products for public consumption. / Master of Science

Yes / Interpolymer complex formation of poly(acrylic acid) with other macromolecules can occur via several mechanisms that vary depending on the pH. At low pH the protonated acid functional group can form bonds with both donor and acceptor moieties, resulting in desolvated structures consisting of two polymers. Complexes were formed in dilute solutions of PAA, functionalised with acenaphthylene, with a range of other polymers including: poly(NIPAM); poly(ethylene oxide) (PEO); poly(dimethylacrylamide) (PDMA); poly(diethyl acrylamide) (PDEAM) poly(vinyl alcohol) (PVA) and poly(vinyl pyrolidinone) (PVP). Fluorescence anisotropy was used to demonstrate complex formation in each case by monitoring the reductions in segmental motion of the chain as the complexes formed. Considerations of the molecular structures of the complexing moieties suggest that solvation energies and pKas play an important role in complex formation.

Thesis advisor: Babak Momeni / Thesis advisor: Charles Hoffman / Aflatoxins (AFs) are secondary fungal metabolites that contaminate common food crops and are harmful to humans and animals. The ability to remove AFs from feed commodities will improve health standards and counter the economic drain inflicted by AF contamination. Strategies to mitigate AF contamination fall into three categories: physical, chemical, and biological. In this thesis, I explore the identification of degraders and degradation mechanisms, as well as their enhancement, within the context of chemical and biological strategies. Known chemical strategies have used strong acids and bases to remove contaminating AF, but these methods often lead to ecological waste issues downstream. Chapter 3 investigates the application of weaker acidic and alkaline conditions to remove two types of AFs, AFB1 and AFG2. I find that a weakly alkaline environment is sufficient to degrade AF, providing an alternative solution for chemical decontamination. Biodetoxification is a promising solution to AF contamination because of its low cost and few undesired environmental side-effects. Microbes possess a rich potential for removing toxins and pollutants from the environment. Despite the fairly wide availability of this potential, identifying suitable candidates and improving them remain challenging. In Chapter 2, I explore the use of computational tools to discover strains and enzymes that detoxify harmful toxins. Of focus is the detoxification of mycotoxins by biological enzymes. Existing computational tools can be used to address questions in the discovery of new detoxification potential, the investigation the cellular processes that contribute to detoxification, and the improvement of detoxification potential in discovered enzymes. I showcase open bioremediation questions where computational researchers can contribute and highlight relevant existing and emerging computational tools that could benefit bioremediation researchers. In Chapter 4, I screen several environmental isolates for their AF detoxification ability, using AFG2. I used different carbon sources (glucose and starch) as isolation and culturing media to examine the effect of the environment on degradation ability. Overall, I find that starch medium expedites the screening process and generally improves the performance of isolates, making this a promising method for identifying new degraders and enhancing their performance. Chapter 5 highlights the characterization of degradation by two promising Rhodococcus species, R. erythropolis and R. pyridinivorans. While previous work has identified their degradation ability, further investigation into degradation mechanisms has been understudied. Here, I explore the characterization of degradation mechanisms toward enzyme identification. Finally, the appendix starts to broach the question of enhancing degradation of known degrading enzymes, the example here is laccase from the fungus Trametes versicolor. Using molecular dynamic and quantum mechanics simulations to identify mutations of interest in increasing the affinity of laccase toward AF, I create five mutants to test their degradation against the performance of wildtype. These mutants show a range of improvements against AF and showcase the efficacy of this approach to enhancement. Together, this body of work highlights the importance of understanding AF degradation for the creation of new strategies of AF mitigation. My thesis provides a framework for developing AF decontamination strategies, from identifying degraders and unlocking their mechanisms to enhancing their performance. / Thesis (PhD) — Boston College, 2024. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Le cytosquelette des cellules est une structure composite et versatile qui leur confère des propriétés mécaniques extrêmement complexes. En particulier, le cortex d'actine qui s'assemble de manière dynamique sous la membrane cellulaire fournit la force nécessaire aux déformations et au mouvement de la cellule : la polymérisation de l'actine permet aux filaments en formation de pousser la membrane et les moteurs moléculaires génèrent des forces contractiles. L'utilisation de systèmes biomimétiques permet d'isoler des modules particuliers du cytosquelette pour les étudier indépendamment de façon simplifiée. Une expérience de reconstitution du cortex d'actine in vitro a été mise au point dans ce but. Les protéines et métabolites nécessaires pour la polymérisation de l'actine sont ainsi introduits dans un liposome et la réaction est localisée à la membrane, en y greffant l'activateur de la polymérisation de l'actine, sur le modèle du cortex cellulaire. Une fois la polymérisation déclenchée, nous sommes arrivés à reproduire un gel d'actine à la membrane, formant une coque. Les propriétés mécaniques de ce système simplifié sont alors étudiées par des expériences qui caractérisent leur dynamique d'étalement sur des surfaces. Les résultats sont comparés à ceux obtenus sur des cellules, et reproduisent une bonne partie des comportements cellulaires. On utilise également ces liposomes dans une situation physiologique: l'internalisation de la toxine de Shiga dans les cellules et nous montrons que la toxine est internalisée dans un système aussi épuré que des liposomes comportant un cortex reconstitué, prouvant le rôle important de l'actine dans ce processus.

In this work we studied protein adsorption on chemically well-controlled surfaces. The focus is put on linking physico-chemical properties of surfaces (hydrophobicity/charge) to the structural properties of the adsorbed proteins. To this end, alkyl thiols differing by their end group were used to build self-assembled monolayers on gold substrates (SAM) that serve as templates for protein adsorption or covalent grafting. SAM surfaces before and after protein adsorption were characterized with a combination of techniques. Ex situ analysis were carried out, in air with polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS), or in vacuum using X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). ToF-SIMS results were analyzed statistically in principal component analysis (PCA) to reveal preferential orientations based on amino acids fragments distributions. Protein adsorption was also followed directly in situ (i.e. in the liquid phase) with quartz crystal microbalance with dissipation monitoring (QCM-D). Two model proteins - β-Lactoglobulin (βLG) and bovine serum albumin (BSA) - were first studied. They are both model globular proteins with different structural properties (βLG is hard while BSA is soft). Different orientations were proposed for both proteins on each SAM surface. A more complex case was then studied with the adsorption and grafting of a monoclonal antibody on the SAM. Again differences in orientations were determined and correlated to biorecognition measurements. In conclusion, this thesis establishes a methodology for the direct label free determination of protein orientation on surfaces.

Ce travail s'est intéressé à la conception et à l'élaboration de nouveaux agents structurants de silice constitués d'assemblages induits et réversibles de copolymères originaux : les copolymères séquencés doublement hydrophiles (DHBC). Un des systèmes étudiés est constitué d'un DHBC neutre-anionique PAPEO-b-PAA ou poly(acrylate methoxy poly(oxyde d'éthylène))-b-poly(acide acrylique). La séquence PAA est un polyacide faible dont le degré d'ionisation dépend du pH. En solution aqueuse et pour un pH bien choisi, l'association de ce copolymère à une polybase faible, de charge opposée à celle du PAA, (typiquement un oligochitosane) conduit à la formation de micelles complexes de polyions (PIC) sphériques de type cur/couronne. Ces micelles peuvent dans un premier temps conduire à la formation de matériaux mésostructurés hybrides hautement organisés. Dans un second temps, en jouant notamment sur les conditions de pH et de force ionique, il est possible de « contrôler » le taux d'extraction des espèces organiques pour obtenir des matériaux poreux fonctionnels capables de piéger des espèces de charge opposée à la fonctionnalité. Si des matériaux hybrides organisés sont obtenus, c'est parce que les équilibres des interactions mises en jeu entre les espèces organiques et inorganiques y sont favorables. Si une interaction polyamine/silice s'exerce aux dépens de l'interaction polyamine/DHBC, elle peut limiter le processus de mésostructuration par les micelles. Lorsqu'un DHBC neutre-cationique PEO-b-PDMAEMA ou poly(oxyde d'éthylène)-b-poly(méthacrylate de 2-(diméthylamine)éthyle) est utilisé en présence d'un polymère anionique tel que le PVS ou poly(acide sulfonique de vinyle), il joue un double rôle dans la synthèse des matériaux siliciques : une partie gère la croissance des particules de silice en interagissant avec les silicates et l'autre partie qui est complexée par des PVS joue le rôle d'agent structurant en apportant une mésostructuration au matériau. Enfin, une approche très prometteuse a permis d'encapsuler des principes actifs hydrosolubles chargés dans un matériau en les utilisant comme agent complexant du DHBC. / This study focused on the design and development of new structuring agents of silica constituted of induced and reversible assemblies of original copolymers, the double hydrophilic block copolymers (DHBC). The first system studied consists of a neutral-anionic DHBC PAPEO-b-PAA ou poly(acrylate methoxy poly (ethylene oxide))-b-poly (acrylic acid). The PAA block is a weak polyacid with a degree of ionization depending on the pH. In aqueous solution and in a right pH range, the association of this copolymer with a weak polybase, an oppositely charged polyamine, such as an oligochitosan, leads to the formation of polyion complex micelles (PIC) with a core/corona structure. These micelles can direct the structure of highly organized inorganic materials with different types of mesostructures. In a second step, by adjusting the conditions of pH, ionic strength, it is possible to "control" the extraction of organic species to get functional porous materials able to trap species of charge opposite to the functionality. Organized materials are obtained because of a favourable balance of the interactions between organic and inorganic species. If a polyamine/silica interaction occurs at the expense of the interaction polyamine/DHBC, the mesostructuring process by the micelles is limited. A neutral-cationic DHBC PEO-b-PDMAEMA poly(ethylene oxide)-b-poly(2-(dimethylamine)ethyl) associated with an anionic PVS poly(vinyl sulfonic acid) polymer can play a dual role in the synthesis of silica materials: firstly managing the growth of silica particles by interacting with the silicates and secondly acting as a structuring agent in association with PVS, confering a mesostructuration to the material. Finally, a very promising approach allowed to encapsulate water-soluble and charged drugs in a material by using as silica complexing agent a complex between the drug and a DHBC.

Forest fires can have a great impact on the relationship between soil organic matter (SOM) and soilbulk density (SBD). SOM will reduce with increased fire intensity, which ultimately leads to more compaction of the soil. The compaction rate might increase to the limit of where root growth will be absent thus leading to actions to restore the soil. This study investigates changes in the relationship between SOM and SBD in spodosol and histosol in Seglingsberg, located in South-central Sweden, where a forest fire occurred summer 2014.  In addition, changes of pH values in the two types of soilwere examined in order to receive information about the chemical states of the different soil types.A total of 29 samples at depths of 0-17 cm were received from one day of fieldwork and these were later analysed concerning the pH, the SBD and the SOM content. The results showed an increase of pH-values in the fire-exposed area compared to pH values measured at the reference site (pH ~5). More importantly, the results from the SBD and SOM analyses indicated that there was in fact an inversely proportional relationship between the two soil parameters. In addition, high pH values were measured at the same subareas of which the highest SBD- and the lowest SOM values were obtained.Statistical analyses were applied on the results in order to conclude if the soil property changes caused by the fire were significantly different from normal conditions or not. The results from the statistical analyses revealed that 25% of the fire-exposed sites had changed significantly. However, more samples should have been taken while in field, since lack of data is thought to have had a great impact on the final results.Although there were no strong statistical evidence for the hypothesis, it is clear that the forest firein Västmanland year 2014 affected both the SOM, SBD and pH values in the soils. / Under sommaren år 2014 utbröt en omfattande skogsbrand i Västmanlands län, vilket medföljdedramatiska konsekvenser för framförallt ett flertal skogsbolag, men även för boende i området. Med skogsbränder följer negativa såväl som positiva konsekvenser, där de positiva framförallt gäller med avseende på arter som har evolverat i samband med bränder. Förutom ovannämnda konsekvenser så finns det risk för att markförhållandena ändras till följd av en skogsbrand. I denna studie undersöktes hur  markegenskaper  i  torv-  samt  podsoljordar  hade  förändrats  med  avseende  på  pH,  halten  avorganiskt material samt packningsgrad. Fältstudien genomfördes i ett drabbat brandområde strax norrom Seglingsberg, Surahammars kommun. Totalt togs 29 stycken jordprover inom fem stycken transekter i området. Av dessa kunde 25 stycken användas till alla tre analyserna. Resultaten från jordprovsanalyserna användes därefter till att genomföra statistiska undersökningar. Detta för att se hur stor spridningen var mellan och inom de fem transekterna samt för att kunna avgöra om jordproverna visade sig vara signifikant förändrade från ursprungsförhållanden.Resultaten från jordprovsanalyserna visade att det fanns mest organiskt material kvar i de östra delarna av området, medan det var kraftigt reducerat ju längre nordväst jordproverna hade hämtats. I samband med att markens organiska material hade reducerats kunde man även bevittna att jordtäcket hade blivit mer kompakterat. Resultaten från pH-analysen pekar också på att förändringarna varit som störst i de nordvästliga delarna. pH-analysen bevisade att markkemin ser annorlunda ut än innanbranden, då värdena ibland låg 2 enheter för högt än vad man vanligen brukar observera i podsol- ochtorvjordar. Då pH-skalan är logaritmisk innebär detta en minskad försurning med 100 gånger. Resultaten från alla jordprovsanalyser tyder att branden varit som mest intensiv i den nordvästra delen av undersökningsområdet, i området bestående av ungskog.Även om resultaten från jordprovsanalyserna pekade på att branden orsakat tydliga mark- förändringar, visade majoriteten av de statistiska undersökningarna inte på signifikanta förändringar. Det är därför inte möjligt att generalisera resultaten och således applicera dessa på hela brandområdet i Västmanland.Eftersom att naturligt förekommande skogsbränder är relativt få till antalet i Sverige, finns därmed få studier tillgängliga inom ämnesområdet. Det finns en upplaga av studier inom kontrollerade och anlagda brandfält, men i och med att dessa förhållanden är fixerade, så påverkas markegenskaperna sällan avsevärt. Forskningsrapporter indikerar på ett mer extremt klimat i framtiden, som förmodas leda till en ökad omfattning samt ett ökat antal naturligt förekommande skogsbränder. Om denna prognos stämmer är det viktigt att undersöka markförhållandena, då en skogsbrand kan ha direktavgörande effekt på återväxten.

L'objectif de cette étude a été de préciser l'implication des processus rhizosphériques dans lesinteractions pour l'acquisition de phosphore (P) entre une céréale et une légumineuse enassociation. Nous avons proposé comme mécanisme de facilitation l'acidification de la rhizosphèredes espèces en association induite par la fixation de N2 de la légumineuse dans le cas de sol neutres àalcalins. Ainsi, l'étude s'est focalisée sur l'influence des changements de pH induits par les racines.Les effets de la disponibilité initiale en P du sol ainsi que de la distance entre les racines des espècesassociées ont également été testés. Les expérimentations ont été menées au champ ainsi qu'enconditions contrôlées. Le blé dur et différentes légumineuses ont été cultivés en culturemonospécifique ou en association sur un même sol pour toutes les expérimentations. Le sol neutreutilisé (Luvisol) présentait trois niveaux contrastés de fertilisation phosphatée et provenait desparcelles d'un essai de longue durée. Nous avons pu démontrer que la manipulation des interactionsrhizosphériques entre une céréale et une légumineuse en association pouvait être optimisée pourpermettre l'augmentation de la disponibilité en P dans la rhizosphère des espèces associées, etencore plus dans le cas de sols présentant une faible valeur initiale de disponibilité. Lescaractéristiques du sol ont eu un rôle clé dans la détermination des processus rhizosphériquesimpliqués. Dans notre cas, les changements de pH induits par les racines ont permis d'augmenter defaçon substantielle la disponibilité en P suite à une acidification, mais aussi à une alcalinisation de larhizosphère. Ainsi la légumineuse, mais aussi la céréale sont susceptibles de faciliter l'acquisition del'espèce associée. Les interactions relatives au pH peuvent influencer la disponibilité en P sur unedistance de plusieurs millimètres et ainsi améliorer l'acquisition de P des plantes à proximité del'espèce facilitatrice.Mots clés : culture associée, facilitation, rhizosphère, disponibilité, phosphore, pH, racine / The aim of the study was to elucidate the implication of rhizospheric processes on plant speciesinteractions for phosphorus (P) acquisition between a cereal and a legume, when intercropped. Weproposed that root-induced acidification of the rhizosphere by the intercropped legume due to N2-fixation as a mechanism of facilitation in neutral to alkaline soils. Thus, the study focused on rootinducedchanges of pH. The effects of initial soil P availability and distance between roots ofintercropped species were also tested. In order to achieve our goals, experiments in the field and incontrolled condition for several cropping devices were conducted. Durum wheat and differentlegumes were grown as sole crops and intercropped on the same soil for all the experiments. Weused a neutral soil (Luvisol) presenting three contrasted fertilization rates from the field of a longtermP fertilizer trial. We demonstrated that cereal-legume can be used to enhance P availability, andeven more so in low P soils, through managing rhizospheric interactions to optimize P acquisition ofintercropped species. Soil characteristics are also a key factor determining the influence of thoserhizospheric processes. In our studied soil root-induced changes of pH substantially enhanced Pavailability in the rhizosphere either through acidification or alkalization. Thus the legume but alsothe cereal may facilitate the acquisition of the intercropped species. Interactions involving pH canaffect P availability over distance of several millimeters and ultimately enhance P acquisition ofplants in the surrounding of the facilitative species.Key words: intercrop, facilitation, rhizosphere, availability, phosphorus, pH, root

Intracellular pH affects nearly all biochemical processes in yeast, the processes regulating the cytosolic pH includes function of many transport proteins. In this work, the impact of selected sodium transporters on cytosolic pH has been studied in two yeast species: Saccharomyces cerevisiae and Zygosaccharomyces rouxii including wild-type and mutants with affected sodium transport. Measurements of cytosolic pH and buffering capacity have been performed using fluorescent protein probe pHluorin - a pH sensitive derivate of green fluorescence protein. Several procedures for calibration of pHluorin fluorescence response have been compared and the importance of a proper correction of the calibration curve has been demonstrated. It has been shown that cytosolic pH is influenced by the function of Nha1 transport protein in S. cerevisiae as well as in Z. rouxii but not by Sod2-22 transporter in Z. rouxii. It has been demonstrated that the buffering capacity of cytosol decrease in the presence of glucose in all strains studied.