Fucosylated and Sulfated Glycans Investigated using Cryogenic Infrared Spectroscopy

Unusual monosaccharides (fucose), covalent modifications of glycans (sulfation) and terminal sequences play important biological roles in physiology and pathology of living organisms. Furthermore, in an evolutionary sense, uncommon structures are often the result of selection pressures and can be the source to a deeper understanding of the evolution of glycosylation.157 At the same time, fucosylated glycans and sulfated glycans still challenge standard mass spectrometry (MS)-based analytical workflows in glycan analysis. MS emerged throughout the last decade as the most widely used analytical technique in glycan analysis. As a stand-alone technique, it is limited in glycan analysis due to the presence of isomers. Isomerism in glycans arises from their composition, connectivity, configuration, and branching. Therefore, MS is often coupled to orthogonal techniques such as liquid chromatography (LC) and ion mobility spectrometry (IM-MS). Most recently, the combination of cryogenic IR spectroscopy in the gas phase with MS proved beneficial for the identification of smaller glycans. At low measurement temperatures, the IR spectrum of small glycans provides a unique fingerprint to the underlying chemical structure and conformation.In this thesis, cryogenic IR spectroscopy as an addition to the MS-based analytical toolbox was used to shed light on the migration of fucose residues in MS experiments. This elusive rearrangement reaction is not restricted to tandem MS workflows but is recently found to occur in intact ions without extensive activation. Here, the role of the proton in fucose migration reactions was investigated for the two glycan epitopes Lewis x and blood group H type 2. A systematic study of adduct ions and functional groups with competing proton affinities demonstrated that the proton can be selectively mobilized and demobilized. Planning MS-based experiments of fucosylated glycan cations certainly needs an effective strategy to circumvent the presence of a mobile proton in order to avoid erroneous sequence assignments.In a multidimensional approach, IR spectroscopy, IM-MS, RDD and computational modelling were combined to decode the rearrangement product and the reaction mechanism. The trisaccharides Lewis x and blood group H type 2 were found to migrate to a third chemical structure, in which the fucose moiety is most likely 1,6-linked to galactose. The barrier is much higher for blood group H type 2 compared to Lewis x and it is feasible that the latter is never detected in its original chemical structure in the mass spectrometer. These results generalize fucose migration to a universal issue in any mass spectrometer to which even various orthogonal MS-based techniques can be blind.In the second part of this thesis, cryogenic IR spectroscopy in combination with computational modelling was employed for the structural analysis of sulfated glycosaminoglycans (GAGs). Diversity in the chemical structure of linear and acidic GAGs arises from the GAG class, sulfation, epimerization and acetylation. Using messenger tagging IR spectroscopy, sulfated mono- and disaccharides have been characterized successfully recently. In the present thesis, the prominent anticoagulant pentasaccharide fondaparinux which carries eight sulfate functional groups was investigated using cryogenic IR spectroscopy in helium nanodroplets as a proof-of-concept. The spectroscopic fingerprint features unique absorption bands in the mid-IR range for the sulfate functional groups. With this knowledge, a systematic set of all naturally occurring sulfation variations in chondroitin and dermatan sulfate (CS/DS) further demonstrated the capabilities of cryogenic IR spectroscopy for their differentiation. Moreover, from their IR fingerprints in combination with computational modelling, conformational diversity arising from sulfation and charge density distribution could be derived. In a different study, the IR fingerprints of four heparan sulfate (HS) diastereomers revealed a modularity in their chemical structure which was explained, using computational modelling, from their unique hydrogen bonding patterns. The knowledge of the preferred hydrogen bonding pattern could aid e.g. the development for labelling strategies in IM-MS. The results show that the high resolution in the optical fingerprints of GAGs allows to unambiguously resolve their diversity arising from GAG class, sulfation and epimerization. The results exemplify the importance of gas- phase cryogenic IR spectroscopy to enhance future analytical workflows for GAG sequencing. A fully MS-based workflow could involve the ionization of an intact GAG chain and combine tandem MS with IM-MS and cryogenic IR spectroscopy of respective fragments to unambiguously characterize a GAG chain in a single MS experiment.In the last part, cryogenic IR spectroscopy was combined with random forest modelling to extract vibrational features that are characteristic to structural features in GAGs. The selected structural features included the GAG class and sulfation and therefore, almost fully characterize the underlying chemical structure. In a proof-of-concept study, a prediction score of >97% could be achieved for HS tetra- and hexasaccharides based on a training set of only 21 spectra. Especially for certain marker motifs, such as 3-O-sulfation in cancer cells, this workflow could prove beneficial. With machine learning algorithms, the need for comprehensive spectral databases could be circumvented for the identification of unknowns. Overall, the results show that MS-based IR spectroscopy certainly has the potential to leave the framework of academic basic research and add as a valuable addition to the MS-based analytical toolbox.Weinig voorkomende monosachariden (fucose), covalente modificaties van glycanen (sulfering) en terminale sequenties spelen belangrijke rollen in de fysiologie en pathologie van levende organismen. Weinig voorkomende structuren zijn in evolutionaire zin vaak het resultaat van selectiedruk en kunnen derhalve een dieper inzicht leveren in de evolutie van glycosylering. Gefucosyleerde glycanen en gesulfoneerde glycanen vormen echter nog steeds een uitdaging voor standaard workflows in glycaananalyse. Massaspectrometrie (MS) heeft zich in het laatste decennium ontwikkeld tot de meest gebruikte techniek voor glycaananalyse, maar is beperkt door de aanwezigheid van isomeren. Isomeren van glycanen zijn het gevolg van hun samenstelling, connectiviteit, configuratie en vertakking. MS wordt daarom vaak gekoppeld aan complementaire technieken zoals vloeistofchromatografie (LC) en ion- mobiliteitsspectrometrie (IM-MS). Gedurende de laatste jaren is de combinatie van cryogene infrarood (IR)-spectroscopie in de gasfase met MS van grote waarde gebleken voor de identificatie van kleinere glycanen. Bij lage meettemperaturen geeft het IR spectrum van kleine glycanen een unieke vingerafdruk van de onderliggende chemische structuur en conformatie.In dit proefschrift is cryogene IR-spectroscopie in combinatie met MS- gebaseerde analytische technieken gebruikt om licht te werpen op de migratie van fucose in MS-experimenten. Deze ongrijpbare migratiereactie is niet beperkt tot tandem MS workflows, maar is recentelijk ook waargenomen in intacte ionen zonder uitgebreide activering. De rol van het proton in fucose- migratiereacties is onderzocht voor de twee glycaanepitopen Lewis x en bloedgroep H type 2. In een systematische studie van adductie-ionen en functionele groepen met concurrerende protonaffiniteiten is aangetoond dat het proton selectief gemobiliseerd en gedemobiliseerd kan worden. Het meten van gefucosyleerde glycaan-kationen met MS vereist een effectieve strategie om de aanwezigheid van een mobiel proton te omzeilen om foutieve sequentie- toewijzingen te voorkomen.In een multidimensionele benadering zijn IR spectroscopie, IM-MS, radical- directed dissociation (RDD) MS en computationele modellering gecombineerd om het migratieproduct en het reactiemechanisme te ontcijferen. De trisachariden Lewis x en bloedgroep H type 2 blijken te migreren naar een chemische structuur, waarin fucose hoogstwaarschijnlijk 1,6-gekoppeld is aan galactose. De barriËre is veel hoger voor bloedgroep H type 2 dan voor Lewis x en het is goed mogelijk dat de laatste nooit in zijn oorspronkelijke chemische structuur gedetecteerd is in de massaspectrometer. Uit deze resultaten blijkt dat fucose-migratie een universeel probleem is in elke massaspectrometer en dat ook het gebruik van verschillende complementaire MS-gebaseerde technieken dit probleem niet geheel kan oplossen.In het tweede deel van dit proefschrift is cryogene IR spectroscopie in combinatie met computationele modellering gebruikt voor de structurele analyse van gesulfoneerde glycosaminoglycanen (GAG9s). De verscheidenheid in de chemische structuur van lineaire zure GAG9s komt voort uit de GAG klasse, sulfatie, epimerisatie en acetylatie. Met behulp van messenger tagging IR spectroscopie zijn recentelijk met succes gesulfoneerde mono- en disachariden gekarakteriseerd. In dit proefschrift is het anticoagulant pentasaccharide fondaparinux, dat acht sulfaatgroepen bevat, onderzocht met behulp van cryogene IR spectroscopie in helium nanodruppels om het werkingsprincipe van de meting aan te tonen. De spectroscopische vingerafdruk toont unieke absorptiebanden in het midden-IR bereik voor de sulfaatgroepen. Het meten van een systematische set van alle natuurlijk voorkomende sulfatievariaties in chondroÔtine- en dermatan-sulfaat (CS/DS) heeft de differentiatie mogelijkheden met behulp van cryogene IR spectroscopie verder aangetoond. Uit de IR-vingerafdruk in combinatie met computationele modellering kan bovendien conformationele diversiteit als gevolg van sulfatie en ladingsdichtheidsverdeling worden afgeleid. In een andere studie onthullen de IR-vingerafdrukken van vier heparansulfaat (HS) diastereomeren een modulariteit in hun chemische structuur die verklaard is met behulp van computationele modellering door hun unieke waterstofbrugpatronen. De kennis van het geprefereerde waterstofbindingspatroon zou bijvoorbeeld kunnen helpen bij de ontwikkeling van labelingstrategieÎn in IM-MS. De resultaten laten zien dat de hoge resolutie in de optische vingerafdrukken van GAG9s het mogelijk maakt om eenduidig de diversiteit op te lossen dievoortkomt uit GAG klasse, sulfatie en epimerisatie. De resultaten illustreren het belang van gas-fase cryogene IR spectroscopie om toekomstige analytische workflows voor GAG sequencing te verbeteren. Een volledig op MS gebaseerde workflow zou de ionisatie van een intacte GAG-keten kunnen omvatten en tandem MS met IM-MS en cryogene IR-spectroscopie van de respectieve fragmenten kunnen combineren om een GAG-keten eenduidig te karakteriseren in ÈÈn enkel MS-experiment.In het laatste deel van het proefschrift is cryogene IR-spectroscopie gecombineerd met random forest modellering om vibratie patronen die kenmerkend zijn voor structurele eigenschappen in GAG9s aan te tonen. De geselecteerde structurele eigenschappen omvatten de GAG-klasse en sulfatie en karakteriseren derhalve bijna volledig de onderliggende chemische structuur. In een proof-of-concept studie is een voorspellingsscore van >97% bereikt voor HS tetra- en hexasachariden op basis van een trainingsset van slechts 21 spectra. Vooral voor bepaalde markermotieven, zoals 3-O-sulfatie in kankercellen, zou deze workflow nuttig kunnen blijken. Met algoritmen voor machine learning zou de noodzaak voor het gebruik van uitgebreide spectrale databanken voor de identificatie van onbekende GAG9s kunnen worden omzeild. Concluderend kan gesteld worden dat de resultaten zoals beschreven in dit proefschrift aantonen dat IR-spectroscopie op basis van MS zeker het potentieel heeft om het stadium van het academisch basisonderzoek te verlaten en een waardevolle aanvulling vormt op MS gebaseerde analytische technieken

To Be A Wife

Carolyn Fairchild, an attractive woman of forty-eight, stepped from the elevator and walked down the carpeted hall to her apartment..

The Surprising Views of Montesquieu and Tocqueville about Juries: Juries Empower Judges

Both Montesquieu and Tocqueville thought that an independent judiciary was key to maintaining a moderate government of ordered liberty. But judicial power should not be exercised too openly, or the people would view judges as tyrannical. In Montesquieu\u27s and Tocqueville\u27s view, the jury was an excellent mask for the power of judges. Both Montesquieu and Tocqueville thought that popular juries had many weaknesses in deciding cases. But, as Tocqueville made clear, the firm guidance of the judge in instructions on law and comments on evidence could prevent juries from going astray and make the institution a free school for democracy. The Article explores Montesquieu\u27s legacy concerning judges and juries in the arguments of both the Federalists and the Anti-Federalists. It also examines the American antecedents of Tocqueville\u27s idea of the jury as a school for democracy

The Worldwide Popular Revolt Against Proportionality in Self-Defense Law

This article examines popular dissatisfaction with the proportionality standard in self-defense law, which holds that the prevention of harm cannot be achieved by causing harm that is disproportionate. Legal elites, such as prosecutors, judges, and legal scholars, have long championed versions of this standard. But there is an increasingly widespread movement in the United States and Europe to modify elite notions of proportionality. Common to these movements is the desire to replace complicated balancing tests with clearer rules, which would limit the discretion of prosecutors and judges, and to permit use of deadly force against attackers in more situations. Fueling the movements is the belief that government is not able or willing to adequately protect its citizens. While these reform movements are occurring in many countries, the article focuses on three places in particular: Florida, Britain, and Belgium. For each place, the author discusses events that led to dissatisfaction with existing rules, political debates surrounding reform, and detailed legislative action. Efforts in Florida and Britain show an approach to reform that centers on presumptions that deadly force may be used in certain situations, such as a forcible entry into a home. Efforts in Belgium show another approach, which is in effect an expansion of provocation doctrine: anyone who exceeds the bounds of proportionality because of emotion (fear, anxiety, or panic) caused by attack or threat of attack is not criminally liable. The paper then analyzes the two types of efforts. The author concludes that emotions caused by attack are proper grounds for mitigation, but not for complete exoneration. Reforms that emphasize presumptions may be justified, particularly presumptions about the use of force against those who forcibly intrude into the home

How the Creation of Appellate Courts in England and the United States Limited Judicial Comment on Evidence to the Jury

The practice of judicial comment on the evidence has traditionally been the main form of jury control. Previous scholarly work has focused on the loss of the power in state courts, and has attributed the decline of judicial comment to a strict separation of functions between judge and jury and to regional differences in legal culture. This article examines two jurisdictions in which the power of comment long remained strong, at least in theory: the High Court of England, with its predecessors, and the federal courts in the United States. In both jurisdictions, judicial power to comment has been limited and in practice reduced, in the federal courts severely. The article reveals that this limitation developed with the advent of courts of appeal with separate personnel and especially of appeals in criminal cases.Lack of appeal, or limited appeal, has been a distinctive trait of common law systems, particularly in criminal cases. There was no appeal as of right in criminal cases until 1907 in England, and 1889 in the federal courts. In the federal system, the early movements to allow appeals in criminal cases and to limit judicial comment on evidence focused on controlling a particular judge: Isaac Parker, U.S. District Judge for the Western District of Arkansas, who presided over more than 100 trials for capital crimes occurring in the Indian Territory from 1875 to 1896

The Rise of Directed Verdict: Jury Power in Civil Cases Before the Federal Rules of 1938

Jury practice in the state and federal courts evolved dramatically in the nineteenth and early twentieth century. Around the time of the ratification of the Bill of Rights in 1791, important legal thinkers praised the civil jury as a bulwark against judicial tyranny. By the advent of the Federal Rules of Civil Procedure in 1938, many commentators regarded the civil jury as an antiquated nuisance. Diminishment of the jury and open exercise of judicial power, encouraged in the Federal Rules by procedures such as summary judgment, would not have been possible without earlier changes in jury practice. Two major changes were the rise of directed verdict procedure and the related judgment notwithstanding the verdict. These mechanisms allowed a judge to give a binding instruction to a jury, or to enter a judgment contrary to the jury’s decision. This Study reveals that railroads revolutionized the law of jury control. Changes in directed verdict were part of a larger program of jury reform beginning in the mid-nineteenth century in England, the states, and the federal government. Because of growing numbers of complicated personal injury suits against railroads, and because of perceived jury bias in those cases, many judges sought to control juries more efficiently. Directed verdicts began to replace new trials. Opposition arose, but the overall trend was toward greater judicial control of juries. The striking changes in jury practice described in this Article suggest difficulties in maintaining a consistent jury trial right by constitutional requirement

The Intersection of Two Systems: An American on Trial for an American Murder in the French Cour D\u27Assises

This study discusses a murder case in France\u27s trial court for the most serious crimes, the Cour d\u27assises. The case was highly unusual because the person on trial was an American, accused of having murdered other Americans in the United States. For reasons given below, cases in which crimes committed in the United States are tried abroad are likely to become more common. This study describes how such a case proceeds, including some of the difficulties that can arise from combining two investigations controlled by very different systems of procedure. An advice section is given for American prosecutors and defense advisers involved in such cases. More broadly, the study sheds light on the differences between the U.S. and continental legal systems, in part building on existing work in the area of comparative criminal procedure and drawing on French sources. The study emphasizes the effects of judicial control over trial on presentation of oral testimony, especially that of the defendant and experts. There are drawbacks to the French approach to oral testimony, such as less vigorous probing of testimony by the parties. There are also advantages, including allowing more information to be known to the fact-finders; permitting a more flexible order of presentation; and fostering dignitary values by letting witnesses speak in their natural voices and by achieving a deeper understanding of the defendant as a unique human being

How the Creation of Appellate Courts in England and the United States Limited Judicial Comment on Evidence to the Jury

The practice of judicial comment on the evidence has traditionally been the main form of jury control. Previous scholarly work has focused on the loss of the power in state courts, and has attributed the decline of judicial comment to a strict separation of functions between judge and jury and to regional differences in legal culture. This article examines two jurisdictions in which the power of comment long remained strong, at least in theory: the High Court of England, with its predecessors, and the federal courts in the United States. In both jurisdictions, judicial power to comment has been limited and in practice reduced, in the federal courts severely. The article reveals that this limitation developed with the advent of courts of appeal with separate personnel and especially of appeals in criminal cases.Lack of appeal, or limited appeal, has been a distinctive trait of common law systems, particularly in criminal cases. There was no appeal as of right in criminal cases until 1907 in England, and 1889 in the federal courts. In the federal system, the early movements to allow appeals in criminal cases and to limit judicial comment on evidence focused on controlling a particular judge: Isaac Parker, U.S. District Judge for the Western District of Arkansas, who presided over more than 100 trials for capital crimes occurring in the Indian Territory from 1875 to 1896

The Intersection of Two Systems: An American on Trial for an American Murder in the French Cour D\u27Assises

This study discusses a murder case in France\u27s trial court for the most serious crimes, the Cour d\u27assises. The case was highly unusual because the person on trial was an American, accused of having murdered other Americans in the United States. For reasons given below, cases in which crimes committed in the United States are tried abroad are likely to become more common. This study describes how such a case proceeds, including some of the difficulties that can arise from combining two investigations controlled by very different systems of procedure. An advice section is given for American prosecutors and defense advisers involved in such cases. More broadly, the study sheds light on the differences between the U.S. and continental legal systems, in part building on existing work in the area of comparative criminal procedure and drawing on French sources. The study emphasizes the effects of judicial control over trial on presentation of oral testimony, especially that of the defendant and experts. There are drawbacks to the French approach to oral testimony, such as less vigorous probing of testimony by the parties. There are also advantages, including allowing more information to be known to the fact-finders; permitting a more flexible order of presentation; and fostering dignitary values by letting witnesses speak in their natural voices and by achieving a deeper understanding of the defendant as a unique human being