VictorJuiz/thesis huggingface dataset:
Biochemical Characterization of Escherichia coli PgaB, an Enzyme Essential for Biofilm Formation
Keywords: PNAG
deacetylase
biofilm
polysaccharide intercellular adhesin

The formation of bacterial biofilms requires an extracellular matrix to facilitate adherence of bacteria to the surface they colonize. Carbohydrate polymers, known as exopolysaccharides, form a key component of most biofilm matrices. A wide variety of medically-important biofilm forming bacterial strains, including S. epidermidis, S. aureus, E. coli, B. pertussis, and Y. pestis generate the same β-1,6-N-acetyl glucosamine (PNAG) homopolymer as a key biofilm matrix exopolysaccharide. In E. coli, as well as in the other bacterial strains, the PNAG undergoes partial enzymatic de-N-acetylation, which is essential for surface attachment and subsequent biofilm formation. In vivo studies implied that the enzyme responsible for carrying out de-N-acetylation in E. coli is PgaB, an enzyme with sequence homologues in many Gram negative species capable of forming biofilms. In this work, the first biochemical characterization of PgaB is presented. We confirmed the activity of PgaB on β-1,6-GlcNAc oligosaccharides. The activity of PgaB is specific for the β-1,6 linkage and no de-N-acetylation of β-1,4-GlcNAc oligosaccharides was detected. Enzyme activity is dependent on the degree of substrate polymerization, as the second order rate constant for pentasaccharide substrate was determined to be four times higher than that of the tetrasaccharide substrate. Oligosaccharide sequencing studies indicate that there may be a pattern in the de-N-acetylation of substrates by PgaB. The central residue is modified in mono-de-N-acetylated pentasaccharide substrate, while di-de-N-acetylated hexasaccharide substrate shows modification mainly at the third and fifth residues from the non-reducing terminus of the substrate. Activity studies revealed that PgaB is activated by Ni2+ as well as by Fe2+, which is uncommon for deacetylase enzymes. Metal coordination to active site residues His184 and His189 was confirmed by mutagenesis studies, which also indicated that the metal likely plays a catalytic role. The results of these metal dependence studies support the observed binding of nickel and iron to the active site in PgaB crystal structures. The characterization studies presented in this thesis allow us to gain a better understanding of the de-N-acetylation aspect of the PNAG biosynthetic process and will serve as a basis for enzyme inhibitor design.
Constructive Efforts: The American Red Cross and YMCA in Revolutionary and Civil War Russia, 1917–24
Keywords: Humanitarianism
American Red Cross
American YMCA
Russian Revolution
Russian Civil War
Progressive Era
American-Russian relations
American foreign relations

This dissertation is about American Red Cross and YMCA work in revolutionary and civil war Russia. It focuses on the most significant phases of these organizations’ efforts in terms of the numbers of personnel involved and the funds expended: Moscow and Petrograd, 1917–18; northern Russia during the Allied military intervention, 1918–19; and Siberia and the Russian Far East, from 1918 through the early 1920s. By drawing on dozens of often underused archival collections this study is able to discuss these “constructive efforts” in much fuller detail than have existing works. The activities of the Americans who worked in Russia, rather than those who made policy from afar, are of primary interest. The concern here, beyond the what, where, and who, is why: Why did American relief or social service work occur? The answers, of which there are several, include a desire to provide assistance to suffering populations. But the humanitarian impulse was often not the one that carried the day when decisions about policy and practice were taken. Military concerns were important, especially while the Great War still raged on the western front, and while Allied and American soldiers fought Russian Bolsheviks. American relief workers also saw themselves as contributing directly to relations between Russia and Russians on the one hand, and the United States, the Allies, and the American people on the other. They were moved to carry out their work because they saw the importance of it for the present and future of relations between the two countries. Americans in Russia also took advantage of the presence of soldiers, civilian refugees, and former prisoners of war from a variety of European countries to spread the good word about all things American. Ultimately, Americans viewed revolutionary Russia through the lens of modernization. With American help, the future could be bright. With the right leadership in place to oversee their education, honest, hardworking, and intellectually curious peasants (as they were described by contemporary observers) could be turned into modern citizens. The Russian project failed to achieve its promise, but for a time Americans retained their optimism about Russia’s future.
Pairing Form with Function: The Oligomeric Size and Configuration of G Protein-coupled Receptors
Keywords: G protein-coupled receptors
Oligomer
Resonance energy transfer
Signaling
Fluorescence
Modeling

The quaternary status of G protein-coupled receptors (GPCRs) is important, unknown and controversial. Estimates of size from numerous pharmacological, biochemical and biophysical studies range from monomers to octamers. Accounts of stability vary from constitutive oligomers to a spontaneous, ligand-regulated interconversion between monomers and dimers. In the present investigation, the oligomeric size of GPCRs in live Chinese hamster ovary (CHO) cells has been examined by two methods. Both are based on the efficiency of Förster resonance energy transfer (FRET) between fluorophore-tagged receptors, as determined from emission spectra via spectral deconvolution. In the first, the apparent FRET efficiency (Eapp) was measured for cells expressing eGFP- and eYFP-tagged M2 muscarinic receptors at different ratios of acceptor to donor. Eapp then was related to the pair-wise efficiency (Ep) according to a model that enumerates all pathways for the transfer of energy between single donors and acceptors within an oligomer of given size (n). Each value n returned a distinct and well-defined value of Ep. Fluorescence lifetime imaging provided an independent estimate of Ep that was in close agreement with the model-based value when n = 4, identifying the M2 receptor as a tetramer. In the second approach, the M1 and M2 muscarinic receptors and the β1 and β2 adrenergic receptors were tagged with GFP2 and eYFP, and the value of Eapp was estimated for each pixel in the image of a cell. The distributions of Eapp from 34–40 cells expressing each receptor were compared with those predicted for populations of dimers, trimers and tetramers, the latter configured as a square and a rhombus. In each case, the combined data were well described in terms of a rhombus. Distributions obtained for the M2 and β2 receptors were not affected by agonists or inverse agonists, nor was there evidence for appreciable numbers of dimers or larger oligomers. Taken together, the results suggest that GPCRs of Family 1 exist largely or wholly as constitutive tetramers.
Functional Domains and Target Genes of the Hindsight Zinc-finger Protein
Keywords: Drosophila
Hindsight
Zinc-finger
transcriptional regulation
nervy
RREB-1

The Drosophila hindsight (hnt) gene encodes a C2H2-type zinc-finger (ZNF) protein crucial for epithelial morphogenesis. The human HNT homologue, RREB1, functions as a transcriptional modulator and regulates several tumor suppressor genes. HNT’s functional motifs, target genes and its regulatory abilities have not been elucidated. Here I showed that the C-terminal region of HNT containing the last five of 14 ZNFs (ZNF 10-14) binds in vitro to DNA-elements similar to those identified for RREB-1. I then mapped HNT’s endogenous binding sites on polytene chromosomes and focus on two, at 4C and 60C, which are associated with the hnt and nervy (nvy) loci, respectively. Sequence analysis of the bound fragments shows conservation of motifs similar to those bound by HNT in vitro. Data from both hnt loss- and gain-of-function experiments show that HNT attenuates the transcription of the hnt and nvy genes in several tissues and developmental stages. I show that the identified HNT C-terminal DNA binding domain ZNF 10-14 is not required for these regulatory functions. I further delineate the minimal functional motifs of HNT in transcriptional regulation and show that its ninth ZNF in isolation has a repressive activity and is sufficient to confer many regulatory functions of HNT. On the other hand, mutation of ZNF 9 in the context of the full-length protein indicates that it is not necessary for HNT functions. Interestingly, ZNF 9 has been lost from HNT vertebrate homologues. I propose two redundant mechanisms of transcriptional regulation by HNT: one is mediated by the potential protein-interaction abilities of ZNF 9; another is through cooperation of other ZNF motifs of HNT; the DNA binding abilities conferred by the C-terminal five fingers may be essential for the latter mechanism.
Regulation of FGF Receptor 1 by Nedd4-1
Keywords: FGFR1
Nedd4-1
Ubiquitin
Endocytosis
Receptor Tyrosine Kinase
Protein Microarray

The ubiquitin system plays a pivotal role in regulating protein degradation, endocytosis and numerous other cellular functions. E3 ubiquitin ligases, which mediate the final step in the ubiquitylation reaction cascade, are responsible for substrate recognition and ubiquitin attachment to them, underscoring the importance of identifying their substrates. Nedd4 family members are E3 ubiquitin ligases comprised of a C2-WW-HECT domain architecture. This thesis was aimed at first globally delineating the substrate specificity of the closely related Nedd4 family members in humans, hNedd4-1 (Nedd4) and hNedd4-2 (Nedd4L), and second, to follow up on one of the novel hits identified, the FGFR1, and study in detail how it is regulated by its E3 ligase hNedd4-1. To globally identify substrates for Nedd4 proteins, a high throughput proteomic screening technology using protein microarrays was employed. Despite the obvious homology in their domain architecture, the results presented here suggest that these Nedd4 family members may function non-redundantly, since they demonstrate a selective preference towards substrate ubiquitylation. Our focus on FGFR1, a substrate identified for hNedd4-1, has revealed an important functional role for this ubiquitin ligase in promoting FGFR1 endocytosis and downregulation of its signaling activity. The evidence presented indicates that this interaction has important consequences for developmental processes that are dependent on FGF signaling: human neural stem cell differentiation and zebrafish embryonic patterning and brain development. We demonstrate that the WW3 domain of Nedd4-1 recognizes a novel, non-canonical binding surface (peptide2) within the juxtamembrane region of FGFR1, and we are currently in the process of solving the 3 dimensional structure of the hNedd4-1 WW3: FGFR1 peptide2 complex using X-ray crystallography. Furthermore, in characterizing the interaction between hNedd4-1 and FGFR1, we have provided evidence for a novel mechanism for regulating the catalytic activity of hNedd4-1 by FGFR1 activation. This involves the formation of hNedd4-1 dimers upon removal of the autoinhibitory C2 domain from the HECT domain. Dimerized hNedd4-1, in turn, exhibits enhanced interactions with FGFR1 and enhanced receptor ubiquitylation. From these data, we proposed a negative feedback inhibitory model for FGFR1 downregulation, whereby activated receptor enhances the activation of its suppressor, hNedd4-1, to ensure timely termination of receptor signaling.
Langue, espace et (re)composition identitaire dans les oeuvres de Mehdi Charef, Tony Gatlif et Farid Boudjellal
Keywords: Francophone Literature
Francophone Cinema
Beur writing
Identity
Mehdi Charef
Tony Gatlif
Farid Boudjellal
Border crossing

My dissertation critically assesses the evolution of Beur productions (1980s - 21st century) with reference to the socio-cultural and economic conditions of their development. I am interested in identity (re)construction in the work of three Algerian origin artists in France: Mehdi Charef, Tony Gatlif and Farid Boudjellal. I examine texts and films like Le thé au Harem d’Archi Ahmed (1983), Petit Polio (1999), Je suis né d’une cigogne (1999) and Exils (2004). My interest lies in analyzing the impact that these “minor” artists and their chosen art genres have on mainstream cultural productions. Besides focusing on the second generation immigrants in France (the Beurs), the authors challenge also the canonical “Frenchness” or “Europeanness” beyond borders, thus promoting the concept of cultural hybridity. I argue that constant displacement, change of political, social and cultural contexts have significantly transformed the dynamics between the center and the periphery. Furthermore, I provide evidence that “French” identity has undergone a continuous transformation over time and has become a substantially diverse and lively construct. I stand for a new approach to the fixed French identities, one that includes a pluriethnic and multilingual francophone body, and for a redefinition of cultural boundaries reshaping the relationship between self and otherness. My thesis has two major sections (language and space) divided respectively into four chapters. The first two chapters look at different ways in which the Franco-Maghrebi literature and cinema forge new ways of expression within French language and culture. The first chapter is mainly focused on the importance of mastering French by the Beur artists, while in the second chapter I insist on more elaborate stylistic dimensions such as postcolonial irony in the given texts. The last two chapters critically analyze how the artistic productions of Charef, Gatlif and Boudjellal situate themselves in the French socio-cultural context by trespassing borders of a cultural, literary, linguistic and geographical order. This multiple border crossing leads to a movement of these texts to different locations within the main culture. At the esthetic level, this movement between different borders promotes the innovation and the creativity that the Beur artists bring to France.
On the Metapolitics of Decay: Walter Benjamin's Will to Happiness
Keywords: Walter Benjamin

This dissertation analyzes the early work of Walter Benjamin (ca. 1916 – 1926). The period under consideration falls between Benjamin’s break from the German Youth Movement (which also coincides with the beginning of the Great War) and his turn to Marxism. Benjamin’s life and work during this period is characterized by, on the one hand, an intensified interest in theological concepts and, on the other hand, the apparent refusal of concrete political engagement. It is the claim of the dissertation that what Benjamin elaborates – in the absence of a concrete political program and with the aid of theological concepts – is a metaphysical conception of politics: what I call a metapolitics of decay. This metapolitics is informed by a certain theological understanding of transience: the decay that attends to a creation which has “fallen” from its original condition. While Benjamin’s metapolitics is oriented towards redemption – to the lossless consummation of historical life – it pursues this goal, not by circumventing transience, but by concentrating on the decay of nature – and by extension, of history. The metapolitical limit upon concrete politics, however, does not foreclose the possibility of the latter. In 1919, in a text posthumously named the “Theologico-Political Fragment,” Benjamin does in fact spell out what I call a politics of transience. One of the major historical and conceptual trajectories that the dissertation traces, therefore, is the movement from the metapolitics of decay to the politics of transience. The political significance of transience and decay reveals itself in the profane and melancholic fixation upon the decay of nature and of history. And yet it is only with the concept of happiness that both the metapolitical and the political dimensions of Benjamin’s work become most clear. Happiness (Glück), which is manifestly not the bliss (Seligkeit) of the prelapsarian condition, is no escape from the melancholy situation of historical life. It remains definitively profane and capable of taking an “elegiac” form. But it is precisely by way of its profanity and its melancholy that happiness comes to signify the idea of redemption. The will to happiness, for Benjamin, is a (weak) messianic force.
Dual Mobile Robot: Adaptable Mobility System
Keywords: Mobile robot
Track-wheel Mobile Robot
Adaptable Design
Product Adaptability

This thesis presents an adaptive and reconfigurable mobile robot: the Dual Mobile Robot (DMR). It is driven by two adaptive track-wheel driving modules that combine wheels and tracks to allow real-time interchangeability according to terrain condition. The DMR can automatically convert from a wheel-based robot into a track-based robot by rotating the track-wheel driving modules by 90 degrees, either only tracks or wheels contact with the ground without any interference. It can be driven as a wheel-based robot when operating over a paved road to achieve higher speed and low energy consumption, and as a track-based robot over uneven terrain. In addition, unlike most state-of-the-art mobile robot designs that have an integrated architecture, this design provides a modular architecture which allows modifications and upgrades to be performed via simple replacements or local changes of modules. To establish the modular architecture, this research utilized a unique design paradigm, “Design for product adaptability”. A function-based design process for product adaptability has been conducted in the conceptual design stage. By following the design process, two types of design alternatives of the DMR have been created. After the best product configuration was chosen through evaluation and prioritization, the selected configuration has been implemented by detail design. The DMR prototype was developed and tested to demonstrate its adaptability and advanced mobility functions in real-world environments. The experimental results successfully validated the hypothesis of the proposed robot with its track-wheel interchangeable ability, significantly exceeding the capability of other existing systems.
Dynamin is Required for the Maintenance of Enveloping Layer Integrity and Epiboly Progression in the Zebrafish Embryo
Keywords: zebrafish
epiboly
morphogenesis
endocytosis
dynamin
calpain

During early development, a series of regulated cell movements is required to set up the adult body plan of an organism. Collectively referred to as gastrulation, these coordinated cell movements organize the germ layers and establish the major body axes of the embryo. One such coordinated cell movement, epiboly, describes the thinning and spreading of a multilayered cell sheet to cover the embryo during gastrulation. The zebrafish embryo has emerged as a vital model system to study the cellular and molecular mechanisms that drive epiboly. In the zebrafish, the blastoderm undergoes epiboly to engulf the yolk cell and close the blastopore at the vegetal pole. This is achieved through the coordinated movement of the deep cells, which make up the embryo proper, and two extra-embryonic tissues, the enveloping layer and yolk syncytial layer. Epiboly is essential to the development of most organisms; however, the cellular and molecular mechanisms driving epiboly are poorly understood. Here I report the findings of two distinct projects which addressed the cellular and molecular basis for epiboly in the zebrafish. One cellular mechanism thought to be involved in driving epiboly is the removal of yolk cell membrane ahead of the advancing blastoderm margin. Using a combination of drug- and dominant-negative based approaches to inhibit Dynamin, a key component of the endocytic machinery, I demonstrated that marginal yolk cell endocytosis is dispensable for the successful completion of epiboly. Instead, I found that Dynamin primarily acts in the blastoderm where it maintains integrity of the enveloping layer (EVL) during epiboly. Dynamin maintains EVL integrity through regulation of the Ezrin/Radixin/Moesin (ERM) family of proteins and the activity of the small GTPase Rho A. With the goal of identifying genes involved in regulating epiboly, I characterized the calpain family of calcium-dependent cysteine proteases in the zebrafish and examined the developmental expression patterns of these genes. My study provided insight into the evolution of this large gene family. Furthermore, I found that most members of this family are expressed in the early embryo, suggesting that they may play a role in regulating early developmental processes such as epiboly.
Modulation of microRNA Functions by the 3’ Untranslated Regions of CD44 and Tumour Suppressor Candidate 2
Keywords: microRNAs
3' Untranslated Region
Breast Cancer

Recently, the importance of non-coding regions of the genome, which were once presumed to be “junk” DNA, has been revealed. The non-coding 3’ untranslated region (3’UTR) plays a significant role in the regulation of microRNA (miRNA) functions. The 3’UTR is hypothesized to function in the feed-back regulation of miRNA functions, since it can bind and inactivate multiple miRNAs. In this study, the effects of the exogenous over-expression of two 3’UTRs, CD44 and Tumour Suppressor candidate 2 (TUSC2), on miRNA functions in breast cancer carcinogenesis were investigated. A series of cell function assays in human and mouse breast cancer cell lines, MT-1, MDA-MB-231 and 4T1 showed phenotypic changes caused by the 3’UTRs. A variety of cell function characteristics were affected, including cell proliferation, colony formation, cell survival, angiogenesis, tumour growth, cell migration, invasion and adhesion. These results were hypothesized to occur due to the interaction of the 3’UTRs with multiple miRNAs. The 3’UTRs were able to antagonize cytoplasmic miRNAs, as demonstrated by luciferase activities. In the case of the CD44 3’UTR, downstream target mRNAs, CDC42, Col1a1 and FN1 had an increased expression along with CD44. When the 3’UTR of TUSC2 was over-expressed, there was an increased translation of TUSC2 and the downstream targets, tissue inhibitor of metalloproteinases (TIMP) -2 and -3.The 3’UTRs of these target mRNAs can bind and target multiple miRNAs in common with the TUSC2 3’UTR, which were confirmed with luciferase activity assays and correlated with a series of siRNA and miRNA assays. During the study of the TUSC2 3’UTR, a potential pseudogene, TUSC2P, was discovered. Interestingly, TUSC2P mRNA expression was found to be decreased in cancer cells compared to normal cells and had similar cell functional characteristics as that of the TUSC2 3’UTR. These findings suggest that the over-expressed non-coding transcript can serve as a competitor for miRNA binding, which freed the potential targets of the miRNAs and led to an up-regulation of multiple protein levels. The non-coding transcript can thus be used as a functional miRNA inhibitor that is capable of modulating multiple miRNAs, which can be applied towards treating cancer in the form of gene therapy.