Current drugs used to treat Congestive Heart Failure target the renin-angiotensin and adrenergic systems. Studies showed increased mortality rates in patients treated with a combination of these medications. Angiotensin-AT1 and ?2-Adrenergic receptors were shown to form receptor heteromers. Blockade of one receptor in the complex can affect the signal transmitted by the other; suggesting that ligand-based therapy is not as selective as we might think. Modulating receptor trafficking after synthesis might prove to be a valid therapeutic strategy. Unfortunately, little is known about receptor assembly and transport from Endoplasmic Reticulum to Plasma Membrane. The objectives of this study are to identify the proteins that participate in the assembly of AT1R-?2AR heteromer and the regulators of the anterograde trafficking of G-Protein Coupled Receptors. This thesis introduces the role of important targets in those poorly understood processes. The identification of such targets could lead to developing better drugs with fewer adverse effects.

Nuclear receptors (NRs) are modular ligand-activated transcription factors that control a broad range of physiological processes by regulating the expression of essential genes involved in cell physiology, differentiation, and metabolism. These receptors are implicated in a number of diseases and due to their profound role in development and disease progression and their modularity, much emphasis is being put forth into nuclear receptor based drug discovery and engineering these receptors to bind novel small molecules Chemical Complementation (CC) is a yeast three-hybrid genetic selection system that was developed to aid in the discovery of these engineered receptors by linking the survival of a yeast cell to a small molecules ability to activate the receptor. Due to several advantages, to include faster growth times and higher transformation efficiencies, we have attempted to extend chemical complementation from yeast to E. coli. The bacterial chemical complementation system (BCC) was designed, based on a bacterial two hybrid system, to parallel yeast CC system. However, bacterial chemical complementation did not produce ligand dependent activation due to heterologous protein expression. In a second project designed to further NR based protein engineering and drug discovery, CC was used to evaluate a library of charge reversal variants rationally designed to gain a better understanding of nuclear receptor function and structure and to produce orthogonal ligand receptor pairs. A library of retinoic acid receptor (RARα) variants were developed based on five residues in the binding pocket known to stabilize the natural negatively charged ligand, all-trans retinoic acid (atRA). We altered the binding selectivity of the receptor to bind positively charged retinoid ligands. We were able to engineer two triple variants capable of activating with the positively charged retinoid but not the natural atRA ligand, however they do not activate as well as RARα wild-type does with atRA. In a third project we characterized covalently linked tamoxifen and histone deacetylase inhibitor based dual inhibiting compounds as breast cancer therapeutics. Several dual inhibiting compounds were found to decrease the proliferation of ER positive breast cancer cells better than tamoxifen alone, the HDACi alone, or noncovalently linked HDACi and tamoxifen.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed June 23, 2008). Includes bibliographical references.

Dans le cadre des approches constructionistes, cette thèse étudie les verbes labiles de l’anglais, qui peuvent manifester des configurations syntaxiques variées sans changer de forme. L’étude de la complémentation de ces verbes montre que leur catégorisation en familles sémantiques est pertinente mais pas suffisante pour expliquer leur comportement. La thèse défend une approche syncrétique de la complémentation du verbe qui rend compte de son importante productivité et de ses limites parfois arbitraires. Une analyse montre que les verbes ont tous une configuration syntaxique par défaut, qui n’est pas signifiante et qui permet simplement au verbe d’exprimer ses arguments de façon non marquée, en accord avec certains principes de cohérence conceptuelle. À l’inverse, lorsque la complémentation du verbe a un apport sémantique identifiable, l’existence de schémas de complémentation pleinement signifiants est postulée. Il s’agit d’ensembles de compléments dont le sens est distinct de celui du verbe auquel ils sont associés et se retrouve de façon régulière avec des verbes de catégories diverses. Il est démontré que les schémas de complémentation doivent être considérés comme des unités linguistiques de plein droit de l’anglais. Cela implique qu’en synchronie, ces schémas sont emmagasinés par les locuteurs plutôt que le résultat d’un processus d’analogie avec des constructions existantes. Leur statut d’unité linguistique permet d’étudier leur sémantisme de la même façon que des unités lexicales plus classiques. S’ils sont en majorité polysémiques, certains schémas ont des emplois difficiles à relier sémantiquement et doivent donc être considérés comme des homonymes. / Within a constructionist framework, this thesis studies English labile verbs, which can enter into various syntactic configurations without changing form. A study of their complementation shows that categorizing them into semantic families is relevant but not sufficient to explain their behavior. The thesis defends a syncretic approach to verb complementation to that accounts for its important productivity and its sometimes arbitrary limits. It is shown that all verbs have a default syntactic configuration, which is not meaningful and which simply allows the verb to express its arguments in an unmarked way, in accordance with certain principles of conceptual coherence. Conversely, when the complementation of the verb has an identifiable semantic contribution, the existence of fully meaningful patterns of complementation is posited. These are defined as sets of complements, whose meaning is distinct from that of the verb with which they are associated and is found regularly with verbs of diverse categories. It is shown that patterns of complementation should be considered fully-fledged English linguistic units. This implies that synchronically, these patterns are mentally stored by speakers rather than the result of a process of analogy with existing constructions. Their status as linguistic units makes it possible to study their meaning in the same way as more classical lexical units. Although most of them are polysemic, some patterns of complementation exhibit uses that are difficult to link semantically and must therefore be viewed as homonyms.

The Sco proteins, present in all kind of organisms, are regarded as one of the key players in the cytochrome c oxidase (COX) assembly. However, experimental and structural data, such as the presence of a thioredoxin-like fold, suggest that Sco proteins may also play a role in redox homeostasis. Our current studies in S. cerevisiae have strongly suggested an antioxidant role to Sco2 protein (ySco2p). While the single deletion of SCO2 does not result in a distinctive phenotype, the concomitant deletion of superoxide dismutase 1 (SOD1) leads to an increased sensitivity to oxidative stress generating agents (paraquat, menadione, plumbagin) compared to the respective single mutants. Since S. cerevisiae is a good model to functionally characterize genes from more complex organisms, identification of such a phenotype has paved the way to test whether the Sco2 homologues from other organisms are able to substitute for the function of ySco2p. The Sco homologues from Homo sapiens, Schizosaccharomyces pombe, Arabidopsis thaliana, Drosophila melanogaster and Kluyveromyces lactis were integrated into the genome of the double deletion mutant. The functional complementation was tested by both growth and biochemical ROS assays. All homologues except for K. lactis K07152 and A. thaliana HCC1 were able to complement the phenotype, indicating their role in antioxidant defense. Interestingly, pathogenic human SCO2 point mutations failed to restore this function. The observation of non-functional homologues despite of the high sequence similarity to ySco2p strengthened our hypothesis on the importance of conserved aminoacid(s) for the defensive role. For this purpose, selected homologues were aligned and the conservation was judged not only based on identity but also similarity (e.g. charge, hydrophobicity). Interestingly, alignment results have pointed out an aminoacid site (located 15 aminoacids downstream of CxxxC motif) that a positively charged lysine is found only in the non-functional homologues. Subsequent mutagenesis analyses verified the functional importance of this aminoacid site (gain and loss of functions) and revealed the detrimental effect of positive charge on antioxidant function. In order to explain the observed functional change, further effort will be put into the calculations of the electrostatic potential and identifications of protein-protein interactions.:Contents List of figures x List of tables xii Abbreviations xiii 1 Introduction 1 1.1 ROS production 1 1.2 Oxidative stress 2 1.3 Antioxidant response 3 1.4 The thioredoxin fold: From structure to function 6 1.5 Sco proteins 7 1.5.1 Structural similarity of Sco proteins to antioxidant enzymes 8 1.5.2 Current knowledge about Sco proteins of S. cerevisiae 9 1.6 Background studies 10 1.7 Using yeast as a model 11 1.7.1 Cross-species complementation studies 11 1.7.2 Yeast model for human mitochondria studies 12 1.8 Aim of the study 12 2 Materials & Methods 14 2.1 Materials 14 2.1.1 Chemicals and Reagents 14 2.1.2 Equipments 16 2.1.3 Kits 17 2.1.4 Antibodies 18 2.1.5 Plasmid 18 2.1.6 Primers 19 2.1.7 S. cerevisiae strains 22 2.1.8 Media 22 2.2 Methods 24 2.2.1 Cultivation of S. cerevisiae cells 24 2.2.1.1 Culture conditions 24 2.2.1.2 Preparation of glycerol stocks 24 2.2.2 Molecular Biology Methods 24 2.2.2.1 S. cerevisiae genomic DNA isolation 24 2.2.2.2 RNA isolation 25 2.2.2.2a Cultured mammalian cells (HEK293) 25 2.2.2.2b Drosophila melanogaster 25 2.2.2.3 RNA purity and concentration determination 25 2.2.2.4 Reverse transcription 25 2.2.2.5 Polymerase chain reaction 25 2.2.2.5a Standard PCR 25 2.2.2.5b Overhang PCR 26 2.2.2.5c Overlap extension PCR 27 2.2.2.5d Site-directed mutagenesis by overlap extension PCR 27 2.2.2.6 DNA agarose gel electrophoresis 28 2.2.2.7 DNA gel extraction and clean-up 29 2.2.2.8 DNA sequencing 29 2.2.2.9 Southern blotting 29 2.2.2.9a DNA preparation 29 2.2.2.9b Blotting 30 2.2.2.9c Preparation of a DIG-labelled probe 30 2.2.2.9d Hybridization of the DIG-labelled probe to DNA 30 2.2.2.9e Detection of hybridized DIG-labelled URA3 probe 31 2.2.2.10 Yeast transformation 32 2.2.2.11 Growth assay 32 2.2.3 Protein methods 33 2.2.3.1 Isolation of crude mitochondria from yeast 33 2.2.3.2 SDS-PAGE 33 2.2.3.3 Protein transfer 34 2.2.3.4 Colloidal Coomassie gel staining 34 2.2.3.5 Protein detection 35 2.2.3.6 Stripping the membrane and reprobing 35 2.2.4 Biochemical methods 35 2.2.4.1 Methylene Blue staining 36 2.2.4.2 Quantification of ROS 36 2.2.4.2a Amplex Red staining 36 2.2.4.2b Lipid peroxidation assay 36 2.2.5 Bioinformatics 37 2.2.6 Statistical Analysis 37 3 Results 40 3.1 Selection of homologues by bioinformatic analysis 40 3.2 Generation of recombinant strains 42 3.3 Confirmation of site-specific integration by check PCR 44 3.4 Verification of single site integration by Southern Blotting 44 3.5 Analysis of the functional homology between selected homologues and ySCO2 45 3.5.1 Complementation assay in solid media 45 3.5.2 Complementation assay in liquid media 47 3.6 Determination of cell viability 48 3.7 Quantification of ROS 51 3.7.1 Quantification of extracellular H2O2 51 3.7.2 Quantification of lipid peroxidation 53 3.8 Investigation of the expression and subcellular localization of homologues 55 3.9 Investigation of the impact of pathogenic hSCO2 mutations on its antioxidant role 58 3.10 Mutational analysis of ySCO2 60 3.11 Identification of functionally important residues 61 3.12 Prediction of salt bridges 65 3.13 Alanine mutagenesis 66 4 Discussion 68 4.1 Functional homology between the selected homologues and ySCO2 68 4.1.1 A. thaliana homologues, HCC1 & HCC2 68 4.1.2 H. sapiens homologues, hSCO1 & hSCO2 69 4.1.3 D. melanogaster homologue, SCOX 70 4.1.4 Yeast homologues, K07152 & SpSCO1 70 4.2 The localization and expression pattern of homologues 71 4.3 The impact of pathogenic hSCO2 mutations on its antioxidant role 72 4.4 Mutational analysis of ySCO2 73 4.5 Attempts to understand the underlying reason(s) behind charge-related functional change 74 4.6 Potential mechanisms associated with the antioxidant action of ySco2p 78 5 Summary 81 6 References 84

Dynein is a minus end directed molecular motor required for numerous cellular processes during intracellular transport and mitosis. Pac1/LIS1 and Bik1/CLIP-170 are two proteins required for targeting dynein to cytoplasmic microtubule plus ends in budding yeast. The lab previously proposed a model whereby Pac1/LIS1 binds to the motor domain of dynein heavy chain, Dyn1/HC, forming a complex that interacts with the +TIP protein Bik1/CLIP170 at plus ends. This project focused on using Bimolecular Fluorescence Complementation (BiFC) to visualize protein-protein interactions among dynein pathway components in vivo. Budding yeast, Saccharomyces cerevisiae is an ideal system to manipulate dynein as it is a non-essential protein in this system. The BiFC assay fuses two non-fluorescent halves of Venus, a YFP-derivative, to proteins of interest. If an interaction between the proteins occur, the two halves are brought to close proximity and the fluorophore is reconstituted. Cells co-expressing Dyn1-VN with Pac1-VC or Bik1-VC exhibited fluorescent foci associated with microtubule plus ends, the cell cortex and spindle pole bodies (SPBs). Additionally, cells co-expressing Pac1-VC with Bik1-VN exhibited fluorescent foci associated with microtubule plus ends. Cells coexpressing Tub1-VC and Bik1-VN or Dyn1-VN have BiFC signal indicating that both interact with the microtubule directly. Pac-1 coexpressed with Tub1 had no signal above background. These data support that these three components associate at microtubule plus ends. Dyn1 and Pac1 interact with Bik1 at microtubule plus ends. Bik1 serves as a docking platform for the two, but dynein is still able to interact with microtubules, while Pac1 is not.

No description available.

Mitglieder der CCC1 like Proteinfamilie sind Metalltransportproteine und Homologe zum vakuolären Eisen- und Mangantransporter CCC1 aus S. cerevisiae. VIT1-Homologe als CCC1-like Proteine pflanzlicher Herkunft können eine Anwendung in der Eisen-Biofortifikation finden. Bei der Überexprimierung von TaVIT2 in Weizen wurde von der 4 fachen Erhöhung des Eisen- und Mangangehalts im Mehl berichtet (Connorton et al., 2017). Die Überexprimierung von OsVIT1 oder OsVIT2 in Reis hat zur Erhöhung sowohl vom Eisengehalt als auch von Zink- und Mangangehältern geführt (Zhang et al., 2012). In dieser Arbeit wurden 6 CCC1-like Proteine aus A. thaliana bezüglich ihrer Substratspezifität untersucht. VIT1 und fünf VIT-Like Proteine (VTLs) wurden an ihren N- und C Termini modifiziert und im Hefekomplementationsverfahren auf ihre Fähigkeit Eisen, Mangan oder Zink zu transportieren untersucht. Ein einfacher Algorithmus zur Modifizierung von Membranproteinen durch Entfernung mehrerer AS-Reste vom N- bzw. vom C-Terminus ist beschrieben. Es sollte untersucht werden, ob diese Modifizierung einen Einfluss auf die Hefekomplementation hat. Native und modifizierte VTLs wurden mittels GFP-Fusion in den Zelllokalisierungsstudien untersucht. Es sollte festgestellt werden, ob Modifikationen die Zelllokalisierung beeinflussen. Eine Lokalisierung an der Vakuolenmembran wurde für VIT1, VTL1-4 und für einige modifizierte VTL4-Konstrukte nachgewiesen. Dagegen zeigte das VTL5-Konstrukt vorwiegend eine Plasmamembranlokalisierung. Die Entfernung von 21 AS vom N Terminus von VTL4 bzw. von 23 AS vom N-Terminus von VIT1 hatten keinen Effekt auf die Komplementation der Δccc1 Mutante. Dagegen zeigten die entsprechenden Konstrukte keine Komplementation der Δpmr1 Mutante. Es wurden modifizierte Konstrukte von AtVIT1 und AtVTL4 gefunden, die nicht in der Lage waren Mangan und Zink zu transportieren und dadurch für Eisen spezifisch gewesen wären. Diese wären gute Kandidaten für eine eisenselektive Biofortifikation. / CCC1 like protein family are metal transport proteins with high homology to the S. cerevisiae vacuolar iron and manganese transporter CCC1. The plant members of this family such as VIT1-homologue might find an application in iron biofortification of crops. An almost 4-fold increase in the iron content in flour of TaVIT2 overexpressing wheat was demonstrated (Connorton et al., 2017), but the overexpressing plants showed a significant increase in manganese content as well. OsVIT1 or OsVIT2 overexpressing in rice (Zhang et al., 2012) lead to increased iron, zinc and manganese. In this thesis six member of A. thaliana CCC1-like proteins were investigated for their substrate specificity. VIT1 itself and five other VIT1-like proteins (called VTLs) were modified at their N- and C-termini and investigated for iron, manganese and zinc transport capacity via a yeast complementation assay. A simple algorithm to modify membrane proteins by removing AA rests from their N- and / or C-terminal regions is presented, and the effects of these modifications on the functionality of the modified protein by the yeast complementation test are reported. The cell localization of native and modified VTLs was studied using fusion to GFP to determine if modifications would alter the subcellular localization in yeast. VTL1-4 as well as VIT1 were localized to the vacuolar membrane of yeast cell. VTL5 was predominantly localized to the plasma membrane of the yeast cell. Some modified VTL4 constructs could still be localised to the vacuolar membrane. Removal of 21 AA from the N-terminus of AtVTL4 as well as removal of 23 AA from the N-terminus of AtVIT1 didn't affect the complementation of the Δccc1 mutant. In contrast no complementation of Δpmr1 mutant was seen with the same constructs. Certain modified forms of AtVIT1 and AtVTL4 were unable to transport either manganese or zinc. We propose that they might be iron-specific, and thus would be candidates for a selective iron biofortification.

Trypanosoma brucei and related protists are distinguished from all other eukaryotes by an unusual mitochondrial genome known as kinetoplast DNA (kDNA) that is a catenated network composed of minicircles and maxicircles. Replication of this single nucleoid involves a release, replicate, and reattach mechanism for the thousands of catenated minicircles and requires at least three DNA polymerase (POLIB, POLIC and POLID) with similarity to E. coli DNA polymerase I. Like other proofreading replicative DNA polymerases, POLIB has both an annotated polymerase domain and an exonuclease domain. Predictive modelling of POLIB indicates that it has the canonical right hand polymerase structure with a unique and large 369 amino acid insertion within the polymerase domain (thumb region) homologous to E. coli RNase T. The goal of this study was to evaluate whether the polymerase domain is necessary for the essential replicative role of POLIB. To study the structure-function relationship, an RNAi-complementation system was designed to ectopically express POLIB variants in T. brucei that has endogenous POLIB silenced by RNAi.Control experiments expressing an ectopic copy of POLIB wildtype (IBWTPTP) or polymerase domain mutant (IBPol-PTP) in the absence of RNAi did not impact fitness in procyclic cells despite protein levels being 5 - 8.5 fold higher than endogenous POLIB levels. Immunofluorescence detection of the tagged variants indicated homogenous expression of the variants in a population of cells and negligible changes in kDNA morphology. Lastly, Southern blot analyses of cells expressing the IBWTPTP or IBPol-PTP variants indicated no changes in free minicircle species. A dually inducible RNAi complementation system was designed and tested with the IBWTPTP and IBPol-PTP variants. Inductions of POLIB RNAi accompanied by ectopic expression of either variant using the standard 1 mg/ml tetracycline resulted in low protein levels of both variants while knockdown of the endogenous POLIB mRNA was greater than 85%. Increasing the tetracycline concentration to 4 mg/ml improved expression levels of both variants. However, levels of the ectopically expressed variants never exceeded that of endogenous POLIB. Using the 4 mg/ml induction conditions, complementation with IBWTPTP resulted in a partial rescue of the POLIB RNAi phenotype based on fitness curves, quantification of kDNA content and Southern blot analysis of free minicircles. IBWTPTP complementation resulted in gradual increase of IBWTPTP protein levels over the 10 day induction, and a small kDNA phenotype instead of the progressive loss of kDNA normally associated with POLIB RNAi. Additionally, the loss of free minicircles was delayed. Complementation with the IBPol-PTP variant produced more consistent levels of IBPol-PTP protein although still below endogenous POLIB levels. Loss of fitness was similar to POLIB RNAi alone. However, a small kDNA phenotype emerged early after just four days of complementation and persisted for the remainder of the induction. The majority of the IBRNAi + IBPol-PTP population (70%) contained small kDNA compared to the parental POLIB RNAi or IBWTPTP complementation that had only 45% and 50% small kDNA, respectively. Lastly, the pattern of free minicircle loss closely resembled POLIB RNAi alone. Together, these data suggest that the dually inducible system results in a partial rescue with the IBWTPTP variant. Rescue with IBPol-PTP variant results in a noticeably different phenotype from either POLIB RNAi alone or IBWTPTP complementation indicating that the POLIB polymerase domain is likely essential for the in vivo role of POLIB during kDNA replication.

This thesis investigates control infinitives and ECM-infinitives in the history of Swedish. Both constructions are non-finite, based on infinitives with or without complements, but share some properties and functions with finite subordinate clauses. Control infinitives (to-infinitives) are headed by the infinitive marker att (which in some cases may be omitted) and have invisible PRO-subjects (“controlled” by, i.e. co-referential with, the subject or object of the matrix), whereas ECM-infinitives are headed by overt subjects, distinguished by their “exceptional case marking” (ECM) from the matrix verb, and never contain the infinitive marker. According to the proposed analyses, conducted within the theoretical framework of generative grammar, control infinitives are CPs, taking the infinitive marker as a non-finite complementizer in C, but lack the TP of the I-domain, whereas ECM-infinitives have no C-layer but, nevertheless, a (sort of) TP. The historical investigation shows that control infinitives have developed more clause like properties over time. In Old Swedish (1220–1526), they only rarely contained e.g. negations or auxiliaries. It is not until the seventeenth century that these elements have come into use in the same way as in modern Swedish. This is accounted for by assuming that the control infinitive in Old Swedish was a recent innovation that did not initially make any use at all of the I-domain. The ECM-infinitives, on the other hand, are taken to have the same structure and function in Old Swedish as in Modern Swedish, as their use and properties have not changed significantly. In addition, the status of the infinitive marker has changed through the history of Swedish. Etymologically a preposition, it is here analysed as a verb phrase element in Early Old Swedish, not as a (non-finite) complementizer as in Modern Swedish. In early Modern Swedish (1526–1732), the preposition till is used in much the same function as att giving rise to two new infinitive markers: till att and till. This development of new infinitive markers is also accounted for in the thesis.