Structural Basis for Feed-Forward Transcriptional Regulation of Membrane Lipid Homeostasis in Staphylococcus aureus

11 p.

The Politics of Internet Regulation in Brazil : dynamics, factors, and actors in broadband policy

In May 2010, Brazil joined the roll of nations with a National Broadband Plan. The Decree nº 7,175/2010 had implemented a program that aimed to offer 30 million permanent broadband accesses until 2014 and established its main goals, such as accelerating economic and social development, promoting digital inclusion, reducing social and regional inequalities, promoting a generation of employment and income, and expanding electronic government services. However, the broadband access in Brazil is limited, expensive, and centralized in the main urban centres. Despite the fast growth in the past years due to mobile internet access, the market is still concentrated in the local incumbent operators that currently provide mobile services, landline services and Paid-TV services, resulting in a high level of market verticalization. The following dissertation investigates the constraint of broadband access development, the dynamics, the actors, and the factors that have delayed the roll-out of broadband services in Brazil. The study also promotes reflections about the challenge posed by the media, by costumers associations and by public opinion as critical observers of the policy making process. This research examines on the political influence towards regulation to determine the way policy will benefit interest groups. Many interviews have been conducted in order to understand the forces which have been acting in the telecommunications in Brazil after privatization, in 1998. This study aims to provide a better understanding of telecommunications regulatory process in Brazil, in order to help the country finding an adequate policy which can lead to the implementation of a broadband roll-out. The universal broadband access is the only way to benefit the whole society in Brazil with a satisfactory level of education and create more jobs and economic development regarding the plenty use of Information and Communications Technology (ICT).

Novel mechanisms for regulation of cell survival and migration by ceramide 1-phosphate

201 p. : gráf.

The growth and survival rate of Oreochromis niloticus fries fed (private) with varying percentages of Leucaena leucocephala leaf meal based diets

A-ten-week feeding trial was carried out to evaluate the growth and survival rate of Oreochromis niloticus fed with varying percentage levels of Leucaena leucocephala leaf meal based diets. The substitution rates of L.leucocephala leaf meal for groundnut cake in the various diets were 0% - Diet 1, 25% - Diet 2, 50% - Diet 3 and 75% - Diet 4. Ten fries with an average weight of 0.44g were stocked at the rate of 10 fish per bowl and fed at 5% body weight. Diet 1 with 0% inclusion of Leucaena leaf meal gave a significant difference (P>0.05) in growth and survival rate compared with diets 2, 3 and 4. The water quality parameters recorded were appropriate for fish culture

Regulation in Uganda's Lake Victoria fishery: historical and contemporary conditions

This paper sets out to explore how Uganda's lake Victoria fishery has been managed. It explores the management of the fishery during the protectorate period, and argues that the apparent success of regulation during this time may be attributed to the very heightened controls arising from Sleeping Sickness Controls. Once these were removed, entry into the fishery was rapid and uncontrolled, and the resultant impact on fish stocks was quickly felt. With its huge area, considerable shoreline, and innumerable islands, the lake Victoria fisheries service was quickly overwhelmed and disbanded as a result. In the early independence years, the Republic's government focused on developing the fishery, plans thwarted by turmoil of, and following, Idi Amin's reign. More recently, the fishery has prospered from Uganda's entry into the Nile perch fillet export market, which ahs adversely affected stocks. We present and comment on recently collected data that considers fishers' impressions of the status of the fishery, regulations and future managerial possibilities, and comment on these in the light of recent changes to Uganda's fisheries administration

The growth and survival rate of (private) Oreochromis niloticus fingerlings fed with varying percentages of Leucaena leucocephala leaf meal based diets

A-ten-week feeding trial was carried out to evaluate the growth and survival rate of Oreochromis niloticus fed with varying percentage levels of Leucaena leucocephala leaf meal as a substitute for groundnut cake. The levels in the various diets were 0% - Diet 1, 25% - Diet 2, 50% - Diet 3 and 75% - Diet 4. Ten fingerlings with an average weight of 0.44g were stocked at the rate of 10 fish per bowl and fed at 5% body weight. Diet 1 with 0% inclusion of leucaena leaf meal gave a significant difference (P>0.05) in growth and survival rate compared with diets 2, 3 and 4. The water quality parameters recorded were appropriate for fish culture

Brain type II calcium and calmodulin-dependent protein kinase: characterization of a brain-region specific isozyme and regulation by autophosphorylation

A variety of molecular approaches have been used to investigate the structural and enzymatic properties of rat brain type ll Ca^(2+) and calmodulin-dependent protein kinase (type ll CaM kinase). This thesis describes the isolation and biochemical characterization of a brain-region specific isozyme of the kinase and also the regulation the kinase activity by autophosphorylation.

The cerebellar isozyme of the type ll CaM kinase was purified and its biochemical properties were compared to the forebrain isozyme. The cerebellar isozyme is a large (500-kDa) multimeric enzyme composed of multiple copies of 50-kDa α subunits and 60/58-kDa β/β’ subunits. The holoenzyme contains approximately 2 α subunits and 8 β subunits. This contrasts to the forebrain isozyme, which is also composed of and β/β'subunits, but they are assembled into a holoenzyme of approximately 9 α subunits and 3 β/β ' subunits. The biochemical and enzymatic properties of the two isozymes are similar. The two isozymes differ in their association with subcellular structures. Approximately 85% of the cerebellar isozyme, but only 50% of the forebrain isozyme, remains associated with the particulate fraction after homogenization under standard conditions. Postsynaptic densities purified from forebrain contain the forebrain isozyme, and the kinase subunits make up about 16% of their total protein. Postsynaptic densities purified from cerebellum contain the cerebellar isozyme, but the kinase subunits make up only 1-2% of their total protein.

The enzymatic activity of both isozymes of the type II CaM kinase is regulated by autophosphorylation in a complex manner. The kinase is initially completely dependent on Ca^(2+)/calmodulin for phosphorylation of exogenous substrates as well as for autophosphorylation. Kinase activity becomes partially Ca^(2+) independent after autophosphorylation in the presence of Ca^(2+)/calmodulin. Phosphorylation of only a few subunits in the dodecameric holoenzyme is sufficient to cause this change, suggesting an allosteric interaction between subunits. At the same time, autophosphorylation itself becomes independent of Ca^(2+) These observations suggest that the kinase may be able to exist in at least two stable states, which differ in their requirements for Ca^(2+)/calmodulin.

The autophosphorylation sites that are involved in the regulation of kinase activity have been identified within the primary structure of the α and β subunits. We used the method of reverse phase-HPLC tryptic phosphopeptide mapping to isolate individual phosphorylation sites. The phosphopeptides were then sequenced by gas phase microsequencing. Phosphorylation of a single homologous threonine residue in the α and β subunits is correlated with the production of the Ca^(2+) -independent activity state of the kinase. In addition we have identified several sites that are phosphorylated only during autophosphorylation in the absence of Ca^(2+)/ calmodulin.

Policy analysis of shoreline restoration options on private shorelines of Puget Sound

Puget Sound shorelines have historically provided a diversity of habitats that support a variety of aquatic resources throughout the region. These valued natural resources are iconic to the region and remain central to both the economic vitality and community appreciation of Puget Sound. Deterioration of upland and nearshore shoreline habitats, have placed severe stress on many aquatic resources within the region (PSAT, 2007). Since a majority of Washington State shorelines are privately owned, regulatory authority to legislate restoration on private property is limited in scope and frequency. Washington States’ Shoreline Management Act (RCW 90.58) requires local jurisdictions to plan for appropriate future shoreline uses. Under the Act, future development can be regulated to protect existing ecological functions, but lost functions cannot be restored without purchase or compensation of restored areas. Therefore, questions remains as to the ecological resilience of the region when considering cumulative effect of existing/ongoing shoreline development constrained by limited shoreline restoration opportunities. In light of these questions, this analysis will explore opportunities to promote restoration on privately owned shorelines within Puget Sound. These efforts are intended to promote more efficient ecosystem management and improve ecosystem-wide ecological functions. From an economics perspective, results of past shoreline management can generally be characterized as both market and government failure in effectively protecting the publics’ interest in maintaining healthy shoreline resources. Therefore coastal development has proceeded in spite of negative externalities and market imbalances resulting in inefficient resource management driven by the individual ambitions of private shoreline property owners to develop their property to their highest and best use. Federally derived property rights will protect continuation of existing uses along privately owned shorelines; therefore, a fundamental challenge remains in sustainable management of existing shoreline resources while also restoring ecological functions lost to past mistakes in an effort to increase the ecologic resiliency within the region. (PDF contains 5 pages)

Engineering nucleic acid mechanisms for regulation and readout of gene expression : conditional Dicer substrate formation and sensitive multiplexed northern blots

Nucleic acids are most commonly associated with the genetic code, transcription and gene expression. Recently, interest has grown in engineering nucleic acids for biological applications such as controlling or detecting gene expression. The natural presence and functionality of nucleic acids within living organisms coupled with their thermodynamic properties of base-pairing make them ideal for interfacing (and possibly altering) biological systems. We use engineered small conditional RNA or DNA (scRNA, scDNA, respectively) molecules to control and detect gene expression. Three novel systems are presented: two for conditional down-regulation of gene expression via RNA interference (RNAi) and a third system for simultaneous sensitive detection of multiple RNAs using labeled scRNAs.

RNAi is a powerful tool to study genetic circuits by knocking down a gene of interest. RNAi executes the logic: If gene Y is detected, silence gene Y. The fact that detection and silencing are restricted to the same gene means that RNAi is constitutively on. This poses a significant limitation when spatiotemporal control is needed. In this work, we engineered small nucleic acid molecules that execute the logic: If mRNA X is detected, form a Dicer substrate that targets independent mRNA Y for silencing. This is a step towards implementing the logic of conditional RNAi: If gene X is detected, silence gene Y. We use scRNAs and scDNAs to engineer signal transduction cascades that produce an RNAi effector molecule in response to hybridization to a nucleic acid target X. The first mechanism is solely based on hybridization cascades and uses scRNAs to produce a double-stranded RNA (dsRNA) Dicer substrate against target gene Y. The second mechanism is based on hybridization of scDNAs to detect a nucleic acid target and produce a template for transcription of a short hairpin RNA (shRNA) Dicer substrate against target gene Y. Test-tube studies for both mechanisms demonstrate that the output Dicer substrate is produced predominantly in the presence of a correct input target and is cleaved by Dicer to produce a small interfering RNA (siRNA). Both output products can lead to gene knockdown in tissue culture. To date, signal transduction is not observed in cells; possible reasons are explored.

Signal transduction cascades are composed of multiple scRNAs (or scDNAs). The need to study multiple molecules simultaneously has motivated the development of a highly sensitive method for multiplexed northern blots. The core technology of our system is the utilization of a hybridization chain reaction (HCR) of scRNAs as the detection signal for a northern blot. To achieve multiplexing (simultaneous detection of multiple genes), we use fluorescently tagged scRNAs. Moreover, by using radioactive labeling of scRNAs, the system exhibits a five-fold increase, compared to the literature, in detection sensitivity. Sensitive multiplexed northern blot detection provides an avenue for exploring the fate of scRNAs and scDNAs in tissue culture.

Proteomic analysis of the Cdc48/Ubx network identifies a role for Ubx2 in the regulation of lipid biosynthesis

Cdc48/p97 is an essential, highly abundant hexameric member of the AAA (ATPase associated with various cellular activities) family. It has been linked to a variety of processes throughout the cell but it is best known for its role in the ubiquitin proteasome pathway. In this system it is believed that Cdc48 behaves as a segregase, transducing the chemical energy of ATP hydrolysis into mechanical force to separate ubiquitin-conjugated proteins from their tightly-bound partners.

Current models posit that Cdc48 is linked to its substrates through a variety of adaptor proteins, including a family of seven proteins (13 in humans) that contain a Cdc48-binding UBX domain. As such, due to the complexity of the network of adaptor proteins for which it serves as the hub, Cdc48/p97 has the potential to exert a profound influence on the ubiquitin proteasome pathway. However, the number of known substrates of Cdc48/p97 remains relatively small, and smaller still is the number of substrates that have been linked to a specific UBX domain protein. As such, the goal of this dissertation research has been to discover new substrates and better understand the functions of the Cdc48 network. With this objective in mind, we established a proteomic screen to assemble a catalog of candidate substrate/targets of the Ubx adaptor system.

Here we describe the implementation and optimization of a cutting-edge quantitative mass spectrometry method to measure relative changes in the Saccharomyces cerevisiae proteome. Utilizing this technology, and in order to better understand the breadth of function of Cdc48 and its adaptors, we then performed a global screen to identify accumulating ubiquitin conjugates in cdc48-3 and ubxΔ mutants. In this screen different ubx mutants exhibited reproducible patterns of conjugate accumulation that differed greatly from each other, pointing to various unexpected functional specializations of the individual Ubx proteins.

As validation of our mass spectrometry findings, we then examined in detail the endoplasmic-reticulum bound transcription factor Spt23, which we identified as a putative Ubx2 substrate. In these studies ubx2Δ cells were deficient in processing of Spt23 to its active p90 form, and in localizing p90 to the nucleus. Additionally, consistent with reduced processing of Spt23, ubx2Δ cells demonstrated a defect in expression of their target gene OLE1, a fatty acid desaturase. Overall, this work demonstrates the power of proteomics as a tool to identify new targets of various pathways and reveals Ubx2 as a key regulator lipid membrane biosynthesis.

Characterization and developmental regulation of a gene expressed specifically in the skeletogenic lineage of the sea urchin embryo

The sea urchin embryonic skeleton, or spicule, is deposited by mesenchymal progeny of four precursor cells, the micromeres, which are determined to the skeletogenic pathway by a process known as cytoplasmic localization. A gene encoding one of the major products of the skeletogenic mesenchyme, a prominent 50 kD protein of the spicule matrix, has been characterized in detail. cDNA clones were first isolated by antibody screening of a phage expression library, followed by isolation of homologous genomic clones. The gene, known as SM50, is single copy in the sea urchin genome, is divided into two exons of 213 and 1682 bp, and is expressed only in skeletogenic cells. Transcripts are first detectable at the 120 cell stage, shortly after the segregation of the skeletogenic precursors from the rest of the embryo. The SM50 open reading frame begins within the first exon, is 450 amino acids in length, and contains a loosely repeated 13 amino acid motif rich in acidic residues which accounts for 45% of the protein and which is possibly involved in interaction with the mineral phase of the spicule.

The important cis-acting regions of the SM50 gene necessary for proper regulation of expression were identified by gene transfer experiments. A 562 bp promoter fragment, containing 438 bp of 5' promoter sequence and 124 bp of the SM50 first exon (including the SM50 initiation codon), was both necessary and sufficient to direct high levels of expression of the bacterial chloramphenicol acetyltransferase (CAT) reporter gene specifically in the skeletogenic cells. Removal of promoter sequences between positions -2200 and -438, and of transcribed regions downstream of +124 (including the SM50 intron), had no effect on the spatial or transcriptional activity of the transgenes.

Regulatory proteins that interact with the SM50 promoter were identified by the gel retardation assay, using bulk embryo mesenchyme blastula stage nuclear proteins. Five protein binding sites were identified and mapped to various degrees of resolution. Two sites are homologous, may be enhancer elements, and at least one is required for expression. Two additional sites are also present in the promoter of the aboral ectoderm specific cytoskeletal actin gene CyIIIa; one of these is a CCAA T element, the other a putative repressor element. The fifth site overlaps the binding site of the putative repressor and may function as a positive regulator by interfering with binding of the repressor. All of the proteins are detectable in nuclear extracts prepared from 64 cell stage embryos, a stage just before expression of SM50 is initiated, as well as from blastula and gastrula stage; the putative enhancer binding protein may be maternal as well.

Characterization of the human SCF ubiquitin ligases - structure, function, and regulation

The SCF ubiquitin ligase complex of budding yeast triggers DNA replication by cata lyzi ng ubiquitination of the S phase CDK inhibitor SIC1. SCF is composed of several evolutionarily conserved proteins, including ySKP1, CDC53 (Cullin), and the F-box protein CDC4. We isolated hSKP1 in a two-hybrid screen with hCUL1, the human homologue of CDC53. We showed that hCUL1 associates with hSKP1 in vivo and directly interacts with hSKP1 and the human F-box protein SKP2 in vitro, forming an SCF-Iike particle. Moreover, hCUL1 complements the growth defect of yeast CDC53^(ts) mutants, associates with ubiquitination-promoting activity in human cell extracts, and can assemble into functional, chimeric ubiquitin ligase complexes with yeast SCF components. These data demonstrated that hCUL1 functions as part of an SCF ubiquitin ligase complex in human cells. However, purified human SCF complexes consisting of CUL1, SKP1, and SKP2 are inactive in vitro, suggesting that additional factors are required.

Subsequently, mammalian SCF ubiquitin ligases were shown to regulate various physiological processes by targeting important cellular regulators, like lĸBα, β-catenin, and p27, for ubiquitin-dependent proteolysis by the 26S proteasome. Little, however, is known about the regulation of various SCF complexes. By using sequential immunoaffinity purification and mass spectrometry, we identified proteins that interact with human SCF components SKP2 and CUL1 in vivo. Among them we identified two additional SCF subunits: HRT1, present in all SCF complexes, and CKS1, that binds to SKP2 and is likely to be a subunit of SCF5^(SKP2) complexes. Subsequent work by others demonstrated that these proteins are essential for SCF activity. We also discovered that COP9 Signalosome (CSN), previously described in plants as a suppressor of photomorphogenesis, associates with CUL1 and other SCF subunits in vivo. This interaction is evolutionarily conserved and is also observed with other Cullins, suggesting that all Cullin based ubiquitin ligases are regulated by CSN. CSN regulates Cullin Neddylation presumably through CSNS/JAB1, a stochiometric Signalosome subunit and a putative deneddylating enzyme. This work sheds light onto an intricate connection that exists between signal transduction pathways and protein degradation machinery inside the cell and sets stage for gaining further insights into regulation of protein degradation.

Negative regulation of transcription factors by Srb10 cyclin-dependent kinase

The ubiquitin-dependent proteolytic pathway plays an important role in a broad array of cellular processes, inducting cell cycle control and transcription. Biochemical analysis of the ubiquitination of Sic1, the B-type cyclin-dependent kinase (CDK) inhibitor in budding yeast helped to define a ubiquitin ligase complex named SCF^cdc4 (for Skp1, Cdc53/cullin, F-box protein). We found that besides Sic1, the CDK inhibitor Far1 and the replication initiation protein Cdc6 are also substrates of SCF^cdc4 in vitro. A common feature in the ubiquitination of the cell cycle SCF^cdc4 substrates is that they must be phosphorylated by the major cell cycle CDK, Cdc28. Gcn4, a transcription activator involved in the general control of amino acid biosynthesis, is rapidly degraded in an SCF^cdc4-dependent manner in vivo. We have focused on this substrate to investigate the generality of the SCF^cdc4 pathway. Through biochemical fractionations, we found that the Srb10 CDK phosphorylates Gcn4 and thereby marks it for recognition by SCF^cdc4 ubiquitin ligase. Srb10 is a physiological regulator of Gcn4 stability because both phosphorylation and turnover of Gcn4 are diminished in srb10 mutants. Furthermore, we found that at least two different CDKs, Pho85 and Srb10, conspire to promote the rapid degradation of Gcn4 in vivo. The multistress response transcriptional regulator Msn2 is also a substrate for Srb10 and is hyperphosphorylated in an Srb10-dependent manner upon heat stress-induced translocation into the nucleus. Whereas Msn2 is cytoplasmic in resting wild type cells, its nuclear exclusion is partially compromised in srb10 mutant cells. Srb10 has been shown to repress a subset of genes in vivo, and has been proposed to inhibit transcription via phosphorylation of the C-terminal domain of RNA polymerase II. Our results suggest a general theme that Srb10 represses the transcription of specific genes by directly antagonizing the transcriptional activators.

Signal transduction, regulation, and developmental logic of EGFR signalling in C. elegans

RTKs-mediated signaling systems and the pathways with which they interact (e.g., those initiated by G protein-mediated signaling) involve a highly cooperative network that sense a large number of cellular inputs and then integrate, amplify, and process this information to orchestrate an appropriate set of cellular responses. The responses include virtually all aspects of cell function, from the most fundamental (proliferation, differentiation) to the most specialized (movement, metabolism, chemosensation). The basic tenets of RTK signaling system seem rather well established. Yet, new pathways and even new molecular players continue to be discovered. Although we believe that many of the essential modules of RTK signaling system are rather well understood, we have relatively little knowledge of the extent of interaction among these modules and their overall quantitative importance.

My research has encompassed the study of both positive and negative signaling by RTKs in C. elegans. I identified the C. elegans S0S-1 gene and showed that it is necessary for multiple RAS-mediated developmental signals. In addition, I demonstrated that there is a SOS-1-independent signaling during RAS-mediated vulval differentiation. By assessing signal outputs from various triple mutants, I have concluded that this SOS-1-independent signaling is not mediated by PTP-2/SHP-2 or the removal of inhibition by GAP-1/ RasGAP and it is not under regulation by SLI-1/Cb1. I speculate that there is either another exchange factor for RASor an as yet unidentified signaling pathway operating during RAS-mediated vulval induction in C. elegans.

In an attempt to uncover the molecular mechanisms of negative regulation of EGFR signaling by SLI-1/Cb1, I and two other colleagues codiscovered that RING finger domain of SLI-1 is partially dispensable for activity. This structure-function analysis shows that there is an ubiquitin protein ligase-independent activity for SLI-1 in regulating EGFR signaling. Further, we identified an inhibitory tyrosine of LET-23/ EGFR requiring sli-1(+)for its effects: removal of this tyrosine closely mimics loss of sli-1 but not loss of other negative regulator function.

By comparative analysis of two RTK pathways with similar signaling mechanisms, I have found that clr-1, a previously identified negative regulator of egl-15 mediated FGFR signaling, is also involved in let-23 EGFR signaling. The success of this approach promises a similar reciprocal test and could potentially extend to the study of other signaling pathways with similar signaling logic.

Finally, by correlating the developmental expression of lin-3 EGF to let-23 EGFR signaling activity, I demonstrated the existence of reciprocal EGF signaling in coordinating the morphogenesis of epithelia. This developmental logic of EGF signaling could provide a basis to understand a universal mechanism for organogenesis.

Regulation of mitochondrial division by the Drp1 receptors

Mitochondria can remodel their membranes by fusing or dividing. These processes are required for the proper development and viability of multicellular organisms. At the cellular level, fusion is important for mitochondrial Ca2+ homeostasis, mitochondrial DNA maintenance, mitochondrial membrane potential, and respiration. Mitochondrial division, which is better known as fission, is important for apoptosis, mitophagy, and for the proper allocation of mitochondria to daughter cells during cellular division.

The functions of proteins involved in fission have been best characterized in the yeast model organism Sarccharomyces cerevisiae. Mitochondrial fission in mammals has some similarities. In both systems, a cytosolic dynamin-like protein, called Dnm1 in yeast and Drp1 in mammals, must be recruited to the mitochondrial surface and polymerized to promote membrane division. Recruitment of yeast Dnm1 requires only one mitochondrial outer membrane protein, named Fis1. Fis1 is conserved in mammals, but its importance for Drp1 recruitment is minor. In mammals, three other receptor proteins—Mff, MiD49, and MiD51—play a major role in recruiting Drp1 to mitochondria. Why mammals require three additional receptors, and whether they function together or separately, are fundamental questions for understanding the mechanism of mitochondrial fission in mammals.

We have determined that Mff, MiD49, or MiD51 can function independently of one another to recruit Drp1 to mitochondria. Fis1 plays a minor role in Drp1 recruitment, suggesting that the emergence of these additional receptors has replaced the system used by yeast. Additionally, we found that Fis1/Mff and the MiDs regulate Drp1 activity differentially. Fis1 and Mff promote constitutive mitochondrial fission, whereas the MiDs activate recruited Drp1 only during loss of respiration.

To better understand the function of the MiDs, we have determined the atomic structure of the cytoplasmic domain of MiD51, and performed a structure-function analysis of MiD49 based on its homology to MiD51. MiD51 adopts a nucleotidyl transferase fold, and binds ADP as a co-factor that is essential for its function. Both MiDs contain a loop segment that is not present in other nucleotidyl transferase proteins, and this loop is used to interact with Drp1 and to recruit it to mitochondria.