Melanin is the major component of skin-, hair-, and eye pigmentation in mammals. Synthesis of melanin takes place in specialized organelles in melanocytes, called melanosomes. As melanosomes mature during pigment synthesis, they are transported towards the tips of dendrites in the melanocyte, and eventually transferred to neighbouring keratinocytes to distribute pigment throughout the skin. A large number of proteins regulate melanin synthesis and distribution. Over one hundred genes have been associated with coat colour mutations in mice, and many of these genes have also been identified in human pigmentation disorders. Other proteins involved in pigmentation are part of pathways that are not unique to pigmentation alone, such as the Ras/ERK pathway. In mouse B16 cells, cAMP stimulation leads to the upregulation of melanin synthesis and dendrite extension. However, cAMP also activates the Ras/ERK pathway in these cells, which, upon prolonged stimulation, leads to an inhibition of melanin synthesis and dendrite extension. Here I show that the protein CNrasGEF, which was previously identified in our lab, is responsible for cAMP-dependent Ras activation in B16 cells, and therefore a part of the negative regulatory pathway of melanogenesis. In order to find other proteins involved in pigmentation pathways, I have developed a method to detect melanosomes using Cellomics KineticScan (KSR) high-content image analysis. This system could potentially be used in a high-throughput RNA interference screen to identify proteins that affect melanosome formation or transport. However, in a pilot study it appeared that knockdown levels achieved upon transient transfection of knockdown constructs from a mouse shRNAmir library against selected targets were in many cases not sufficient to detect an effect on melanocytes, either by confocal microscopy, or by Cellomics KSR analysis. Further reduction of expression levels is necessary before this system can be scaled up to high-content/high-throughput identification of proteins involved in pigmentation.

<p>The peritrophic matrix (PM) lines the insect midgut and is composed of chitin and protein. It is required for organization of digestion and for protection of epithelial cells from mechanical damage, pathogens, and toxins. The PM of <i>Mamestra configurata</i> (Lepidoptera: Noctuidae), bertha armyworm, a serious pest of cruciferous oilseed rape, was studied. The multilayered PM is delaminated from the anterior midgut epithelium during molting Phase II by periodic pulses and degraded during the molting Phase I stage. These events are controlled by chitin synthase-B, and chitinolytic enzymes, such as chitinase and β-<i>N</i>-acetylglucosaminidase. Eighty-two PM proteins were identified and classified as: i) peritrophins, ii) enzymes and iii) other proteins. Peritrophins were further classified as simple, binary, complex and repetitive according to their structural organization and phylogenetic analysis of peritrophin A domains. The expression of most genes encoding PM proteins was specific to the midgut and independent of larval feeding status, developmental stage, or PM formation.</p> <p>This study includes the first report of chitin deacetylase (CDA) activity in the insect midgut suggesting that the PM may contain chitosan. Digestive enzymes, such as insect intestinal lipases (IILs) and serine proteases were also associated with the PM. The IIL genes differed in their expression during larval development; however, serine protease genes were expressed continuously and serine protease activity was present in the midgut of feeding and nonfeeding stages. <i>M. configurata</i> IIM4, a complex peritrophin, was susceptible to degradation by Mamestra configurata nucleopolyhedrovirus-A challenge, as the first evidence of IIM degradation by an alphabaculovirus enhancin. <i>M. configurata</i> IIM2, a binary peritrophin, was unaffected by baculoviral challenge and such resistance of an IIM has not been reported previously. The current study is also the first demonstration of silencing by RNA interference (RNAi) of any gene encoding a PM protein, in this case <i>M. configurata</i> CDA1 (McCDA1) and McPM1. In addition, both <i>in vitro</i> and <i>per os</i> feeding experiments revealed <i>McCDA1</i> silencing starting at 24 or 36 hours posttreatment, as one of the most successful demonstrations of RNAi in a lepidopteran.</p>

<p>Dengue fever and dengue hemorrhagic fever are estimated to afflict 50-100 million people annually and are caused by one of the four serotypes of dengue virus. Dengue virus is carried and transmitted to humans by mosquitoes of the Aedes genus. Given the broad geographic distribution of Aedes mosquitoes, it has been estimated that nearly half the world's population is at risk of contracting the disease. Currently, no vaccine or specific antiviral treatment is available to combat this emerging menace. </p><p>A greater understanding of how dengue virus interacts with its insect and human hosts will facilitate the intelligent design of specific antivirals to combat the disease and enable the selective breeding of mosquitoes resistant to the virus. Although the genomes of the two primary mosquito vectors have been sequenced, the molecular tools necessary for conducting a systematic genetic analysis of host factors required for DEN infection are not yet available. These tools do however exist in the closely related fruit fly, Drosophila melanogaster. By using a strain of dengue virus that was adapted to propagate in fruit fly cells, we completed a full genetic screen for host factors required for efficient dengue virus propagation. When homologues of these host factors were assayed in a human cell line, over half were also shown to be required for efficient viral propagation. This indicates that while the virus is utilizing many of the same pathways in both of its hosts, the interaction with the insect vector has unique features that may contribute to the observed lack of pathogenesis in mosquitoes.</p> / Dissertation

LRBA (LPS Responsive Beige-like Protein Kinase A anchor) gene expression is induced by the mitogen LPS and is a member of the WBW gene family member which is comprised of genes that are involved in cellular proliferation and differentiation. This work provides evidence for the over-expression of LRBA in certain cancers, and that LRBA promoter activity and endogenous LRBA mRNA levels are negatively regulated by the tumor suppressor p53 and positively regulated by E2F transactivators. Furthermore, we demonstrate that inhibition of LRBA expression or function leads to decreased proliferation of cancer cells and that LRBA plays a role in the EGFR signal transduction pathway. In addition to the findings of LRBA's role in carcinogenesis, this work also shows evidence of the knockdown of the SH2-containing Inositol 5' Phosphatase (SHIP) in both mouse and human cells. Furthermore, we provide evidence that SHIP-1 is involved in the AKT signal transduction pathway in human Natural Killer cells.

RNA interference (RNAi) is an evolutionarily conserved process that regulates gene expression. Host cells and viruses interact in many ways, including through miRNAs and RNAi. Viral miRNAs are encoded when viruses, specially including the the polyoma and herpes families, are transcribed in the nucleus. Some viral miRNAs function to regulate host or viral gene expression. Most viral miRNAs’ functions are not known, however, in great detail. A miRNA can be encoded late during infection, as it is by SV40, a model polyomavirus. This downregulates early viral gene expression by directing mRNA RISC-mediated cleavage. As more polyomaviruses are discovered that are associated with human disease, it becomes more important to understand their function and to uncover whether these emerging viruses encode miRNAs. The work presented here shows the discovery of several viral miRNAs in human polyomaviruses—JCV, BKV, and MCV. In addition, I found that viral miRNAs have the evolutionarily conserved function of negatively regulating viral early gene transcripts at a late stage in the infection. During viral replication, viruses utilize the miRNA components of RNAi. However, in invertebrate organisms RNAi also actively defends against viral infection. It is still being debated, though, whether RNAi plays an antiviral role in mammalian cells. Should it be true that RNAi is an antiviral response in mammalian cells, then what is predicted by such a scenario is inconsistent with my studies. I have found that RNAi is strongly inhibited in the early stages after viral infection. Studies with a chemical mimic of viral infection (poly I:C) imply that the innate cellular immune response is responsible for this inhibition. I investigated the molecular changes, in response to viral infection, (e.g. poly ADP-ribosylation of Ago2) in the RNA-induced silencing complex (RISC). I determined that the inhibition of RNAi is brought about by components of the innate response. Completion of this study details a previously unknown “cross talk” between RNAi and the host innate immune response in mammalian cells. Furthermore, I found mir-17 family attenuates a subclass of interferon-stimulated genes. An understanding of viral miRNA and RNAi offers a clue as to we can use molecular intervention for viral infections. / text

Repair of DNA double-strand breaks is critical to the maintenance of genomic stability, and failure to repair these DNA lesions can cause loss of chromosome telomeric regions, complex translocations, or cell death. In humans this can lead to severe developmental abnormalities and cancer. A central pathway for double-strand break repair is homologous recombination (HR), a mechanism that operates during the S and G2 phases of the cell cycle and primarily utilizes the replicated sister chromatid as a template for repair. Most knowledge of HR is derived from work carried out in prokaryotic and eukaryotic model organisms. To probe the HR pathway in human cells, we performed a genome-wide siRNA-based screen; and through this screen, we uncovered cellular functions required for HR and identified proteins that localize to sites of DNA damage. Among positive regulators of HR, we identified networks of pre-mRNA-processing factors and canonical DNA damage response effectors. Within the former, we found RBMX, a heterogeneous nuclear ribonucleoprotein (hnRNP) that associates with the spliceosome, binds RNA, and influences alternative splicing. We found that RBMX is required for cellular resistance to genotoxic stress, accumulates at sites of DNA damage in a poly(ADP-ribose) polymerase 1-dependent manner and through multiple domains, and promotes HR by facilitating proper BRCA2 expression. Screen data also revealed that the mammalian recombinase RAD51 is commonly off-targeted by siRNAs, presenting a cautionary note to those studying HR with RNAi and highlighting the vulnerability of RNAi screens to off-target effects in general. Candidate validation through secondary screening with independent reagents successfully circumvented the effects of off-targeting and set a new standard for reagent redundancy in RNAi screens.

Down syndrome is one of the most common genetic disorders, resulting in a range of neurological and neuromuscular disabilities. Although the presence of specific disabilities varies among individuals with Down syndrome, all individuals with Down syndrome are born with hypotonia (low muscle tone) and over half with congenital heart defects. Later in life, all individuals demonstrate intellectual disabilities to varying degrees, while many also develop early-onset Alzheimer’s disease. While the cause of Down syndrome is known to be a triplication of the 21st chromosome, it is unknown how this extraneous genetic material causes the development of these phenotypes. We have begun research into the biological basis of these disabilities using the tiny nematode, Caenorhabditis elegans as a genetic model. We used the technique RNA interference (RNAi), which allows us to study the in vivo function of genes by knocking down their expression one at a time in a living, behaving animal. We have used this technique to systematically study the in vivo function for genes involved in Down syndrome. To this end, we identified and knocked down C. elegans genes with sequence similarity to 67% of genes on the human 21st chromosome genes. We used these RNAi-treated worms to investigate the neuromuscular function of human chromosome 21 gene equivalents by assaying locomotion and pharyngeal pumping in a blinded screen. We used locomotion as a measure of neurological and neuromuscular function, while we used pharyngeal pumping as a model for cardiac function. We also performed an aldicarb screen to examine the role of some of these genes in the function of the synapse. Our experiments have provided valuable insight into the in vivo function of the vast majority of genes on the human 21st chromosome. This will be vital to identify genes that are potentially involved in eliciting Down syndrome-related phenotypes, laying the groundwork for further studies into the neurobiology of Down Syndrome. / text

Kolorektale Karzinome sind durch ein spezifisches Muster chromosomaler Aberrationen charakterisiert, die im Verlauf der Tumorprogression akkumulieren. Obwohl die meisten Tumoren Sequenzgewinne oder Amplifikationen von Chromosom 13q aufweisen, sind die Zielgene dieser Aberration nach wie vor unbekannt. Um potentielle Onkogene bzw. therapeutische Zielgene auf Chromosom 13q zu identifizieren, wurde eine hochauflösende Analyse dieser Region durchgeführt. Dazu wurden 25 primäre Kolonkarzinome (UICC-II/III) und 15 kolorektale Zelllinien mittels Array-CGH untersucht. Zusätzlich wurden die Genexpressionsprofile dieser Tumoren und Zelllinien mittels Whole-Genome-Mikroarrays bestimmt. 67 Gene wiesen sowohl eine vermehrte Kopie-Anzahl als auch ein erhöhtes Expressionsniveau auf. Die Expressionsmuster dieser Gene wurden dann in 25 Kolonkarzinom-Zelllinien mittels Real-Time-PCR validiert, wobei 44 der 67 Gene eine deutliche Überexpression auch in den Zelllinien zeigten. Das Ausschalten von 13 dieser 44 Gene in der Kolonkarzinom-Zelllinie SW480 führte zu einer Reduktion der Zellviabilität von 20-60%. Diese 13 Gene könnten somit potentielle Onkogene oder mögliche therapeutische Zielgene darstellen. In Folgeexperimenten versuchen wir daher, die der Viabilitätsreduktion zugrundeliegenden Signalwege zu entschlüsseln.

Die Spinozerebelläre Ataxie Typ 3 (SCA3) oder Machado-Joseph-Krankheit (MJD) gehört zur Gruppe der neurodegenerativen Polyglutaminerkrankungen (PolyQ-Erkrankungen) und ist die häufigste autosomal-dominante zerebelläre Ataxie weltweit. Ein in der Länge hochvariabler Polyglutaminabschnitt ist vermutlich die Ursache für die Toxizität der ansonsten nicht verwandten Proteine, welche die PolyQ-Erkrankungen verursachen. Abgesehen von dem verlängerten Polyglutaminbereich scheinen die physiologische Funktion und der zelluläre Kontext dieser Proteine und ihrer Interaktionspartner entscheidend für die spezifische Pathogenese und den Krankheitsverlauf zu sein. Diese Arbeit soll dazu beitragen, genetische Interaktoren zu identifizieren, welche die PolyQ-Toxizität verstärken oder vermindern, um somit die molekularen Krankheitsmechanismen zu entschlüsseln, die durch die Trinukleotid-Wiederholungen ausgelöst werden.  Dafür wurde ein humanes, von Ataxin-3 abgeleitetes Transgen in den Facettenaugen von Drosophila exprimiert.  Die daraus resultierende Degeneration der Photorezeptoren induziert einen Raue-Augen-Phänotyp (Rough Eye Phenotype, REP) in adulten Fliegen. Um genetische Modifikatoren des REP zu identifizieren, wurde die Expression bestimmter Gene (Fliegengene mit einem humanen Ortholog, insgesamt ca. 7.500) augenspezifisch per RNAi vermindert. Mögliche Veränderungen im beobachteten REP sind dann höchstwahrscheinlich auf den RNAi-vermittelten Knockdown der Genexpression zurückzuführen. Damit wären die stummgeschalteten Kandidatengene zur Modifizierung der PolyQ-induzierten Neurotoxizität fähig.  Die auf diese Weise identifizierten Genprodukte sind in verschiedene biologische Prozesse involviert und stehen stellvertretend für unterschiedlichste molekulare Funktionen. Für eine Auswahl von Kandidatengenen wurden zusätzliche Untersuchungen angestellt, um die Art und das Ausmaß der Interaktionen zu bestimmen. Dabei wurden neue Modifikatorengene analysiert, welche z. B. in die Methylierung von tRNA oder den Sphingolipid-Metabolismus involviert sind. Diese Ergebnisse können neue Erkenntnisse bei der Aufklärung der Pathogenese der MJD und anderer PolyQ-Erkrankungen hervorbringen und gleichzeitig zum Verständnis der Rolle von Ataxin-3 und seinen Modulatorproteinen beitragen.

The encysted embryos (cysts) of the crustacean Artemia franciscana have several molecular mechanisms to enable anhydrobiosis – life without water. This study examines the function of group 1 Late Embryogenesis Abundant (LEA) proteins, hydrophilic unstructured proteins which accumulate in the stress-tolerant cysts of A. franciscana. Group 1 LEA proteins were knocked down in cysts using RNA interference. Cysts without group 1 LEA proteins exhibited low survival following desiccation and/or freezing, suggesting a role for these proteins in tolerance of low water conditions. In contrast, cysts with or without group 1 LEA proteins responded similarly to hydrogen peroxide exposure , indicating little to no function in reducing damage due to oxidative stress. This is the first in vivo functional study of group 1 LEA proteins in an animal, and may have applied significance in aquaculture, where Artemia is an important feed source, and in the cryopreservation of cells for therapeutic applications.