Growth factors can regulate a variety of cellular processes by activating receptors on the cell surface. Many of the receptors belong to the receptor tyrosine kinase family, including the platelet-derived growth factor receptor and the epidermal growth factor receptor and its related family members, such as ErbB2. Constitutively activated receptor tyrosine kinases and their downstream signaling pathways (such as the Ras-mitogen activated protein kinase pathway and the phosphatidylinositol 3 kinase-Akt pathway) are frequently observed in cancer cells. Therefore, understanding the degradation mechanism of receptors is important and may facilitate the development of new prognostic or treatment strategies for cancer. Ankyrin105 is the smaller isoform of ankyrin3 and is localized to late endosomes and lysosomes. Our laboratory has previously shown that ankyrin105 can bind to the phosphatidylinositol 3 kinase regulatory subunit p85, stimulate lysosomal-mediated degradation of the platelet-derived growth factor receptor and differentially affect its signaling pathways in NIH 3T3 cells. To determine whether ankyrin105 can induce degradation of multiple receptor tyrosine kinases in a similar manner, we extended these studies to include the epidermal growth factor receptor and its downstream signaling in this project. Hemagglutinin-tagged ankyrin105 was introduced into COS-1, HEK293T, MCF10A, MDA-MB-231 and AU565 cells, respectively. We demonstrated that overexpression of ankyrin105 did not enhance the epidermal growth factor receptor degradation or downregulation of its signaling pathways in these selected cell lines. AU565 cells, which expressed relatively high levels of both epidermal growth factor receptor and ErbB2, were susceptible to geldanamycin or herceptin facilitated ErbB2 internalization and degradation, which subsequently promoted the epidermal growth factor receptor degradation. However, ankyrin105 did not further improve geldanamycin-induced epidermal growth factor receptor degradation or impact its downstream signaling pathways. These studies suggest that the influence of ankyrin105 may be receptor-specific (platelet-derived growth factor receptor, but not epidermal growth factor receptor) and/or cell type specific (NIH 3T3 cells, but not COS-1, HEK293T, MCF10A, MDA-MB-231 or AU565 cells).

Ligand-induced internalisation and subsequent downregulation of receptor tyrosine kinases (RTKs) serve to determine biological outputs of their signalling. Intrinsically kinase-deficient RTKs control a variety of biological responses, however, the mechanism of their downregulation is not well understood and its analysis is focused exclusively on the ErbB3 receptor. The Eph group of RTKs is represented by the EphA and EphB subclasses. Each bears one kinase-inactive member, EphA10 and EphB6, respectively, suggesting an important role for these molecules in the Eph signalling network. While EphB6 effects on cell behaviour have been assessed, the mechanism of its downregulation remains elusive. Our work reveals that EphB6 and its kinase-active relative, and signaling partner, EphB4, are downregulated in a similar manner in response to their common ligand, ephrin-B2. Following stimulation, both receptors are internalised through clathrin-coated pits and are degraded in lysosomes. Their targeting for lysosomal degradation relies on the activity of an early endosome regulator, the Rab5 GTPase, as this process is inhibited in the presence of a Rab5 dominant-negative variant. EphB6 also interacts with the Hsp90 chaperone and EphB6 downregulation is preceded by their rapid dissociation. Moreover, the inhibition of Hsp90 results in EphB6 degradation, mimicking its ligand-induced downregulation. These processes appear to rely on overlapping mechanisms, since Hsp90 inhibition does not significantly enhance ligand-induced EphB6 elimination. Taken together, our observations define a novel mechanism for intrinsically kinase-deficient RTK downregulation and support an intriguing model, where Hsp90 dissociation acts as a trigger for ligand-induced receptor removal.

No description available.

<p> Schizophrenia is treated with haloperidol, an antipsychotic drug. Although highly effective in treating the positive symptoms of this disease, extrapyramidal side effects also accompany haloperidol treatment, including parkinsonism. Previous investigations revealed that dopamine receptor blockade by haloperidol was not temporally correlated with the appearance of parkinsonian side effects, which begin approximately 3 weeks after haloperidol treatment. In fact, by using tyrosine hydroxylase as a marker for dopamine, TH-immunoreactivity was significantly decreased 5 minutes after haloperidol administration and further downregulation was seen after 10 minutes. Microglial activation has also been implicated in Parkinson's disease models. Haloperidol also induces maximal microglial activation at 5 minutes after administration, with activation increasing by 2 minutes. In this respect, microglial activation may precede TH downregulation, thereby mediating the downregulation. In order to test this possibility, minocycline, a microglial inhibitor, was administered to Sprague-Dawley rats. Minocycline successfully inhibited microglial activation and showed partial protection over TH levels. Caffeine and nicotine have also been implicated as neuroprotective agents in Parkinson's disease. Epidemiological evidence has indicated that both caffeine and nicotine protect against Parkinson's disease. Therefore, caffeine and nicotine were independently tested and found to both prevent TH downregulation and inhibit microglial activation. Overall, microglial activation has been found to correlate with TH downregulation induced by haloperidol. Minocycline, nicotine and caffeine have all been found to inhibit microglial activation, preventing neurotoxicity associated with haloperidol administration. </p> / Thesis / Master of Science (MSc)

The physiological responses of angiotensin II (Ang II) are mediated across the cell membrane through the angiotensin II type 1 receptor (AT1R), a heptahelical membrane protein coupled to trimeric G-proteins on the cytosolic side. AT1R on binding its ligand, Ang II, leads to downregulation of cell-surface receptor and also its mRNA. We have investigated whether the 3'- and 5'-untranslated regions of the human AT1R mRNA mediate the degradation of hAT1R mRNA by post-transcriptional mechanisms in human adrenocortical carcinoma cell line (H295R cells). Protein kinase C (PKC) activator, phorbol-12-myristate-13-acetate (PMA), showed that the downregulation of hAT1R mRNA is mediated by the PKC pathway. Experiments performed in the presence of cycloheximide and/or Ang II demonstrated that protein translation is essential for hAT1R mRNA downregulation. In vitro cell-free RNA degradation assays did not show any increase in the rate of degradation of in vitro transcribed RNA in the presence of cytoplasmic extract from cells treated with Ang II, which suggested that hAT1R steady state mRNA levels may not be mediated by changes in mRNA degradation rates. Luciferase assay after transient transfection of chimeric plasmids of luciferase and hAT1R-3'-UTR showed that Ang II stabilizes the mRNA rather than increase the rate of degradation. Similar results were observed in Northern blot experiments utilizing beta-globin fusion with 3'-UTR that led to stabilization of the chimeric mRNA. Luciferase fusion constructs with both 5'- and 3'-UTRs demonstrated that UTRs are not involved in the Ang II-mediated degradation of hAT1R mRNA. Experiments using transcriptional inhibitor actinomycin D demonstrated that the hAT1R mRNA is not destabilized in response to Ang II activation in H295R cells. Nuclear run-on assay performed in the adrenocortical carcinoma cells demonstrated that the Ang II-stimulated downregulation of hAT1R is mediated by transcriptional inhibition. The transcription of hAT1R mRNA was reduced by 44 and 70% after Ang II treatment for 1 and 2 hours, respectively. Taken together, these findings suggest that the Ang II-induced downregulation of hAT1R steady state mRNA levels is transcriptionally controlled and is not mediated by post-transcriptional mechanisms.

Association of Mycobacterium avium subspecies paratuberculosis (MAP) with Crohn's disease (CD) and not with ulcerative colitis (UC), two forms of inflammatory bowel disease (IBD), has been vigorously debated in recent years. This theory has been strengthened by recent culture of MAP from breast milk, intestinal tissue and Blood from patients with active Crohn's disease. Culture of MAP from clinical samples remained challenging due to the fastidious nature of MAP including its lack of cell wall in infected patients. The advent of real time PCR has proven to be significant in infectious disease diagnostics. In this study, real time reverse transcriptase PCR (RT-PCR) assay based on targeting mRNA of the IS900 gene unique to MAP has been developed. All variables included in RNA isolation, cDNA synthesis and real time PCR amplification have been optimized. Oligonucleotide primers were designed to amplify 165 bp specific to MAP and the assay demonstrated sensitivity of 4 genomes per sample. In hope this real time RT-PCR may aid in the detection of viable MAP cells in Crohn's disease patients, a total of 45 clinical samples were analyzed. Portion of each sample was also subjected to 12 weeks culture followed by standard nested PCR analysis. The samples consisted of 17 cultures (originated from 13 CD, 1 UC and 3 NIBD subjects), 24 buffy coat blood (originated from 7 CD, 2 UC, 11 NIBD and 4 healthy subjects) and 4 intestinal biopsies from 2 CD patients. Real time RT-PCR detected viable MAP in 11/17 (65%) of iii suspected cultures compared to 12/17 (70%) by nested PCR including 77% and 84% from CD samples by both methods, respectively. Real time RT-PCR detected MAP RNA directly from 3/7 (42%) CD, 2/2 (100%) UC and 0/4 healthy controls similar to results following long term culture incubation and nested PCR analysis. Interestingly, real time RT-PCR detected viable MAP in 2/11 (13%) compared to 4/11 (26%) by culture and nested PCR in NIBD patients. For tissue samples, real time RT-PCR detected viable MAP in one CD patient with the culture outcome remains pending. This study clearly indicates that a 12-hr real time RT-PCR assay provided data that are similar to those from 12 weeks culture and nested PCR analysis. Consequently, use of real time In our laboratory, we previously demonstrated a possible downregulation in the Interferon-gamma receptor gene (IFNGR1) in patients with active Crohn's disease using microarray chip analysis. In this study, measurement of RNA by real time qRT-PCR indicated a possible downregulation in 5/6 CD patients compared to 0/12 controls. The preliminary data suggest that downregulation in INFGR1 gene, and the detection of viable MAP in CD patients provides yet the strongest evidence toward the linkage between MAP and CD etiology.

No description available.

Les microARNs appartiennent à la famille des petits ARNs non-codants et agissent comme inhibiteurs des ARN messagers et/ou de leurs produits protéiques. Les mi- croARNs sont différents des petits ARNs interférants (siARN) car ils atténuent l’ex- pression au lieu de l’éliminer. Dans les dernières années, de nombreux microARNs et leurs cibles ont été découverts chez les mammifères et les plantes. La bioinforma- tique joue un rôle important dans ce domaine, et des programmes informatiques de découvertes de cibles ont été mis à la disposition de la communauté scientifique. Les microARNs peuvent réguler chacun des centaines de gènes, et les profils d’expression de ces derniers peuvent servir comme classificateurs de certains cancers. La modélisation des microARNs artificiels est donc justifiable, où l’un pourrait cibler des oncogènes surexprimés et promouvoir une prolifération de cellules en santé. Un outil pour créer des microARNs artificiels, nommé MultiTar V1.0, a été créé et est disponible comme application web. L’outil se base sur des propriétés structurelles et biochimiques des microARNs et utilise la recherche tabou, une métaheuristique. Il est démontré que des microARNs conçus in-silico peuvent avoir des effets lorsque testés in-vitro. Les sé- quences 3’UTR des gènes E2F1, E2F2 et E2F3 ont été soumises en entrée au programme MultiTar, et les microARNs prédits ont ensuite été testés avec des essais luciférases, des western blots et des courbes de croissance cellulaire. Au moins un microARN artificiel est capable de réguler les trois gènes par essais luciférases, et chacun des microARNs a pu réguler l’expression de E2F1 et E2F2 dans les western blots. Les courbes de crois- sance démontrent que chacun des microARNs interfère avec la croissance cellulaire. Ces résultats ouvrent de nouvelles portes vers des possibilités thérapeutiques. / MicroRNAs belong to the family of small non-coding RNAs and act as down regula- tors of messenger RNAs and/or their protein products. microRNAs differ from siRNAs by downregulating instead of shutting down. In recent years, numerous microRNAs and their targets have been found in mammals and plants. Bioinformatics plays a big role in this field, as software has emerged to find new microRNA targets. Each individual microRNA can regulate hundreds of genes, and it has been shown that microRNA expression profiles can classify human cancers. The need for artificially created mi- croRNAs is then justified, as one could target overexpressed oncogenes and promote healthy cell proliferation. MultiTar V1.0, a tool for creating artificial microRNAs, has been implemented and is available as a web application. The tool relies on structural and biological properties of microRNAs and uses a Tabusearch metaheuristic. A typical biological problem is presented and it is shown that an in-silico microRNA has in-vitro effects. The 3’UTR sequences of E2F1, E2F2 and E2F3 were given as input to the tool, and predicted microRNAs were then tested using luciferase essays, western blots and growth curves. At least one microRNA is able to regulate the three genes with luciferase essays and all of the created microRNAs were able to regulate the expres- sion of E2F1 and E2F2 with western blots. Growth curves were also studied in order to investigate overall biological effects, and reduction in growth was observed for all solutions. Results obtained with the predicted microRNAs and the target genes open a new door into therapeutic possibilities.

Platelet-derived growth factor (PDGF) isoforms, which bind to closely related a- and b-tyrosine kinase receptors, induce migration, proliferation, survival and differentiation of mesenchymal cells. They signal by the active receptor attracting Src homology 2 (SH2) domain containing proteins, which subsequently initiate a set of signaling pathways. The aim of this thesis was to elucidate regulatory mechanisms involved in PDGFRb signaling. In the first two projects we investigated the roles in downregulation of PDGFRb of two related adaptor proteins, i.e. ALG-2 interacting protein X (Alix) and His-domain containing protein tyrosine phosphatase (HD-PTP) functions of. We found that Alix and HD-PTP influence ubiquitination of PDGFRb following PDGF stimulation, by affecting the E3 ligase c-Cbl. Alix enhances complex formation between c-Cbl and PDGFRb, increases c-Cbl phosphorylation and decreases its stability. Interestingly, while both HD-PTP and Alix participate in degradation of PDGFRb, only Alix affects receptor internalization. Moreover, we demonstrated that absence of HD-PTP promotes cell proliferation. In conclusion, we suggest that both Alix and HD-PTP are important adaptor proteins in regulation of PDGFRb downregulation, although the observed differences between their actions suggest that Alix and HD-PTP exert their functions via different mechanisms. The third study explored the importance of tyrosine residue 857 in the activation loop of PDGFRb. We report that, in vitro the tyrosine residue 857 to phenylalanine (Y857F) mutant receptor kinase activity is diminished while in vivo it does not affect the phosphorylation of PDGFRb. The phosphorylation pattern of PDGFRb revealed that most sites in the Y857F mutant receptor were phosphorylated similarly as in the wild-type receptor. However, tyrosine residue 771 was found to be hyperphosphorylated in the Y857F mutant receptor. This may be due to defective phosphorylation and activation of SHP-2, since it has been shown to dephosphorylate the receptor at Y771. In addition, activation of the Erk1/2 and Akt pathways was defective downstream of the Y857F mutant receptor. Interestingly, the Y857F mutant receptor was able to mediate cell migration, but not proliferation. The last study investigated a role of the tyrosine kinase Fer in PDGF signaling. We showed that Fer interacted with and was activated by PDGFRb in a ligand-dependent manner. In cells depleted of Fer, receptor phosphorylation was decreased and phosphorylation of Stat3 was abolished, whereas Stat5, Erk1/2 and Akt were activated normally. Colony formation in soft agar was abolished in cells depleted of Fer, but no effect was seen on cell proliferation and migration. Since Stat3 has been shown to be involved in transformation, we speculate that phosphorylation of Stat3 in Fer-depleted cells, affects the ability of cells to form colonies.

Plasmon-resonant nanoparticle complexes show promising potential for light-triggered, controllable delivery of deoxyribonucleic acids (DNA) for research and therapeutic purposes. For example, the approach of RNA interference (RNAi) ‒ using antisense DNA or RNA oligonucleotides to silence activity of a specific pathogenic gene transcript and reduce expression of the encoded protein ‒ is very useful in dissecting genetic function and holds promise as a molecular therapeutic. Herein, we investigate the mechanism and probe the in vitro therapeutic potential of DNA light-triggered release from plasmonic nanoparticles. First, we investigate the mechanism of light-triggered release by dehybridizing double-stranded (dsDNA) via laser illumination from two types of nanoparticle substrates: gold (Au) nanoshells and Au nanorods. Both light-triggered and thermally induced releases are distinctly observable from nanoshell-based complexes. Surprisingly, no analogous measurable light-triggered release was observable from nanorod-based complexes below the DNA melting temperature. These results suggest that a nonthermal mechanism may play a role in light-triggered DNA release. Second, we demonstrate the in vitro light-triggered release of molecules non-covalently attached within dsDNA bound to the Au nanoshell surface. DAPI (4',6-diamidino-2-phenylindole), a bright blue fluorescent molecule that binds reversibly to double-stranded DNA, was chosen to visualize this intracellular light-induced release process. Illumination through the cell membrane of the nanoshell-dsDNA-DAPI complexes dehybridizes the DNA and releases the DAPI molecules within living cells. The DAPI molecules diffuse to the nucleus and associate with the cell’s endogenous DNA. This work could have future applications towards drug delivery of molecules that associate with dsDNA. Finally, we demonstrate an engineered Au nanoshell (AuNS)-based therapeutic oligonucleotide delivery vehicle, designed to release its cargo on demand upon illumination with a near-infrared (NIR) laser. A poly(L)lysine peptide (PLL) epilayer coated onto the AuNS surface (AuNS-PLL) is used to capture intact, single-stranded antisense DNA oligonucleotide, or alternatively, double-stranded short-interfering RNA (siRNA) molecules. A green fluorescent protein (GFP)-expressing human lung cancer H1299 cell line was used to determine cellular uptake and GFP gene silencing mediated by AuNS-PLL delivery vector. The light-triggered release of oligonucleotides could have broad applications in the study of cellular processes and in the development of intracellular targeted therapies.