Tau, a microtubule associated protein, has been implicated in the formation of neurofibrillary tangles, a hallmark of Alzheimer’s disease.1 Resulting from the hyperphosphorylation of tyrosine, serine and threonine residues, protein kinases play an intricate role in the pathway of NFT formation.2 While a number of serine/ threonine kinases such as Glycogen Synthase Kinase 3 beta, have been implicated in the hyperphosphorylation of tau, and it is now believed that tyrosine phosphorylation plays a role. It appears that the degree to which tau is phosphorylated, and region in which phosphorylation occurs play a critical role in aggregate formation. Here we present the use of Ferrocene bioconjugates to monitor tyrosine-mediated hyperphosphorylation and aggregation. Electrochemistry, electron microscopy, surface plasmon resonance, Time of Flight Secondary Ion Mass Spectrometry and X-Ray Photoelectron Spectroscopy were used to gain insight into the role of tyrosine phosphorylation on hyperphosphorylation.

Tau, a microtubule associated protein, has been implicated in the formation of neurofibrillary tangles, a hallmark of Alzheimer’s disease.1 Resulting from the hyperphosphorylation of tyrosine, serine and threonine residues, protein kinases play an intricate role in the pathway of NFT formation.2 While a number of serine/ threonine kinases such as Glycogen Synthase Kinase 3 beta, have been implicated in the hyperphosphorylation of tau, and it is now believed that tyrosine phosphorylation plays a role. It appears that the degree to which tau is phosphorylated, and region in which phosphorylation occurs play a critical role in aggregate formation. Here we present the use of Ferrocene bioconjugates to monitor tyrosine-mediated hyperphosphorylation and aggregation. Electrochemistry, electron microscopy, surface plasmon resonance, Time of Flight Secondary Ion Mass Spectrometry and X-Ray Photoelectron Spectroscopy were used to gain insight into the role of tyrosine phosphorylation on hyperphosphorylation.

Alzheimer¡¦s disease (AD), one of the most common dementia, is characterized by the formation two types of aggregation in the brain: senile plaques and neurofibrillary tangles (NFTs). NFTs are composed of hyperphosphorylated Tau. Tau protein mainly expressed in brain and was identified as one of the microtubule-associated proteins (MAPs). Hyperphophorylation on Tau affects its binding to tubulin and capacity to promote microtubule assembly. A number of proline-directed kinase capable of phosphorylating PHF-Tau have been identified, including Glycogen Synthase Kinase-3£] (GSK-3£]). Here we demonstrated that GSK3£] can co-purify with PHFs and can co-localize with Tau in vitro in N18 cells. To examine the phosphorylation mechanism of Tau by GSK-3£], N-terminal, C-terminal, T231A, T231E, 154~441, S396A, S400A, S404A, S413A and S396A S400A mutants of Tau were used, respectively. We were able to demonstrate that phosphorylation on Thr231 and Ser404 in Tau may play important roles for GSK3£] phosphorylation and its functional regulation. Most importantly, we have proved that T231P motif is necessary and critical for Tau phosphorylation by GSK3£]. Moreover, we used T231E, S396E and S400E mutants of Tau to understand the functional regulation of Tau by GSK3£] phosphorylation by tubulin assembly assay. Surprisingly, we observed all of these Tau mutants can promote tubulin assembly and form tubulin bundles in N18 cells. It has been proved that Pin1 WW domain can bind to Cdc2-phosphorylated Thr-231-Pro motif of Tau and restore the ability of Tau to promote tubulin assembly. In this study, we also studied whether Pin1 can regulate GSK3£]- phosphorylated Tau. The results show that Pin1 WW domain can bind to phosphorylated Thr-231 of Tau by GSK3£] and restore the ability of Tau to promote tubulin assembly.

The peri-implantation period in mammals is critical with respect to survival of the conceptus and establishment of pregnancy. During this period of pregnancy, reciprocal communication between ovary, conceptus, and endometrium is required for successful implantation and placentation. Therefore, studies were conducted to indentify and characterize novel endometrial genes important for implantation and conceptus development in the ovine uterus. The first and second studies defined the uterine expression of seven members of the cathepsin (CTS) family of lysosomal proteases, and a secreted inhibitor of CTSL called cystatin C (CST3) during the peri-implantation period. In addition, regulation of CTS and CST3 by progesterone (P4) and interferon tau (IFNT) was evaluated. CTSL was the most abundant CTS in the ovine ovine uterus and was also coordinately expressed with CST3 in the endometrial epithelia and conceptus trophectoderm. CTSL and CST3 were found to be novel P4-induced and IFNT-stimulated genes in the luminal epithelial cells of the ovine endometrium. The third study identified radical S-adenosyl methionine domain containing 2 (RSAD2) and interferon-induced with helicase C domain 1 (IFIH1) in the ovine uterus. Results of this study indicated that IFNT induces RSAD2 and IFIH1 in a P4-independent manner in the stroma, immune cells, and glands of the ovine endometrium. These two genes are proposed to have biological roles in the establishment of uterine receptivity to the conceptus during implantation. The fourth study characterized endometrial expression of stanniocalcins (STC) during pregnancy. STC1 appeared in the endometrial glands on Day 18 of pregnancy, increased from Days 18 to 80, and remained abundant through Day 120 of gestation. In addition, this study demonstrated that STC1 is induced by P4 and increased by placental hormones, such as placental lactogen (CSH1) and growth hormone (GH), in the ovine endometrial glands. Collectively, these studies identified genes that are expected to be critical to unraveling the mechanism(s) of reciprocal fetal-maternal interactions required for successful implantation and pregnancy. A more complete understanding of these genes will be important for developing therapeutic strategies to prevent, treat and/or diagnose infertility in domestic animals and humans, because they are biomarkers of P4 and/or IFN effects.

This thesis presents the first search for a neutral Minimal Supersymmetric Standard Model Higgs boson decaying to a pair of hadronic taus in proton-proton collisions with a center-of-mass energy of √s = 7 TeV. Results are presented for an integrated lumi- nosity of 1.056 fb−1 using data from the ATLAS detector at the Large Hadron Collider. After signal selection, 245 events are observed. The number of events is consistent the background estimate of 256 ± 26. An exclusion limit for the Higgs boson production is derived as a function of (cross-section)×(branching ratio). This limit is presented as a function of two parameters: mA and tanβ in the mmax scenario. A point in the (mA, h tanβ) phase space is excluded if the signal hypothesis is rejected at the 95% confidence level.

This thesis presents the first search for a neutral Minimal Supersymmetric Standard Model Higgs boson decaying to a pair of hadronic taus in proton-proton collisions with a center-of-mass energy of √s = 7 TeV. Results are presented for an integrated lumi- nosity of 1.056 fb−1 using data from the ATLAS detector at the Large Hadron Collider. After signal selection, 245 events are observed. The number of events is consistent the background estimate of 256 ± 26. An exclusion limit for the Higgs boson production is derived as a function of (cross-section)×(branching ratio). This limit is presented as a function of two parameters: mA and tanβ in the mmax scenario. A point in the (mA, h tanβ) phase space is excluded if the signal hypothesis is rejected at the 95% confidence level.

Title: Study of fake-tau background with the ATLAS experiment Author: Gabriela Martinovicová Institute: Institute of Particle and Nuclear Physics Supervisor: Mgr. Vojtěch Pleskot, Ph.D., Institute of Particle and Nuclear Physics Abstract: The τ-leptons are the important final-state components, not only in the Standard Model processes but also in the processes beyond the Standard Model studied at the ATLAS experiment at CERN. They are characterized by mostly decaying into hadrons with one or three charged particles and, in most cases, with at least one neutral pion in the final-state. Due to their short decay length, only their decay products are observed in the detector. Jets naturally fake hadronically decaying τ leptons, so it is necessary to estimate such a fake-τ background. The Fake Factor method uses a correction factor, called fake factor (FF), measured from the data and applied to the data to estimate the fake-τ background in a given signal region. One of the complications is that FF differs for τ candidates faked by jets derived from quarks or gluons and thus must be measured in the control region with the same fraction of quark jets as in the signal region. The solution to this problem is the universal FF method, which proposed that from the FFs measured in samples with a large difference in the...

Aged HIV-positive (HIV+) individuals represent a large proportion of the HIV population as life spans are extended significantly by successful antiretroviral therapy. Increased age with HIV infection brings a unique set of central nervous system (CNS) complications including more rapid onset and progression of age-related diseases, loss of protein quality control and accumulation of aberrant proteins, such as hyperphosphorylated Tau (hpTau). In this context, we have discovered a new signaling connection between age-related neurodegeneration and HIV involving the PINCH protein. Particularly interesting new cysteine histidine-rich protein (PINCH), an adaptor protein in neuronal cells is involved in cytoskeletal organization, cell migration and cell survival. While some of the pathophysiological aspects of the PINCH-signaling cascade in tauopathy are largely conserved among neurodegenerative diseases such as Alzheimer’s disease, HIV and others, the presence of the HIV protein Tat impacts specific key points in the PINCH pathway that exacerbate CNS cell dysfunction. In virus-infected cells, Tat regulates viral replication. Even though neurons are not permissive to viral infection, the Tat protein can enter all cell types. Our studies show that Tat interferes with key PINCH signaling partners in neurons. PINCH is robustly expressed by neurons and to a lesser degree by astrocytes in HIV infection of the CNS; whereas, in the healthy CNS, PINCH is nearly undetectable. Similarly, protein phosphatase 1α (PP1α), one of the binding partners of PINCH, is increased in the neurons of HIV encephalitis patients and co-localizes with PINCH in neurons. PINCH is a non-catalytic scaffolding protein that binds integrin-linked kinase (ILK), and PP1α and mediates Akt and GSK3β kinase activities, all of which when disrupted contribute to aberrant Tau phosphorylation. In Tat transgenic mice, increased expression levels of PINCH, PP1α and hpTau were observed. Also, in vivo manipulations of expression levels of PINCH altered the levels of hpTau, where overexpression of PINCH increased hpTau levels in Tat transgenic mouse brains and PINCH knockdown decreased hpTau formation. Furthermore, our studies show that Tat increases levels of hpTau and PINCH, interacts with PP1α and changes the subcellular distribution of PINCH and PP1α in vitro. Tat alters the levels of ILK, Akt and GSK3β, which are key kinases associated with hpTau formation. Furthermore, our preliminary data using expression plasmid for PP1α also shows that overexpression of PP1α decreases Tat-induced aberrant hpTau formation. Our studies address HIV replication-independent functions of Tat in neurons linked to PINCH signaling. These studies address a novel pathway through which Tau may be aberrantly phosphorylated. Thus, understanding new pathways of communication among Tat, PINCH, PP1α, ILK and Tau will open new directions for the study of HIV-associated tauopathy and will provide opportunities for therapeutic interventions in age- and disease-related pathologies. Several studies report associations between the PINCH protein and HIV-associated CNS disease. PINCH is detected in the cerebrospinal fluid (CSF) of HIV patients and changes in levels during disease may be indicative of changes in disease status over time. PINCH binds hpTau in the brain and CSF, but little is known about the relevance of these interactions to HIV CNS disease. In this study, PINCH and hpTau levels were assessed in three separate CSF samples collected longitudinally from 20 HIV+ participants before and after initiating antiretroviral therapy, or before and after a change in the current regimen. Correlational analyses were conducted for CSF levels of PINCH and hpTau and other variables including plasma CD4+ T-cell count, plasma and CSF viral burden, CSF Neopterin, white blood cell (WBC) count, and antiretroviral CNS penetration-effectiveness (CPE). Results suggest that in these HIV participants, changes in CSF levels of PINCH appear to correlate with changes in plasma CD4+ count and with changes in CSF hpTau levels, but not with plasma or CSF viral burden, Neopterin, or WBC, or with anti-retroviral regimen CPE. Furthermore, results from our case matched HIV brain-CSF study confirms that higher levels of PINCH and hpTau are detected in HIV encephalitis brains. Additionally, correlation between PINCH and hpTau levels in brain and other clinical parameters such as age at death, date of death suggesting the era of antiretroviral therapy, CPE score and cognition in HIV patients yielded interesting results that are currently being expanded upon by investigators in the Langford laboratory. Thus, these results suggest that PINCH may be involved in Tauopathy associated neurodegeneration in the HIV CNS disease. Therefore, understanding the contribution of PINCH to HIV-associated Tauopathy may provide a new therapeutic avenue for regulating synaptodendritic dysfunction associated with Tau. Moreover, characterizing the clinical significance of PINCH in the CSF may warrant including PINCH as a member of biomarker panel to assess severity or progression of HIV-associated neurocognitive alterations. / Biomedical Neuroscience

L'incidence de maladies neurodegeneratives et systémiques liées au vieillissement, tels la maladie d'Alzheimer (MA) et le diabète, augmente rapidement. Plusieurs études rapportent que les patients souffrant de diabète ont entre 50 et 75 % plus de risque de développer la MA que les gens sains du même âge. La protéine tau hyperphosphorylée est une des composantes majeures des enchevêtrements neurofibrillaires, une composante neuropathologique classique de la MA. L'étude présente examine les modifications de tau dans deux modèles de souris transgéniques développant un diabète de type 2. La phosphorylation de tau est augmentée au niveau de l'hippocampe de ces souris. Le diabète de type 2 provoque aussi la dérégulation de quelques kinases et phosphatases de tau. Cette dérégulation serait résultante de la résistance à l'insuline et l'hypothermie. Le diabète de type 2 provoquerait donc l'accélération de la cascade menant à développer la maladie d'Alzheimer.

Les démences affectent des millions de personnes dans le monde et la forme la plus répandue est la maladie d’Alzheimer (MA). Malgré des décennies de recherches, il n’y a toujours pas de traitement efficace pour contrer cette maladie. Elle est caractérisée par deux marqueurs distincts : les plaques amyloïdes extracellulaires générées par le clivage de la protéine Amyloid Precursor Protein (APP) ainsi que les enchevêtrements neurofibrillaires formés de la protéine tau. Cette dernière est également dérégulée dans une vingtaine de maladies neurodégénératives appelées tauopathies. Plusieurs études ont montré que les niveaux des microARNs (miRs) sont altérés chez des personnes atteintes de maladies neurodégénératives telles que la MA. Notamment, le miR-132 se retrouve à être l’un des plus réduit. Pour mieux comprendre l’implication des microARNs (miRs) dans la progression de la MA et les tauopathies, mon objectif de doctorat a été d’étudier le rôle du miR-132 sur la régulation de tau en utilisant aussi bien des outils in vitro que des méthodes in vivo. Nous avons identifié in vitro un facteur d’épissage, PTBP2 pouvant réguler l’inclusion d’un exon important de tau. Ce facteur est augmenté et corrèle inversement avec l’expression de miR-132 chez un groupe de patients atteints de paralysie supranucléaire progressive (PSP), une maladie tauopathique. À l’aide de tests comportementaux, nous avons également démontré qu’une abolition génétique de miR-132 chez la souris réduisait l’apprentissage et la mémoire qui sont des conséquences connues de la MA. Enfin, nous avons établi que l’absence du miR-132 accélère la phosphorylation et l’agrégation de tau dans un modèle de souris Alzheimer. Nous avons démontré que miR-132 régule directement tau par son 3’Untranslated Region (3’UTR) et que l’expression de miR-132 corrèle avec différents tests cognitifs dans une cohorte de patients atteints de la MA. De plus, nous avons développé une approche thérapeutique prometteuse en utilisant ce miR comme agent de traitement dans le cerveau d’un modèle de souris Alzheimer. Ces travaux ont contribués à la compréhension de la progression des maladies neurodégénératives multifactorielles telles que la MA. / Dementia affects millions of people worldwide and the most common form is Alzheimer’s disease (AD). After more than a century of research, there is no efficient cure for this neurodegenerative disease. There are two pathological hallmarks : senile plaques formed by beta-amyloid peptide deposits and neurofibrillary tangles composed of a hyperphosphorylated and aggregated protein called tau. Tau pathology is also found in twenty neurodegenerative diseases called tauopathies. Studies have shown that miRNA expression profiles are deregulated in post-mortem brain tissues of patients. Of interest, miRNA-132 (miR-132) was the most downregulated. To understand the role of miRNAs in AD, my main goal was to study the involvement of miR-132 in tau regulation using in vitro tools and transgenic mice. We have identified a splicing factor, PTBP2 which affects tau exon inclusion. This factor is upregulated in a subset group of tauopathic patients, (progressive supranuclear palsy (PSP)). The miR-132 level reduction was also correlated with the PTBP2 upregulation in this cohort of patients. In the second study, we have demonstrated that learning, memory formation and retention are altered in a miR-132 knockout mouse model. Finally, we have found that a long-term loss of miR-132 promotes tau hyperphosphorylation and aggregation in AD mice. We have demonstrated that tau is a direct target of miR-132 and their expression levels in human correlate with different cognitive test scores from in AD patients. Finally, we have developed a miR-132-based therapeutic strategy in the AD mouse brain with promising results. Taken together, these results have contributed to the better understanding of complex neurodegenerative diseases such as AD.