Our lab recently described a role for JAK-STAT signaling in the maintenance of quiescence during the murine hair cycle. Research into signaling pathways and cytokines/growth factors involved in the mammalian hair cycle has not focused extensively on the JAK-STAT pathway. In this thesis, I investigated the upstream effector(s) and downstream mechanisms of JAK-STAT signaling in the HFSC during telogen, using a variety of methods, including murine conditional mutants of the JAK-STAT pathway, pharmacological and immunological techniques. The mechanism through which OSM exerts this effect is via JAK-STAT5 signaling downstream of the OSM receptor, which is antagonized by pharmacological JAK inhibition. Conditional epidermal ablation of OSMR or STAT5 during early- and mid-telogen (P42 – P60) shortens the telogen phase significantly, and inhibition of macrophages by way of neutralizing antibodies, small molecule inhibitors, and genetic ablation (with Csf1r-CreER::R26-iDTR mice) during telogen also promotes hair growth. Single-cell RNA sequencing of dermal immune cells across murine telogen identified a distinct subset of TREM2+ macrophages that are enriched for OSM, and gene-set analysis suggests these “trichophages” are similar to the microglia of the central nervous system. I show that this distinct subset of TREM2+ macrophages predominate during early- and mid-telogen, where they produce Oncostatin M (OSM), which is sufficient to maintain quiescence of hair follicle stem cells (HFSCs). Proliferation of HFSCs and hair growth is associated with depletion of this subset of TREM2+ macrophages. Interestingly, macrophage markers and OSM were found to be upregulated in the balding scalp of males with androgenetic alopecia, suggesting that this mechanism is physiologically relevant in the control of human hair cycling.

The aim of this project was to explore the role of the tyrosine kinases JAK1 and JAK3 in cytokine signalling, focusing on interleukin-2 signalling in CD8⁺ effector T lymphocytes. Initial experiments compared the effects of the pan JAK1/JAK3 inhibitor tofacitinib, the selective JAK1 inhibitor GSK186, and the selective JAK3 inhibitor GSK192 on IL-2 control of effector CD8+ cytotoxic T cells (CTL). On the basis of these preliminary data, a detailed analysis of the effect of tofacitinib on effector CD8⁺ T lymphocytes was performed. Phosphorylation events regulated by tofacitinib were identified using mass spectrometry analysis of SILAC (stable isotope labelling with amino acids in cell culture) labelled CTL. Tofacitinib regulated a selective number of phosphorylation sites, with less than 1.2% of the CTL phosphoproteome significantly regulated by tofacitinib treatment following 4hrs tofacitinib treatment. Proteins with downregulated phosphorylation sites were enriched in functions related to the Jak-STAT signalling, regulation of gene expression, and MAPK signalling cascades. Proteins with upregulated phosphorylations were also enriched in functions related to regulation of gene transcription. The proteome of tofacitinib treated CTL was defined by label free mass spectrometry. Approximately 4.5% of the CTL proteome was significantly regulated following 24 hours tofacitinib treatment, suggesting tofacitinib regulates the expression of a selective subset of proteins. Tofacitinib treatment resulted in the downregulation of proteins involved in ribosome biosynthesis, steroid biosynthesis, regulation of transcription and the cell cycle; and the upregulation of proteins with hydrolase activity, and with roles in the lysosome and extracellular exosomes. The phosphoproteomic and proteomic data demonstrates that JAK kinase dependent IL-2 signalling regulates essential processes in CTL by controlling a selective number of phosphorylation events and proteins. Validation of proteins identified as regulated following tofacitinib treatment identified new targets of IL-2 signalling in CTL, including the transcription factor NFIL3. NFIL3 was shown to be upregulated in CD8⁺ T lymphocytes following stimulation with IL-2 and regulated perforin and CD62L expression, suggesting a role in the regulation of CTL effector function.

Identifying the molecular interactions of pathogens in different mosquito species is critical for understanding how mosquitoes transmit diseases. In this study, the role of the Jak-STAT immune signalling pathway in two different mosquito species, (Aedes aegypti L.) and (Culex quinquefasciatus L.) was assessed. Using in silico analysis tools, cell culture, and molecular techniques, changes in gene expression were assessed during lipopolysaccharide (LPS) challenge and West Nile virus (WNV) infection in the two species. It was found that activation of the Jak-STAT pathway occurred more quickly in Ae. aegypti cells compared to Cx. quinquefasciatus cells during LPS exposure. During WNV infections, no significant differences were observed, although preliminary evidence suggests that differential activation of the Jak-STAT pathway may exist between the two species. This research extends our understanding of the mosquito immune system while demonstrating the critical importance of vector-virus interactions across different mosquito species.

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Identifying the molecular interactions of pathogens in different mosquito species is critical for understanding how mosquitoes transmit diseases. In this study, the role of the Jak-STAT immune signalling pathway in two different mosquito species, (Aedes aegypti L.) and (Culex quinquefasciatus L.) was assessed. Using in silico analysis tools, cell culture, and molecular techniques, changes in gene expression were assessed during lipopolysaccharide (LPS) challenge and West Nile virus (WNV) infection in the two species. It was found that activation of the Jak-STAT pathway occurred more quickly in Ae. aegypti cells compared to Cx. quinquefasciatus cells during LPS exposure. During WNV infections, no significant differences were observed, although preliminary evidence suggests that differential activation of the Jak-STAT pathway may exist between the two species. This research extends our understanding of the mosquito immune system while demonstrating the critical importance of vector-virus interactions across different mosquito species.

Made available in DSpace on 2015-07-22T20:49:35Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-05-26 / A via de sinalização Janus Kinase/Transdutores de Sinal e Ativadores de Transcrição (JAK/STAT) é uma das mais importantes vias de sinalização para transdução do sinal usadas pelas citocinas. Entretanto, o papel da via JAK/STAT na lesão de isquemia reperfusão (IR) hepática ainda não foi explorado. Este estudo foca no papel da proteína-quinase JAK2, que está acima de STAT-1 na cascata de sinalização JAK/STAT, e o seu papel no mecanismo da lesão de IR hepática. Isquemia quente parcial (lobos esquerdo e médio) foi induzida no fígado de camundongos C57BL/6 por 90 minutos, seguidos por 6 horas de reperfusão. Os animais foram tratados com o inibidor específico de JAK-2 (Tyrphostin AG490; 40 mg/kg, i.p) ou veículo (DMSO), 60 minutos antes do início da isquemia. O bloqueio de JAK2 resultou em significante redução da lesão hepática e da apoptose de hepatócitos. A análise imunohistoquímica revelou uma importante redução no infiltrado de macrófagos e de polimorfonucleares neutrófilos no tecido hepático dos animais que receberam AG490 anteriormente ao insulto isquêmico, comparado aos animais controle (DMSO). A expressão de citocinas pro-inflamatórias (TNF-α, IL-6, IL-1β) e das quimiocinas (CXCL-10 and CXCL-2) também estava reduzida no grupo tratado com AG490, comparado ao grupo controle. A análise da presença de células apoptóticas, através da técnica de TUNEL no tecido hepático dos camundongos, revelou menor número de hepatócitos em processo de apoptose no grupo tratado com AG490 consistente com o achado de western-blot que revelou menor expressão da proteína caspase-3 clivada em paralelo com o aumento da expressão da proteína anti-apoptótica Bcl-XL, no grupo que recebeu AG490. Para confirmar nossos achados in vivo nós testamos AG490 (75 mM) em culturas de macrófagos derivados da medula óssea (BMM) e em cultura de células da linhagem de hepatoma- CRL1830, ambas estimuladas com LPS (10 ng/ml). Em culturas de macrófagos derivados da medula óssea, AG490 reduziu a expressão gênica das citocinas e quimiocinas pró-inflamatórias (IL-6, IL-12p40, IL-1β, CXCL-10 e iNOS) que encontravam-se elevadas apos estimulação com LPS. Em cultura de células da linhagem de hepatoma- CRL1830, AG490 reduziu a expressão de caspase-3 clivada. Além do que, o bloqueio de JAK2 inibiu a fosforilação de STAT1 e STAT3. Pela primeira vez demonstramos que a sinalização por JAK2 é essencial na fisiopatologia da lesão de IR hepática, já que seu bloqueio seletivo protegue o fígado contra inflamação e apoptose. / The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling is one of the major pathways for cytokine signal transduction. However, the role of the JAK/STAT pathway in liver I/R is not clear. This study focuses on JAK2, which functions upstream of STAT-1 in JAK/STAT, and its role in the mechanism of liver IRI. Partial warm ischemia was produced in the hepatic lobes of C57BL/6 mice for 90min, followed by 6h of reperfusion. Mice were treated with JAK-2 inhibitor (Tyrphostin AG490; 40 mg/kg, i.p) or veicle, 60min prior to ischemic insult. JAK2 blockade resulted in significant reduction of hepatocyte apoptosis and liver injury. Macrophage and neutrophil infiltration, as assessed by immunohistochemistry, was markedly decreased in AG490-treated livers, compared with controls. The expression of proinflammatory cytokines (TNF-α, IL-6, IL-1β) and chemokines (CXCL-10 and CXCL-2) was also significantly reduced in AG490-treated group, compared with controls. AG490-treated livers showed less TUNEL positive cells and reduced cleaved caspase-3 protein expression in parallel with increased Bcl-XL expression. We employed AG490 (75 mM) in primary bone marrow-derived macrophage (BMM) and hepatoma cell (CRL1830) cultures, both stimulated with LPS (10 ng/ml). In BMM cultures, AG490 depressed otherwise LPSinduced pro-inflammatory gene expression programs (IL-6, IL-12b, IL-1β, CXCL-10 and iNOS). In hepatoma cells, AG490 reduced cleaved-caspase-3 expression. Moreover, JAK2 blockade inhibited STAT1 and STAT3 phosphorylation. This is the first report, which documents that JAK2 signaling is essential in the pathophysiology of liver IRI, as its selective blockage ameliorated the disease process and protected livers from inflammation and apoptosis. / TEDE / BV UNIFESP: Teses e dissertações / 103 p.

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Tumour heterogeneity is a key hurdle for the effective treatment of cancer using oncolytic viruses (OVs). A better understanding of the pathways involved in delineating tumour cell resistance and hypersensitivity to OVs is critical in order to guide the development of new therapeutic strategies to enhance OVs. In this thesis, I performed a comparative genetic and epigenetic study of the murine OV-resistant colon cancer cell line CT26.WT and its hypersensitive subclone CT26.lacZ. This study led to the identification of retroviral insertion sites in AKAP7 and TP53RK genes, that are potentially involved in conveying sensitivity to infection by OVs and the dysregulation of the interferon antiviral response in the CT26.lacZ cell line. Gene overexpression and gene silencing experiments suggest a functional role of these proteins in controlling viral growth. Further investigation of these genes and their relationship to antiviral response pathways is warranted and may lead to novel strategies for improving the therapeutic activity of OVs.

Essential thrombocythemia (ET) is a rare myeloproliferative neoplasm affecting 43.7 out of every 100,000 people in the United States. The disease is characterized by abnormally high platelet counts, mutational abnormalities in Janus Kinase 2 (JAK2)/Calreticulin (CALR)/myeloproliferative leukemia virus oncogene (MPL), and increased megakaryocyte production and differentiation. The average age of onset for patients with ET is between 65-70 years, but recent studies have demonstrated a downward trend in the age of diagnosis. Mechanistically, ET mutations cause the dimerization of JAK and upregulation of the JAK-STAT pathways. Common treatment approaches seek to use cytoreduction and platelet inhibition to lower the risk of a thrombotic event. Hydroxyurea and low-dose aspirin have been the gold-standard of treatment for ET patients. This thesis sought to compare the current available therapy with second-line treatments and investigational treatments. Anagrelide is a key second-line treatment for ET that is used in the event of intolerance to hydroxyurea. It acts through cytoreductive mechanisms which result in a decreased platelet count. Major bleeding is a severe adverse event associated with anagrelide. Interferons are another second-line defense in the treatment of ET despite a lack of FDA approval for this indication. Interferons act directly to reduce platelet counts and, unlike other drug classes, mount an immunological response against the JAK2 stem cells to reduce the allelic burden. An immunological approach to ET may be key to the sustained treatment of the disorder without a daily dosing regimen. Despite the promise of interferons, severe adverse effects limit the adherence of many patients to this class of drugs. JAK inhibitors are an investigational drug class that acts directly through the JAK-STAT pathway. JAK inhibitors have shown little efficacy in the treatment of ET and may be better suited for treatment in combination therapies. Telomerase inhibitors are one such investigational drug class that may pair well with JAK inhibitors for the treatment of ET. All of these drug classes were compared to hydroxyurea with respect to their pharmacokinetics, pharmacodynamics, and patient evaluation. Hydroxyurea and low-dose aspirin showed superiority in comparison to other drug classes due to their low toxicity profile and minimum adverse side-effects, high oral bioavailability and wide distribution, high adherence, and production of the most uniform response to reducing thrombotic events and platelet counts. The interferon drug class shows unique potential for the treatment of ET and should be placed above the second-line treatment standard of anagrelide due to its benefits in treatment of younger and pregnant patients. Interferons are the only class of drug for the treatment of ET that did not increase the risk of drug-related leukemogenic transformations. Despite non-adherence due to side-effects and lack of an oral administration, interferons are superior to anagrelide due to their longer dosing interval and immunological attack on JAK2 stem cells. Treatment of ET with anagrelide has shown similar efficacy to hydroxyurea and interferons in platelet reduction and rivals hydroxyurea in the prevention of thrombotic risk. Despite this benefit, the risk of bleeding associated with anagrelide is a significant disadvantage. Hydroxyurea and low-dose aspirin remain the current standard of treatment for patients with ET, although new approaches may soon be available.

Circadian clock in the suprachiasmatic nucleus of the hypothalamus (SCN) regulates daily rhythms in behavior and physiology and is an important part of the mechanisms regulating mammalian homeostasis. SCN are synchronized with a 24hour cycle mainly by light, but they can also be regulated by a variety of nonphotic signals, such as growth factors, opioids, cytokines, or lipopolysaccharide (LPS), which act by inducing the JAK/STAT signaling pathway. STAT family proteins (i.e. signal transducers and activator of transcription) regulate many aspects of cellular physiology, from growth and differentiation to immune response. However, the JAK/STAT signaling pathway has not yet been studied in the SCN and the function of STAT proteins in the SCN has not yet been clarified. In the first part of the thesis, we focused on localization of STAT3 and STAT5 proteins in the rat SCN and determination of rhythm in proteins and mRNA. Our experiments showed the daily rhythm in the levels of STAT3 protein in SCN astrocytes of rat with low but significant amplitude and with maximum in the morning. In addition, we revealed strong but nonrhythmic expression of STAT5A protein in astrocytes and STAT5B protein in nonastrocytic cells of SCN. It was also found that Stat3 mRNA show, similarly to protein, circadian rhythm in...