Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Lenka Burgetová Supervisor: PharmDr. Martina Čečková PhD. Specialized supervisor: Dr. Kathryn Taylor PhD. Title of diploma thesis: Investigating the role of zinc transporter ZIP 6 and STAT3 in mitosis It has been shown that STAT3 (signal transducer and activator of transcription 3) plays a role in the development of cancer. ZIP6 is the downstream target of this transcription factor. Previous research has focused mainly on the activation of STAT3 by tyrosine phosphorylation, while the effect of phosphorylation at a second site, serine 727, remained relatively uninvestigated. In this study, it is proposed that serine-phosphorylated STAT3 is activated throughout mitosis in tamoxifen-resistant (TamR) breast cancer cells and that zinc transporter ZIP6 and serine-phosphorylated STAT3 are involved in a zinc-mediated mitotic mechanism. After using nocodazole to induce mitotic arrest, the expression of tyrosine- phosphorylated STAT3 protein was observed to be reduced while the expression of serine- phosphorylated STAT3 was increased. Zinc supplementation after nocodazole treatment appeared to push cells through mitotic-arrest and cause proteolytic cleavage of STAT3 suggesting a novel...

Alzheimer’s disease (AD), one of the major causes of disability and mortality in Western societies, is a progressive age-related neurodegenerative disorder. Increasing evidence suggests the etiology of AD may involve disruptions of zinc (Zn) homeostasis. We hypothesize that disruption of Zn homeostasis leads to alterations of Zn transporter (ZnT) proteins, resulting in increased production of neurotoxic amyloid beta (Aβ) peptide in AD brain. To address this hypothesis we carried out the following studies. 1. We characterized alterations of ZnT-1, ZnT-4 and ZnT-6 in the brain of preclinical AD (PCAD) subjects, who show no overt clinical manifestations of AD but demonstrate significant AD pathology at autopsy. 2. We identified the presence of ZnT-2 in human brain and compared protein levels in the brains of subjects with PCAD, mild cognitive impairment (MCI), early (EAD), and late-stage AD (LAD) to those in age matched normal control (NC) subjects. 3. We examined the relationship between protein levels of ZnT-1, ZnT-2, ZnT-4, ZnT-6 and Aβ produced by H4 human neuroglioma cells (H4-APP) transfected to overexpress amyloid precursor protein (APP), treated with short interfering RNA (siRNA) against each ZnT. Our data show a significant decrease (P < 0.05) of ZnT-1 and a significant increase of ZnT-6 in hippocampus/parahippo-campal gyrus (HPG) of PCAD subjects. In PCAD cerebellum (CER) the data show a significant increase of ZnT-4 and ZnT-6 compared to NC subjects. Levels of ZnT-2 were also significantly decreased in HPG of PCAD subjects compared to NC subjects. In addition, levels of ZnT-2 were significantly (P < 0.05) elevated in SMTG of PCAD and MCI subjects, compared to NC subjects. ZnT-2 was significantly (P < 0.05) elevated in HPG of EAD and LAD, and in SMTG of LAD brains, but was significantly (P < 0.05) decreased in LAD CER compared to NC subjects. siRNA mediated attenuation of each ZnT protein studied (ZnT-2, ZnT-4 and ZnT-6) led to significantly (P < 0.05) decreased production of Aβ compared to controls. Our results suggest alterations in Zn transport may play a role in Aβ processing and contribute to the neuropathology of AD.

Zinc is the second most abundant transition metal in biological systems. This cation is required for the catalytic activity of hundreds of enzymes which mediate protein synthesis, DNA replication and cell division. Despite the central importance of zinc in cellular homeostasis, the mechanism of zinc uptake, compartmentalization and efflux is unknown. Recently, a family of proteins, called ZIP, has been shown to control zinc uptake. Mutations in one of the genes coding for these proteins (ZIP4) can lead to potentially life-threatening diseases like Acrodermatitis Enteropathica and high levels of ZIP4 have been detected in patients suffering from pancreatic cancer. Therefore our goal is to investigate the mechanism of ZIP4 transport and regulation. It was previously shown that the intracellular loop between transmembrane III and IV (M3M4) of ZIP4 is ubiquitinated in the presence of high intracellular zinc which lead to protein degradation. Our initial hypothesis was that the large intracellular domain of ZIP4 (M3M4) is a sensor which detects the intracellular concentration of zinc and regulates the surface expression of ZIP4. In order to test this hypothesis we expressed and purified the M3M4 domain to examine the ability of M3M4 to bind zinc. Our results have demonstrated that M3M4 binds zinc with a 2:1 zinc:protein stoichiometry with nanomolar affinity. We have also shown that upon binding of zinc, M3M4 undergoes a large conformational change.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-01-09T12:02:26Z No. of bitstreams: 2 Dissertação - Janaina Gomes de Siqueira - 2016.pdf: 2965200 bytes, checksum: 015ab0c28746d09363a65c8817dd4068 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-01-09T12:03:04Z (GMT) No. of bitstreams: 2 Dissertação - Janaina Gomes de Siqueira - 2016.pdf: 2965200 bytes, checksum: 015ab0c28746d09363a65c8817dd4068 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-01-09T12:03:04Z (GMT). No. of bitstreams: 2 Dissertação - Janaina Gomes de Siqueira - 2016.pdf: 2965200 bytes, checksum: 015ab0c28746d09363a65c8817dd4068 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-11-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Histoplasma capsulatum var. capsulatum is a pathogenic fungus that causes the systemic disease known as histoplasmosis. This mycosis presents endemicity in the regions of the Mississippi and Ohio rivers in the United States, also found in several countries of South America. In Brazil, the number of people infected by this fungus has been growing in recent years, especially in immunocompromised people. H. capsulatum is a thermodymorphic microorganism, found in two morphotypes: mycelium at 25ºC and yeast at 37ºC. During the infection process the pathogenic microorganism needs to obtain nutrients so that it grows and develops within the tissue of the host. Among the essential nutrients, zinc is an essential metal ion, used in the maintenance of numerous metabolic pathways. Zinc is used as catalytic and structural cofactor of numerous metalloproteins. Zinc uptake is characterized as a virulence factor in H. capsulatum. In order to study the molecular mechanisms of zinc homeostasis in H. capsulatum, the in silico analysis of zinc homeostasis related proteins. The ZafA transcription factor, which acts on the regulation of genes in response to the zinc level in the medium and the transcription factor PacC that regulates genes in response to pH, were also found. ZrfA and ZrfC membrane transporters were also found ZrfF, ZrcA and ZrcC vacuolar transporters. The transcriptional profile analysis of these genes was measured by RT-PCR in infected macrophages and in vitro. During the infection the ZafA and ZrfA genes were induced, suggesting that these genes were expressed in order to supply the fungal cell with zinc, due to the metal deficiency caused by the infection. In vitro, ZafA, ZrfC, ZrfF, ZrcA and ZrcC were induced at zinc deprivation condition, compared with control condition. According with proteomic data of fungus under conditions of zinc deprivation, it was suggested the induction of proteins related to rescue, defense and virulence, related mainly to oxidative stress, besides other metabolic pathways such as gluconeogenesis. These data suggest that the fungus undergoes a metabolic remodeling in response to the oxidative stress caused by zinc deprivation. / Histoplasma capsulatum var. capsulatum é um fungo patogênico causador da doença sistêmica conhecida como histoplasmose. Essa micose apresenta endemicidade nas regiões do rio Mississipi e Ohio nos Estados Unidos, encontrado também em vários países da América do Sul. No Brasil, o número de pessoas infectadas por este fungo vem crescendo nos últimos anos, principalmente em pessoas imunocomprometidas. H. capsulatum é um micro-organismo termodimórfico, encontrado em dois morfotipos: micélio a 25ºC e levedura a 37ºC. Durante o processo de infecção o micro-organismo patogênico necessita obter nutrientes para que cresça e se desenvolva dentro do tecido do hospedeiro. Dentre os nutrientes essenciais, o zinco é um íon metal essencial, utilizado na manutenção de inúmeras vias metabólicas. O zinco é utilizado como cofator catalítico e ou estrutural de inúmeras metaloproteínas. A captação de zinco é caracterizada como um fator de virulência em H. capsulatum. Com o objetivo de estudar os mecanismos moleculares da homeostase de zinco em H. capsulatum, foi realizada primeiramente a análise in silico das proteínas relacionadas a homeostase de zinco. Foram encontrados o fator de transcrição ZafA que atua na regulação de genes em resposta ao nível de zinco no meio e o fator de transcrição PacC que atua na regulação de genes em resposta ao pH, adicionalmente foram encontrados transportadores de membrana ZrfA e ZrfC, além dos transportadores vacuolares ZrfF, ZrcA e ZrcC. A análise do perfil transcricional destes genes foi mensurada por RT-PCR em macrófagos infectados e in vitro. Durante a infecção os genes ZafA e ZrfA foram induzidos, sugerindo que estes genes foram expressos a fim de suprir a célula fúngica com zinco, devido a deficiência do metal causada pela infecção. In vitro, ZafA, ZrfC, ZrfF, ZrcA e ZrcC foram induzidos na condição de privação de zinco, comparado com a condição controle. De acordo com os dados proteômicos do fungo em condições de privação zinco, foi sugerido a indução de proteínas relacionadas ao resgate, defesa e virulência, relacionadas principalmente ao estresse oxidativo, além de outras vias metabólicas como gliconeogênese. Estes dados sugerem que o fungo sofre um remodelamento metabólico em resposta ao estresse oxidativo causado pela privação de zinco.

Zinc is a metal involved in structure of many enzymes, in the growth and differentiation of plants. Wheat is one of the most consumed cereals. Some wheat cultivars can&amp / #8217 / t deal with zinc deficiency and this situation not only reduces grain yield but also weakens the resistance of cereals to diseases and impairs the nutritional quality of the grain. Some wheat cultivars are not affected by zinc deficiency. In this study, &amp / #8216 / differential display&amp / #8217 / , used for determination differentially expressed genes between two samples, was performed. The most zinc efficient bread wheat cultivar Kira&ccedil / -66 was grown in hydroponics medium and samples were taken at different time periods. RNA isolations were done and differential display technique was performed. After examining the results, differentially expressed bands were selected and sequenced. DNA sequence analysis were done in available databases which showed that three of the bands were fragments of putative zinc transporters. In this study we have found threee putative gene fragments using differential display technique on zinc efficient plants grown under differeing zinc concentrations. These fragments showed homology with zinc transporter, ABC transporter and ADH (Alcohol Dehydrogenase). It is known that all of these three genes are involved in zinc efficiency mechanism. Further studies will be conducted on these gene fragments.

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