A enzima conversora de angiotensina (ECA) catalisa a conversão de angiotensina I (Ang I) no vasoconstritor angiotensina II (Ang II) e hidrolisa a bradicinina (BK). ECA somática (sECA) possui dois domínios homólogos (N e C) que têm 60% de identidade. Embora estas duas regiões tenham homologia grande, o sítio catalítico C-domínio exibe uma atividade três vezes maior do que o N-domínio na hidrolise de Ang I in vivo. Este fato torna interessante o desenvolvimento de novos estudos de inibidores ou a melhoria dos já existentes. O objetivo deste estudo foi obter a região Ala959 até Ser1066 do Cdomínio da sECA (c-sECA), em uma estrutura conformacional semelhante à estrutura nativa. Nós amplificamos a sequência correspondente ao sítio catalítico da c-sECA com 324pb e clonamos esta sequência no vetor pET 28a(+). O segmento (nomeado de pET28_c-sECA) foi expresso em sistema bacteriano. A proteína foi expressa na forma solúvel e a purificação foi feita em uma única etapa utilizando a coluna de afinidade His-tag, a qual produziu a proteína pura. Análises estruturais por dicroísmo circular e fluorescência confirmaram que a proteína recombinante estava na conformação correta, e os ensaios de atividade mostraram que a c-sECA possui atividade enzimática e é inibida por lisinopril. / The angiotensin-converting enzyme (ACE) catalyzes the conversion of angiotensin I (Ang I) to the vasoconstrictor angiotensin II (Ang II) and the hydrolysis of bradykinin (BK). Human somatic ACE (sACE) has two homologous domains (N and C) that show 60% identity. Although these two regions have high homology, the catalytic site of the C-domain exhibits three times more activity than that of the N-domain in the hydrolysis of Ang I in vivo. This fact necessitates the development of new inhibitors or the improvement of existing ones. This study aimed to obtain the Ala959 to Ser1066 catalytic region of C-domain of sACE (c-sACE) in a structural conformation that resembles the native structure. We amplified the 324-bp sequence corresponding to the catalytic site of c-sACE and cloned this sequence into a pET28a(+) vector. The segment (named pET28_c-sACE) was expressed in a bacterial system. The expressed protein segment was soluble, and its purification was performed in one step using a His-tag affinity column. Structural analysis by circular dichroism and fluorescence confirmed that the purified protein is correctly folded, and an activity assay showed that c-sACE possesses enzymatic activity and is inhibited by lisinopril.

A enzima conversora de angiotensina (ECA) catalisa a conversão de angiotensina I (Ang I) no vasoconstritor angiotensina II (Ang II) e hidrolisa a bradicinina (BK). ECA somática (sECA) possui dois domínios homólogos (N e C) que têm 60% de identidade. Embora estas duas regiões tenham homologia grande, o sítio catalítico C-domínio exibe uma atividade três vezes maior do que o N-domínio na hidrolise de Ang I in vivo. Este fato torna interessante o desenvolvimento de novos estudos de inibidores ou a melhoria dos já existentes. O objetivo deste estudo foi obter a região Ala959 até Ser1066 do Cdomínio da sECA (c-sECA), em uma estrutura conformacional semelhante à estrutura nativa. Nós amplificamos a sequência correspondente ao sítio catalítico da c-sECA com 324pb e clonamos esta sequência no vetor pET 28a(+). O segmento (nomeado de pET28_c-sECA) foi expresso em sistema bacteriano. A proteína foi expressa na forma solúvel e a purificação foi feita em uma única etapa utilizando a coluna de afinidade His-tag, a qual produziu a proteína pura. Análises estruturais por dicroísmo circular e fluorescência confirmaram que a proteína recombinante estava na conformação correta, e os ensaios de atividade mostraram que a c-sECA possui atividade enzimática e é inibida por lisinopril. / The angiotensin-converting enzyme (ACE) catalyzes the conversion of angiotensin I (Ang I) to the vasoconstrictor angiotensin II (Ang II) and the hydrolysis of bradykinin (BK). Human somatic ACE (sACE) has two homologous domains (N and C) that show 60% identity. Although these two regions have high homology, the catalytic site of the C-domain exhibits three times more activity than that of the N-domain in the hydrolysis of Ang I in vivo. This fact necessitates the development of new inhibitors or the improvement of existing ones. This study aimed to obtain the Ala959 to Ser1066 catalytic region of C-domain of sACE (c-sACE) in a structural conformation that resembles the native structure. We amplified the 324-bp sequence corresponding to the catalytic site of c-sACE and cloned this sequence into a pET28a(+) vector. The segment (named pET28_c-sACE) was expressed in a bacterial system. The expressed protein segment was soluble, and its purification was performed in one step using a His-tag affinity column. Structural analysis by circular dichroism and fluorescence confirmed that the purified protein is correctly folded, and an activity assay showed that c-sACE possesses enzymatic activity and is inhibited by lisinopril.

Jumonji C (JmjC) domain containing proteins comprise a large family of enzymes that catalyse oxidative reactions. The jumonji domain containing protein 6 (JMJD6) has pleiotropic functions as a lysyl hydroxylase and arginyl demethylase. Previous studies have shown that Jmjd6 is involved in histone modification, mRNA splicing and regulation of polymerase II pause release. A constitutive knockout of Jmjd6 in mice is neonatal lethal and shows defects in macrophage host responses. Recently, JMJD6 was shown to support Foot-and-mouth disease virus replication through interactions with the dead-box RNA helicase Dhx9. This PhD thesis aims to further explore functions of Jmjd6 in macrophages and its roles during viral infections. The hypothesis is that through interactions with RNA helicases, Jmjd6 regulates host responses to foreign nucleic acids and/or has functions as a host factor for replication of DNA and/or RNA viruses. Testing of this hypothesis required the generation of Jmjd6-deficient cell recourses. A new conditional Jmjd6 mouse allele was characterised and a method optimised to knockout the gene in bone marrow-derived macrophages (BMDM) using TAT-Cre recombinase. To study vaccinia (VACV) and influenza A virus (IAV) infections in human cell lines, JMJD6 was depleted using RNA interference or CRISPR-Cas9 gene editing. In BMDM, JMJD6 expression was up-regulated in the late phase of lipopolysaccharide stimulation. The nuclear expression pattern of Jmjd6 in BMDM overlapped with that of DDX41 but not with DHX9, two RNA helicases that have been implicated in sensing of viral DNA and RNA, respectively. Deletion of Jmjd6 in BMDM reduced induction of type I interferon response genes after stimulation with synthetic analogs of viral RNA. To characterise the role of JMJD6 during infection with a DNA virus, Jmjd6-deficient cells were infected with VACV. Knockout of Jmjd6 reduced VACV growth in macrophages but not in HeLa cells. In contrast to HeLa cells, Jmjd6-deficient macrophages displayed abnormal localisations of viral factories and increased cell death, showing that Jmjd6 is specifically required for productive VACV infection in macrophages. To further analyse whether Jmjd6 has pro- or anti-viral functions during RNA virus infection, JMJD6 depleted A549 cells were infected with IAV. JMJD6 depletion in A549 drastically reduced IAV growth from an early stage of infection. Preliminary data indicate that this phenotype is related to a defect in nuclear import of IAV ribonucleoprotein complexes. In summary, this work has identified JMJD6 as a novel pro-viral host factor for VACV and IAV infection and has underpinned its importance for macrophage functions.

Modern lifestyle with its lack of exercise and healthy diet often leads to obesity which is accompanied by a decreasing biological effect of insulin and the onset of hyperinsulinemia, and consequently type 2 diabetes. Persistently high levels of insulin stimulate signalling pathways with growth effects; cells thus become more sensitive to mitogenic effects of all growth factors which may even lead to the loss of control over cell proliferation and the rise of various malignancies. Due to a high degree of structure homology of insulin, IGF-I/II as well as particular IR (existing in "mitogenic" IR-A isoform and "metabolic" IR-B isoform) and IGF-1R, there are a number of cross- interaction among hormones and receptors; nevertheless, the biological response may be different during the binding to a receptor. The determination of the crucial structural regions in insulin and IGF which are responsible for binding to the receptors could lead to the evolution of selective insulin analogues with strengthen metabolic effects, or could lead to the evolution of selective antagonism of IGF which would, in turn, suppress the mitogenic effect. The highest overlap is between insulin and IGF-II since both hormones are able to bind to the isoform A of an insulin receptor (IR-A) with a high affinity, and to activate...