The oxysterol, 25-hydroxycholesterol (25-HC), is important for sterol metabolism and emerging evidence suggests that 25-HC plays a more critical role in immunity and infection. However, the precise antiviral mechanism and the target of 25- HC remains unclear. Here efforts were made to investigate the link between viral infection and the triggering of the 25-HC associated interferon (IFN) response, and how this dynamically alters the endogenous level of 3-hydroxy- 3-methylglutaryl-CoA reductase (HMGCR), a key enzyme that catalyses the production of the precursor of cholesterol and oxysterols. In this thesis I have sought to specifically explore the temporal changes and role of HMGCR in DNA virus (cytomegalovirus) and RNA (Influenza) virus infections. I hypothesise that HMGCR is a target for 25-HC associated IFN-mediated host defence against viral infection. To characterise HMGCR and test this hypothesis, the following objectives were defined: (1). To establish an experimental system to quantitatively study the endogenous HMGCR protein level; (2). To investigate the mechanism of the down-regulation of HMGCR involved in the IFN-mediated innate immune response; (3). To study the behaviour of HMGCR in the influenza virus induced 25-HC associated IFN-mediated innate immune response; (4). To study the behaviour of HMGCR in the cytomegalovirus induced 25-HC associated IFN-mediated innate immune response. Chapter 3, describes establishing an experimental system for the quantification of endogenous HMGCR levels. Different protein detection methods, including a modified western blot protocol and immunostaining, were tested. The results of RNA interference of HMGCR demonstrate that under lipid-deficient condition with the supplementation of mevastatin (an HMGCR inhibitor) the modified western blot protocol specifically detects endogenous HMGCR. This chapter lays the foundational work for the temporal analysis and testing the role of HMGCR in infection. In Chapter 4, the mechanism of the degradation of HMGCR following 25-HC and IFN treatments, in wild-type and Ch25h−/− mouse bone marrow derived macrophages (BMDMs), was investigated. Similar to 25-HC, IFN-γ treatment results in the drop of both the transcript and protein abundance of HMGCR in wild-type BMDMs. Differential temporal analysis of RNA and protein alterations and the use of proteasome inhibitors reveals that both 25-HC and IFN-γ lead to a marked reduction of HMGCR protein via a proteasomal degradation mechanism within early times of treatments. Further, the immediate reduction of HMGCR levels induced by IFN-γ was completely abrogated in Ch25h−/− BMDMs. Hence, the reduction of HMGCR following IFN-γ treatment is due to the de novo synthesis in macrophages of 25-HC. However, the decrease of Hmgcr gene expression was observed in not only wild-type but also Ch25h−/− BMDMs, suggesting additional mechanisms for regulating Hmgcr RNA levels. These results demonstrate the mechanism of the down-regulation of HMGCR resulted from the induction of IFN response during viral infection, is only partially due the de novo synthesis of 25-HC. In chapter 5, influenza A virus was used to investigate the role of HMGCR in the IFN-mediated innate immune response. The inhibition of HMGCR by RNA interference inhibited viral growth, suggesting the requirement of HMGCR for optimal intracellular viral growth. Viral infection in wild-type murine BMDMs reduced the endogenous HMGCR levels. However, the reduction of HMGCR at early times was prevented in Ch25h−/− BMDMs. Intriguingly, the decrease of HMGCR at late time points was still observed in Ch25h−/− BMDMs. These results indicate that the down-regulation of HMGCR with influenza virus infection in BMDMs at early times is completely due to the de novo synthesis of 25-HC; whereas at late times alternative pathways or mechanisms exist. Additionally, human epithelial A549 cells and A549/PIV5-V cells that are deficient in STAT1 were used to study the role of IFN pathway in the down-regulation of HMGCR at late times during viral infection. Results from these studies show that at late times the reduction of HMGCR is due to IFN-independent mechanisms. Chapter 6, extends these investigations to the herpes virus murine cytomegalovirus and infection of BMDMs. HMGCR is known to be essential for cytomegaloviral infections and 25-HC, statin and RNAi inhibition of HMGCR restrict viral growth. 25-HC is shown to reduce HMGCR at immediate early times of infection. However, most notably, the down-regulation of HMGCR was also observed in Ch25h−/− BMDMs at late times with murine cytomegalovirus infected BMDMs. These results confirm that alternative pathways or mechanisms exist, playing roles in the crosstalk between cholesterol metabolism and innate immune response. Collectively, this study characterises the role of HMGCR in the 25-HC associated IFN-mediated host defence against viral infection. Results indicate that, in addition to the IFN-mediated host response, alternative pathways or other mechanisms also result in the down-regulation of HMGCR during viral infection. HMGCR is at the crossroad of different pathways or mechanisms, and is therefore not only targeted by 25-HC. Hence, further questions can be addressed from these results: (1). What are the alternative pathways or mechanisms for the down-regulation of HMGCR? (2). How do these pathways or mechanisms work in hosts’ immune system? Answering these questions can contribute to refining the pathway map of innate immunity and understanding the precise role of HMGCR, or even the sterol biosynthesis pathway, in hosts’ immune response against pathogens.

Thesis (MEng)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Nano-satellites are mostly used in lower earth orbit applications, where communication intervals are limited, often to a combined total of less than one hour per day. With these type of inherent limitations of lower earth orbits, there are also the physical size and equipment restriction of nano-satellites to consider, especially those of the CubeSat specification. It is of critical importance to use the limited time and communication resources as effectively as possible. The network protocol has a huge influence on reliability and throughput of a satellite network. An important requisite for designing, comparing and improving network protocols is a network protocol simulator, that is able to envisage the design results. Simulation can facilitate rapid development and unforeseen discoveries. Very little information is currently available regarding communication protocols used in nano-satellites. This thesis aims to explore and improve the current status of nano-satellite network simulation, as well as to demonstrate the development of an improved communication protocol strategy. It was found that there is a lack of proper simulation tools for satellite networks, which led to the development of SatSim. SatSim is a discrete event network simulation tool, developed in Python, which can be used to develop and analyse network protocols. SatSim was verified by comparing simulation results with other published results, which made use of different software tools and theoretical throughput calculations. AX.25 is one of the most commonly used network protocols in the nano-satellite industry. It was implemented in SatSim and verified with theoretical throughput calculations, as no other simulation data on AX.25 was available. AX.25 was used as a baseline protocol to improve upon. FX.25 was developed by the Stensat Group in an attempt to improve AX.25. FX.25 adds forward error correction to AX.25, by wrapping additional data around the AX.25 frames. This method maintains backward compatibility with AX.25. FX.25 was implemented in SatSim and the simulation results proved that FX.25 was a more reliable protocol than AX.25, as it can communicate at lower elevations and over noisier communication channels. However, the drawback of the additional forward error correction is the increased overhead, which reduces the overall payload data throughput. A modular AX/FX.25 protocol was then implemented in SatSim, to exploit the strengths of both protocols. This hybrid protocol yielded significant improvements to data throughput and can enable future software defined radio or hardware developments. / AFRIKAANSE OPSOMMING: Nano-satelliete word hoofsaaklik gebruik in lae-aard wentelbaan toepassings waar kommunikasietyd beperk is, soms tot minder as een uur per dag. Gepaardgaande met hierdie inherente beperking van lae-aard wentelbane, is daar ook die verminderde omvang en kapasiteit van nano-satelliete, veral ten opsigte van die CubeSat spesifikasie. Effektiewe aanwending van die beperkte tyd en kommunikasie-hulpbronne is dus noodsaaklik. Die keuse van netwerk protokol het ’n beduidende invloed op die betroubaarheid en data deurset van ’n satelliet netwerk. ’n Belangrike voorvereiste vir die ontwerp, vergelyking en verbetering van netwerk-protokolle, is ’n netwerk simulator. Beperkte inligting is tans beskikbaar oor kommunikasie protokolle in nano-satelliet toepassings. Hierdie tesis fokus op die verbetering van nano-satelliet netwerk-simulasie, asook die ontwikkelling van ’n verbeterde netwerk-protokol strategie vir nano-satelliet toepassings. Dit het na vore gekom dat daar ’n leemte is in die beskikbaarheid van simulasie sagteware wat gerig is op die ondersoek van satelliet netwerke. Hierdie waarneming het die ontwikkeling van SatSim genoop. SatSim is ’n diskrete-gebeurtenis netwerk-simulasie sagtewarepakket wat in die Python programmeertaal ontwikkel is om netwerk protokolle te ontwikkel en te analiseer. SatSim was geverifieer deur simulasies te vergelyk met die resultate van ander navorsingspublikasies, wat van verskillende sagtewarepakkette gebruik gemaak het, sowel as teoretiese deursetberekeninge. AX.25 is een van die netwerk protokolle wat mees algemeen in die nano-satelliet bedryf voorkom. AX.25 was geïmplementeer in SatSim en geverifieer met teoretiese deursetberekeninge. AX.25 was gebruik as ’n grondslag om op te verbeter. FX.25 was ontwikkel deur die Stensat Group in ’n poging om op AX.25 te verbeter. FX.25 voeg vorentoefoutkorreksie by tot AX.25, deur addisionele data tot die AX.25 netwerk pakkies te voeg. Hierdie benadering bewerkstellig agteruit-verenigbaarheid met AX.25. FX.25 was geïmplementeer in SatSim en simulasieresultate dui daarop dat FX.25 ’n meer betroubare protokol is as AX.25, omdat dit teen laer elevasiehoeke en oor swakker kommunikasiekanale kan kommunikeer. Die verbeterde betroubaarheid is ten koste van datadeurset, as gevolg van die toevoeging van die vorentoe-foutkorreksiedata. ’n Modulêre AX/FX.25 protokol was geïmplemeteer om te kapitaliseer op die sterk eienskappe van beide protokolle. Hierdie hibriede protokol het ’n beduidende verbetering gelewer ten opsigte van data deurset en kan toekomstige sagteware-gedefinieerde radio en hardewaretoepassings stimuleer.

O diabetes mellitus (DM) é uma doença com prevalência e morbidade elevadas em todo o mundo, sendo que o DM1 é responsável por 5-10% dos casos. A vitamina D, hormônio de ação pleiotrópica, pode melhorar o curso do DM1, embora os mecanismos não estejam completamente elucidados. Dessa forma, ampliar o conhecimento sobre a ação desse hormônio pode auxiliar no prognóstico, bem como na compreensão dos possíveis mecanismos envolvidos na prevenção do DM. Neste trabalho, foram avaliados os efeitos fisiológicos da suplementação de vitamina D (800 UI/dia/sete dias; via oral) em camundongos machos (n=31; linhagem C57BL/6) distribuídos em quatro grupos: Controle + Água (CA; n=9); Controle + Vitamina D (CV; n=9); Diabético + Água (DA; n=6) e Diabético + Vitamina D (DV; n=7). Os camundongos tornados diabéticos (aloxana, 60 mg/Kg, intravenosa), quando comparados aos controles, exibiram redução do peso corporal e concentrações plasmáticas de glicose mais elevadas durante o período experimental de 10 dias (características do estado insulinopênico). Entretanto, a suplementação com vitamina D não alterou essa condição. Camundongos tornados diabéticos, em relação aos controles, exibiram redução do peso corporal (p<0,05) e concentrações plasmáticas de glicose (p<0,001) mais elevadas durante o período experimental. Animais suplementados com vitamina D apresentaram, em relação aos controles, níveis de 25(OH)D mais elevados (CA vs CV, p<0,001; DA vs DV, p<0,001). Níveis séricos maiores de ureia (CA vs DA, p<0,05; CA vs DV, p<0,01; CV vs DA, p<0,05; CV vs DV, p<0,01) e creatinina (CA vs DA, p<0,001; CA vs DV, p<0,001; CV vs DA, p<0,001; CV vs DV, p<0,001), espessamento da cápsula de Bowman, hipertrofia glomerular e destruição de hepatócitos foram observados em camundongos diabéticos em relação aos controles. Entretanto, a suplementação com vitamina D não alterou estas condições. O grupo DA apresentou menor nível sérico de albumina em relação aos grupos CA (p<0,05) e CV (p<0,05); níveis inferiores de hemoglobina (p<0,05) e hematócrito (p<0,05) em relação ao grupo DV; e menor leucometria (p<0,05) e mononucleares sanguíneos (p<0,05) em relação ao grupo CA. Os resultados sugerem que a vitamina D possa influenciar a resposta imunológica em animais diabéticos, modulando hematócrito, hemoglobina, bem como os níveis séricos de albumina / Diabetes mellitus (DM) is a disease with high prevalence and morbidity worldwide, and the DM1 is responsible for 5-10% of cases. The vitamin D hormone pleiotropic action, can improve the course of T1DM, although the mechanisms are not fully elucidated. Thus, better understanding the action of this hormone can aid in prognosis as well as in understanding the possible mechanisms involved in the prevention of diabetes. We evaluated the physiological effects of vitamin D (800 IU/day/seven days, v.o.) in male mice (n=31, C57BL/6 strain) divided into four groups: Control + Water (CW, n=9); Control Vitamin D + (CV n=9); Diabetic + Water (DW, n=6) Diabetic + Vitamin D (VD, n=7). The mice induced-diabetes by alloxan (60 mg/kg, i.v.), when compared to controls, exhibited reduced body weight and plasma glucose concentrations were higher during the experimental period of 10 days (features insulinopenic state). However, vitamin D supplementation did not alter this condition. Diabetic mice, compared to controls, exhibited reduced body weight (p<0,05) and plasma glucose concentrations (p <0.001) higher during the trial period. Animals supplemented with vitamin D showed higher levels of 25 (OH) D than controls (CW vs CV, p <0,001; DW vs DV, p<0,001). Higher serum urea (CW vs. DW, p <0,05; CW vs DV, p <0,01; CV vs DA, p <0,05; CV vs DV, p <0,01) and creatinine (CW vs. DW, p <0,001; CW vs DV, p <0,001; CV vs DW, p <0,001; CV vs DW, p <0,001), thickening of Bowman\'s capsule, glomerular hypertrophy and destruction of hepatocytes were observed in diabetic mice compared to controls. However, vitamin D supplementation did not alter these conditions. The DW group showed lower serum albumin compared to CW (p<0,05) and CV (p<0,05) groups; lower hemoglobin (p<0,05) and hematocrit (p <0,05) compared to the DV group; and lower leukocyte counts compared to CW (p <0,05) and mononuclear blood (p <0,05) compared to the CW group. The results suggest that Vitamin D may influence the immune response in diabetic animals, modulating hematocrit, hemoglobin and serum albumin

A avicultura brasileira assumiu nos últimos anos importante papel dentro do PIB nacional, e a produção de ovos dentro do setor vem tomando cada vez maior importância. No decorrer dos últimos anos vários avanços foram observados no que se refere ao melhoramento genético de poedeiras comerciais, no sentido de maiores produtividade e longevidade. Ambos parâmetros podem ser afetados pela atuação de radicais livres e nutrição adequada. Embora o organismo vivo apresente mecanismos fisiológicos que visam diminuir os danos causados por radicais livres, a suplementação destes pode promover benefícios, desde a reprodução da ave até o desempenho produtivo de poedeiras oriundas de matrizes suplementadas com substâncias antioxidantes, dentre elas a cantaxantina. Recentemente vários estudos têm sido feitos para a nutrição adequada das aves com utilização adequada de aminoácidos, energia, fontes minerais, enzimas, etc., para que atinjam o máximo potencial produtivo. Porém, os níveis vitamínicos têm sido pouco estudados, e normalmente com foco em frangos de corte e/ou níveis mínimos a serem trabalhados para que o animal não entre em deficiência, trabalhos com níveis vitamínicos visando produtividade máxima em poedeiras comerciais não foram encontrados na literatura pesquisada. O presente estudo teve como objetivo avaliar poedeiras comerciais modernas, da linhagem Hisex Brown, em duas situações: origem de matrizes suplementadas com cantaxantina e 25 hidroxicolecalciferol, e níveis vitamínicos altos contra níveis vitamínicos praticados comercialmente. O experimento foi inteiramente casualizado e ocorreu nas dependências da granja experimental da USP de Pirassununga. As aves foram alojadas com 1 dia de idade e mantidas até as 70 semanas, passando pelas fases de cria, recria e produção, em que tiveram os parâmetros zootécnicos observados e comparados. Como resultados, observou-se no trabalho quanto à suplementação das matrizes: melhor peso corporal aos 35 dias, e daí até o final da recria (p<0,05), não apresentando maiores consumos de ração em relação ao grupo controle. Na fase de produção observou-se melhor peso dos ovos e espessura da casca para o grupo tratado e maior resistência da casca para o grupo controle (p<0,05). Quanto aos níveis vitamínicos observou-se: vantagem em peso aos 35 dias, o que não se observou às 17 semanas, e observou-se menor conversão alimentar no grupo controle. Durante a fase de produção, o grupo tratado apresentou resultados melhores em peso de ovos e resistência da casca (p<0,05) / Brazilian poultry has been taken an important part on brazilian economy on the past few years, and the egg production has been taken an important part on brazilian poultry. During the last years, several improvements has been done on the way of better production and longer lifetime of the layers. Both parameters are influenced by free radicals action and the correct use of nutrition. Although life animals have system to avoid damages caused by free radicals, suplementation with antioxidants can bring them benefits, since the reproduction of the breeder, until the production of the layer provided from light breeders suplemented, one of these substances is cantaxantin. Recently, studies have been conduced to determine the appropriate levels of aminoacids, energy, minerals, enzymes, etc., everything to permit the animal, the maximum production. However, vitamin levels haven´t been studied that much, and normally, the goal of theses studies are broilers and/or the minimum necessary to don´t let animals be deficient. This study had the goal to evaluate layers of Hisex Brown lineage, on two situations: the suplementation of the light breeder with cantaxantin and 25 hidroxicolecalciferol, and optimum vitamin nutrition against the ones comercially used. The experiment was entirely randomized designed, and occurred on the experimental houses of USP of Pirrasununga. The day old pullet were housed and maintained there for 70th weeks old, going throw rearing and production period, on which had its parameteres evaluated. As the results, on the work with breeders suplementation better body weight at 35 days old, and than until the end of rearing period (p<0,05), without higher feed consumption then control group. On the production period it was observed higher egg weight and thickness of the shell (p<0,05) for the suplemented breeder group, but better shell resistance for the control group. For the work wtih optimum vitamin nutrition results it was observed improvement of body weight at 35 days old (p<0,05) on the OVN group, but it was not observed until the end of rearing period. During the production time it was not observed any improvement but the weight and resistance of eggs on OVN group (p<0,05)

Um dos objetivos no tratamento de Diabetes mellitus é aumentar a função, proliferação e sobrevivência de células beta pancreáticas, pois o transplante de ilhotas pancreáticas é severamente limitado pela escassez de doadores de órgãos. Nós mostramos que prolactina recombinante humana (rhPRL) apresenta efeitos benéficos em células beta, inibindo a apoptose. Recentemente reportamos o aumento dos níveis de expressão da proteína antiapoptótica HSP27/25, regulada por rhPRL em ilhotas humanas. Nosso objetivo é explorar o papel de HSP27/25 na citoproteção induzida por prolactina em células beta. Métodos: Células MIN6 parentais e silenciadas para HSP25 foram privadas de soro por 24h e cultivadas na presença ou ausência de rhPRL (300ng/mL). Posteriormente foram tratadas na presença ou ausência de uma combinação de citocinas (TNF-&#945;, INF-&#947 e IL-1&#946;) por diferentes períodos de tempo. A apoptose foi avaliada usando-se análise por citometria de fluxo. Os níveis de expressão gênica de HSP27/25 e membros da família BCL-2, da expressão proteica de membros desta mesma família, além de Caspase-9, Smac-Diablo HSP27/25, HSTF1, pP38 e pSTAT1 foram analisados por Westen blot. Ainda, foram avaliadas a atividade de Caspase-8 e -3, por ensaios fluorimétricos. Após o tratamento com rhPRL e citocinas, a proporção de núcleos fragmentados aumentou em células silenciadas para HSP25 (p<0,05), quando comparadas com células controles. A inibição da atividade de Caspase-3 e -8, tanto quanto os níveis de expressão proteica de caspase- 9, por rhPRL e o aumento da razão de expressão Bcl-2/Bax e BclxL/Bax foram revertidos em células silenciadas (p<0,05). Além disso, a cinética dos níveis de expressão de HSP27/25 e HSTF1 foram estudadas em culturas de ilhotas pancreáticas humanas, mostrando que enquanto HSTF1 apresenta um aumento significativo (p<0,01) nos níveis de expressão proteica após 10min de tratamento com rhPRL, HSP27 alcançou seu nível máximo após 2h do tratamento hormonal. Adicionalmente, foi detectado um aumento significante (p<0,05) nos níveis de fosforilação de STAT1 e de p38, após 10min, atingindo o pico de expressão após 30min (p<0,01) do tratamento com rhPRL. Estes estudos demonstram o papel chave de HSP27/25 sobre os efeitos citoprotetores induzidos por rhPRL, desde que a ausência desta proteína aboliu completamente os efeitos benéficos induzidos por PRL sobre a morte de células beta. Ainda, nós fornecemos pela primeira vez, evidências da corregulação de HSP27 e HSTF1 induzidas por rhPRL em células humanas pancreáticas, que pode estar sendo mediada pela ativação de STAT1 e p38. Coletivamente, nossos resultados podem conduzir para a atenuação da morte de células beta por meio da regulação de uma via de proteção endógena, a qual é independente da modulação do sistema imunológico. / One of the goals in Diabetes mellitus treatment is to enhance pancreatic &#946;-cell function, proliferation and survival since transplantation of pancreatic islets is severely limited by shortage of organ donors. We have shown that recombinant human prolactin (rhPRL) inhibits beta-cell apoptosis. We have recently reported PRL-induced up-regulation of the anti-apoptotic HSP25/27 in human islets. Since the function of HSP25/27 in beta-cells has not been directly studied, we set out to explore the role of HSP25/27 in prolactin-induced beta-cell cytoprotection. Methods: Parental and HSP25 knocked-down Min6 cells were serumstarved for 24h and then cultured in the presence or in the absence of rhPRL (300ng/mL) for different time periods. Apoptosis was evaluated using flow cytometry analysis. Levels of Bcl-2 gene family members, caspase-9, Bcl-2, BclxL, Bax, Smac-Diablo, HSP27/25, HSTF1, pStat- 1,pP38 were studied by western blot. Caspase-3 and -8 activity, were evaluated by fluorimetric assays. Upon cytokines and rhPRL treatment, the proportion of fragmented nuclei was increased in HSP25 silenced cells (p<0.05) when compared to control cells. The inhibition of cytokine-induced capase-3 and -8 activity as well as Bcl-2/Bax and BclxL/Bax ratios and caspase-9 protein levels mediated by rhPRL in wild type cells was reverted in knocked-down cells (p<0.05). Moreover, the kinetics of HSP 25/27 and HSTF1 expression levels studied in primary cultures of human pancreatic islets showed that while HSTF1 presented a significant increase (p<0.01) in protein expression level after 10 min of rhPRL treatment, HSP27 reached its maximum expression level upon 2h of hormonal treatment. Additionally, a significant (p<0.05) increase in both P38 and STAT1 phosphorylation levels were detected after 10min of rhPRL treatment reaching the highest levels upon 30 min (p<0.001) of hormonal treatment. Therefore, we demonstrated a key role for HSP25/27 in rhPRL-induced cytoprotective effects, since the lack of this protein completely abolished the beneficial effects induced by PRL on beta-cell death. Moreover, we provided for the first time, evidence for the co-regulation of HSP27 and HSTF1 induced by rhPRL in human pancreatic cells which could be mediated by activated STAT1 and P38. Collectively, our results could lead to the mitigation of beta-cell death through the up-regulation of an endogenous protective pathway which is independent on the modulation of the immune system.

APRS system is described are sent toin my essay, which is used for sending and receiving text messages. The text messages frequenc 144 MHz by using the modulation scheme AFSK. APRS communicate by using the protocol AX 25, which sends and receives the text messages in the area where are carried despatches and control bits as well. The whole area of high frequency part is in one integrated circuit. The control, coding and decoding of the area is the microcontroller’s job.

APRS system is described are sent toin my essay, which is used for sending and receiving text messages. The text messages frequenc 144 MHz by using the modulation scheme AFSK. APRS communicate by using the protocol AX 25, which sends and receives the text messages in the area where are carried despatches and control bits as well. The whole area of high frequency part is in one integrated circuit. The control, coding and decoding of the area is the microcontroller’s job.

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PhD (Microbiology) / Department of Microbiology / Background Conversion of Cholesterol to 25-HydroxyCholesterol (25HC) by Cholesterol 25-hydroxylase (CH25H) has been shown to exert broad antiviral properties. Given its antiviral activities, CH25H is part of an increasingly appreciated connection between type I interferon (IFN-I) and lipid metabolism. Moreover, the details of this connection appear to differ in mouse and human cells. Nevertheless, the molecular basis for the induction of CH25H in humans is not known. Objective Elucidation of signaling and transcriptional events for induction of CH25H expression is critical to design therapeutic antiviral agents. Materials and methods: Wildtype THP-1 monocytic cell-line or THP-1 MyD88 Knockout cell-line were treated with PMA for 72 hours for differentiation into macrophages. Differentiated macrophages or Microglial cells were stimulated with either TLR-agonists, pro-inflammatory cytokine, or interferons, and CH25H mRNAs expression levels were measured by qPCR. Results In this study, we show that CH25H is induced by Zika virus infection or TLR stimulation. Interestingly, CH25H is induced by pro-inflammatory cytokines including 1L- 1, TNF-, and IL-6, and this induction depends on STAT-1 transcription factor. Additionally, we have observed that ATF3 weakly binds to the CH25H promoter, suggesting co-operation with STAT-1. However, ZIKV induced CH25H was independent of type I interferon. Conclusion This study has demonstrated for the first time that pro-inflammatory cytokines such as 1L-1, TNF-, and IL-6 induce CH25H expression. Moreover, this provides further understanding to the connection between innate immunity and sterol metabolism and encourages the exploration of cytokines in antiviral immunity. / NRF

Background: This study examined the association of 25-hydroxyvitamin D (25(OH)D) and C-reactive protein (CRP) with postoperative medical complications and one year mortality of elderly patients sustaining a low-energy cervical hip fracture scheduled for surgery. We hypothesized that vitamin D deficiency and CRP in these patients might be associated with an increased 1-year mortality. Methods: The prospective single-center cohort study included 209 patients with a low-energy medial femoral neck fracture; 164 women aged over 50 years and 45 men aged over 60 years. Referring to 1-year mortality and postoperative medical complications multiple logistic regression analysis including 10 co-variables (age, sex, BMI, ASA, creatinine, CRP, leukocytes hemoglobin, 25(OH)D, vitamin D supplementation at follow-up) was performed. Results: Vitamin D deficiency was prevalent in 87 % of all patients. In patients with severe (<10 ng/ml) and moderate (10–20 ng/ml) vitamin D deficiency one year mortality was 29 % and 13 %, respectively, compared to 9 % in patients with > 20 ng/ml 25(OH)D levels (p =0.027). Patients with a mild (CRP 10–39.9 mg/l) or active inflammatory response (CRP ≥ 40 mg/l) showed a higher one year mortality of 33 % and 40 % compared to 16 % in patients with no (CRP < 10 mg/l) inflammatory response (p = 0.002). Multiple logistic regression analysis identified CRP (OR 1.01, 95 % CI 1.00- 1.02; p = 0.007), but not 25(OH)D (OR 0.97, 95 % CI 0.89-1.05; p = 0.425) as an independent predictor for one year mortality. 20 % of patients suffered in-hospital postoperative medical complications (i.e. pneumonia, thromboembolic events, etc.). 25(OH)D (OR 0.89, 95 % CI 0.81–0.97; p = 0.010), but not CRP (OR 1.01, 95 % CI 1.00-1.02; p = 0.139), was identified as an independent risk factor. Conclusion: In elderly patients with low-energy cervical hip fracture, 25(OH)D is independently associated with postoperative medical complications and CRP is an independent predictor of one year mortality.