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Estudo do polimorfismo dos genes das citocinas TNFα, IFNγ, TGFβ, IL-6, E IL-10 em hansenÃase / Study of polymorphism of genes of cytokines TNFa, IFN, TGFβ, IL-6, IL-10 in leprosyLuana Nepomuceno Gondim Costa Lima 09 January 2009 (has links)
As citocinas desempenham um papel importante na resposta imune do hospedeiro contra o M. leprae. Polimorfismos de genes de citocinas tÃm sido implicados como um fator do hospedeiro influenciando a susceptibilidade para doenÃas infecciosas. O objetivo deste estudo foi verificar a relaÃÃo entre a hansenÃase e os polimorfismos dos genes TNFα (fator de necrose tumoral-α) -308 G→A; IFNγ (interferon-γ) +874 T→A; IL-6 (interleucina-6) -174 G→C; IL-10 -1082 A→T, -819 C→T, -592 A→C e TGFβ (fator de crescmento tumoral-β) cÃdon 10 e cÃdon 25. O estudo foi realizado com moradores do municÃpio de Sobral com 15 anos ou mais, no Estado do CearÃ, durante o perÃodo de marÃo de 2006 a julho de 2008. Os indivÃduos foram divididos em trÃs grupos. O grupo caso Ãndice foi composto por 46 indivÃduos com hansenÃase. Controles internos foram 110 contactantes que residiam no domicÃlio do caso Ãndice e os controles externos foram 83 indivÃduos que nÃo residiam no mesmo domicÃlio do caso Ãndice. Desses indivÃduos foram coletados 3ml para extraÃÃo de DNA atravÃs do âGenomic Prep Blood DNA Isolation Kitâ (GE Healthcare) e para tipificaÃÃo dos polimorfismos dos genes das citocinas atravÃs do âkitâ da âOne-Lambdaâ (Canoga Park, CA, EUA). TambÃm forma coletados 4,9ml de sangue para detecÃÃo de anticorpos IgM para PGL-I utilizando um teste ELISA. Os dados epidemiolÃgicos e clÃnicos foram obtidos a partir de um questionÃrio aplicado à todos participantes, padronizado para o projeto âEpidemiologia da hansenÃase no CearÃ: aprofundamento dos estudos imuno-epidemiolÃgicosâ, ao qual esse estudo està vinculado. Assim, nÃo foram observadas associaÃÃes significantes entre os polimorfismos dos genes das citocinas estudados e a susceptibilidade à hansenÃase. Em relaÃÃo ao gene IL-10, os indivÃduos com o genÃtipo GCC/GCC apresentaram uma tendÃncia a desenvolver a hansenÃase mais precocemente. Em relaÃÃo aos SNPs do gene IFNγ e TGFβ encontramos uma associaÃÃo do genÃtipo T/T do IFNγ e do genÃtipo T/T G/G do TGFβ com uma predisposiÃÃo à doenÃa em indivÃduos vacinados, podendo ser que indivÃduos com esses genÃtipo nÃo sejam beneficiados com a vacina. Em relaÃÃo aos SNPs do gene IL-6 do grupo de controles internos foi observada uma associaÃÃo entre um considerÃvel aumento do genÃtipo C/C e a positividade para o anti-PGL-I. Dessa forma, o estudo do polimorfismo dos genes das citocinas traz um melhor esclarecimento da relaÃÃo entre a genÃtica do hospedeiro e a hansenÃase, complementando estudos sobre a sua transmissÃo e fatores intra e extra-familiares em suas caracterÃsticas imunolÃgicas.
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Mitochondrial DNA polymorphism in American shad (Alosa sapidissima) and its implications for population structureBentzen, Paul January 1988 (has links)
No description available.
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An Analysis of Nucleotide Polymorphism in the Human MT-IIa Gene Promoter RegionStevens, Brett January 1992 (has links)
Previous research has shown varying degrees of renal damage on exposure to equal amounts of cadmium in occupationally exposed mining and factory workers. Further work has shown that in vitro exposure of human peripheral lymphocytes to the same cadmium levels resulted in significant variation in Metallothionein (MT) mRNA transcriptional induction over basal MT mRNA expression in a series of individuals. This variation could account for the differences in renal Cd toxicities identified previously. In this study, the human MT-IIₐ gene was cloned from 12 individuals, and the 5'promoter region was sequenced for each to determine the extent of promoter nucleotide variation. This is of interest since such an analysis has not been done in the past. No study has been done to look at the degree of polymorphism in a particular promoter region. Thus, there are no data on the degree of nucleotide drift or change which can occur in promoter regulatory elements. Such a study could provide insight into whether promoter changes could result in the type of variation described above. It could also give some insight into the degree of variation in sequences in the literature. The results obtained indicated that the human MT-IIₐ promoter region is highly conserved, with only one polymorphic site identified at position 557, between the glucocorticoid responsive element and the fourth metal regulatory element sequences. This suggests that promoter variation is not likely a significant yfactor in MT mRNA induction variability, although further analysis would be needed to show this since only 12 people were analyzed. The results were compared against a study of nucleotide polymorphism in Drosophila melanogaster, which is the only other data on nucleotide variation specifically (Kreitman, 1983; Kreitman and Hudson, 1991). As well, a number of discrepancies were noted from the original published sequences in the literature, suggesting that errors are likely published in genomic sequence which are never identified, except through trial and error. This has potential repercussions when considering the use of such sequence in cloning and sequencing projects, like the sequencing of the human genome, since this would depend on the accuracy of previously published data. / Thesis / Master of Science (MS)
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Time-resolved x-ray scattering using synchrotron radiation applied to the study of a polymorphic transition in carbamazepine.Forbes, Robert T., Edwards, Anthony D., Shekunov, Boris Yu., Grossmann, J,, York, Peter January 2001 (has links)
No / The thermodynamic status of -carbamazepine has been clarified using equilibrium solubility measurements, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), heated X-ray powder diffraction (XRPD), and temperature-controlled X-ray scattering techniques. -Carbamazepine is the least stable of the three well-characterized anhydrous polymorphs of carbamazepine at 25°C. In addition, it was confirmed that -carbamazepine undergoes an exothermic transition to -carbamazepine at 130°C. The novel technique of time-resolved simultaneous small- and wide-angle X-ray scattering has been successfully applied to monitor this transition in situ. It was concluded that -carbamazepine has a monotropic relationship with -carbamazepine.
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Characterisation of the SULT1A1 polymorphism in a South African Tswana population group / y Hlengiwe P. Mbongwa.Mbongwa, Hlengiwe Prosperity January 2010 (has links)
This dissertation brings to the fore the “Characterization of the SULT1A1 polymorphism in a
South Africa Tswana population group.” The primary experimental group studied came from
South African homogeneous Tswana individuals who participated voluntarily in an ongoing
large-scale epidemiological Prospective Urban and Rural Epidemiological (PURE) study the
North-West University (Potchefstroom Campus) participates in, as one of the 16 low- middleand
high-income countries across the world.
The primary aspect investigated was the comprehensive profile of the single nucleotide
polymorphism (SNP) and copy number variation (CNP) of the SULT1A1 gene. Using the PCRbased
RFLP method, SULT1A1 genotypes, and allele frequency distributions in an
experimental group of 1 867 individuals were determined. According to the literature this is by
far the largest and most homogeneous group from which such information has been acquired to
date. The SULT1A1*1, SULT1A1*1/*2 and SULT1A1*2 genotypes were found to be present at
a percentage of 43.76, 47.12 and 9.11 respectively. In comparison to similar studies in other
population groups, results from this study indicate that there are ethnic differences in the
SULT1A1 genotypes incidence. Asian group differs from Caucasian and Tswana groups
because of its exceptionally high prevalence of individuals with the SULT1A1*1 genotype and a
very low incidence of the SULT1A1*2 genotype. The SULT1A1*1 genotype profiles of
Caucasian and Tswana groups were comparable, but notable differences were observed for the
SULT1A1*2 genotype.
Using a quantitative multiplex PCR method for the CNV study, the numbers of copies of the
SULT1A1 gene in the Tswana population were determined, and the results showed 1 to ~5
copies: only 0.65% of the subjects had a single copy, whereas 59.69% of the subjects had 3 or
more copies. This result shows a significant discrepancy between the Caucasian-American
samples, which showed that only 26% from that group had more than three copies. However,
there is a significant relationship with the African-American population, which presented 63%
with 3 or more copies. This finding confirms results from a much smaller African-American
study, and suggests a possible genetic link between the African Tswana and the heritage of the
African-Americans. These findings were submitted for publication to the South African Journal
of Science, as that journal specializes in publication of new knowledge that has a regional focus
on Africa. Simultaneous phenotypic consequences of the SNP and CNP of the SULT1A1 gene, as well as
the thermo-stable and thermo-labile forms of the sulfotransferases were determined. For this,
the formation of [35S]-4-nitrophenyl sulphate from 4-nitrophenol and [35S]-3’-phosphoadenosine-
5’-phosphosulfate ([35S]-PAPS) in platelet homogenates were measured, with the data
normalized to a common platelet count. This investigation required fresh blood for enzyme
activity. These samples came from 98 Caucasian subjects who voluntarily participated in this
part of the study. The experimental data presented a unique challenge to develop a statistical
model to accommodate the complexity of the distribution of the data in the phenotype and
genotype components, which could be achieved by the development of a mixed model. The
model indicated that product formation increased through increasing copy number, but did not
differ for SULT1A1*1 and SULT1A1*1/*2. However, the rate of increase in product for the
thermo-stable forms of the SULTs was greater than that of thermo-labile forms. In contrast, copy
number effect for SULT1A1*2 differed considerably from that of the other two genotypes. Since
genotype is also a significant factor, it was concluded from Tukey post-hoc tests that the
population group means for product formation differ significantly (for all levels). These results
are presently being prepared for publication in an accredited international journal.
Finally, perturbations in 23 biochemical parameters measured in the PURE study were
analyzed as a function of the SULT1A1 SNP and CNP were evaluated. No group separation in
this regard could be found. It could be shown however, that sulfonation of the iodothyronines,
which are endogenous substrates for the SULTs, was influenced by the SULT1A1 genotype.
The relative concentrations in plasma of the sulphonated iodothyronines may be expressed as
T2S > T3S >> T4S, which coincides with the substrate preference of the SULT1A1 enzymes.
This observation may, however, only be qualitatively interpreted as (1) the targeted
metabolomics mass spectrometric method used for the quantitative analysis of these
substances needs further development, and (2) the influence of deiodonation was not taken into
account in these studies. In conclusion, three perspectives are given at the end of the thesis
which might be considered for further investigations. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2010.
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Characterisation of the SULT1A1 polymorphism in a South African Tswana population group / y Hlengiwe P. Mbongwa.Mbongwa, Hlengiwe Prosperity January 2010 (has links)
This dissertation brings to the fore the “Characterization of the SULT1A1 polymorphism in a
South Africa Tswana population group.” The primary experimental group studied came from
South African homogeneous Tswana individuals who participated voluntarily in an ongoing
large-scale epidemiological Prospective Urban and Rural Epidemiological (PURE) study the
North-West University (Potchefstroom Campus) participates in, as one of the 16 low- middleand
high-income countries across the world.
The primary aspect investigated was the comprehensive profile of the single nucleotide
polymorphism (SNP) and copy number variation (CNP) of the SULT1A1 gene. Using the PCRbased
RFLP method, SULT1A1 genotypes, and allele frequency distributions in an
experimental group of 1 867 individuals were determined. According to the literature this is by
far the largest and most homogeneous group from which such information has been acquired to
date. The SULT1A1*1, SULT1A1*1/*2 and SULT1A1*2 genotypes were found to be present at
a percentage of 43.76, 47.12 and 9.11 respectively. In comparison to similar studies in other
population groups, results from this study indicate that there are ethnic differences in the
SULT1A1 genotypes incidence. Asian group differs from Caucasian and Tswana groups
because of its exceptionally high prevalence of individuals with the SULT1A1*1 genotype and a
very low incidence of the SULT1A1*2 genotype. The SULT1A1*1 genotype profiles of
Caucasian and Tswana groups were comparable, but notable differences were observed for the
SULT1A1*2 genotype.
Using a quantitative multiplex PCR method for the CNV study, the numbers of copies of the
SULT1A1 gene in the Tswana population were determined, and the results showed 1 to ~5
copies: only 0.65% of the subjects had a single copy, whereas 59.69% of the subjects had 3 or
more copies. This result shows a significant discrepancy between the Caucasian-American
samples, which showed that only 26% from that group had more than three copies. However,
there is a significant relationship with the African-American population, which presented 63%
with 3 or more copies. This finding confirms results from a much smaller African-American
study, and suggests a possible genetic link between the African Tswana and the heritage of the
African-Americans. These findings were submitted for publication to the South African Journal
of Science, as that journal specializes in publication of new knowledge that has a regional focus
on Africa. Simultaneous phenotypic consequences of the SNP and CNP of the SULT1A1 gene, as well as
the thermo-stable and thermo-labile forms of the sulfotransferases were determined. For this,
the formation of [35S]-4-nitrophenyl sulphate from 4-nitrophenol and [35S]-3’-phosphoadenosine-
5’-phosphosulfate ([35S]-PAPS) in platelet homogenates were measured, with the data
normalized to a common platelet count. This investigation required fresh blood for enzyme
activity. These samples came from 98 Caucasian subjects who voluntarily participated in this
part of the study. The experimental data presented a unique challenge to develop a statistical
model to accommodate the complexity of the distribution of the data in the phenotype and
genotype components, which could be achieved by the development of a mixed model. The
model indicated that product formation increased through increasing copy number, but did not
differ for SULT1A1*1 and SULT1A1*1/*2. However, the rate of increase in product for the
thermo-stable forms of the SULTs was greater than that of thermo-labile forms. In contrast, copy
number effect for SULT1A1*2 differed considerably from that of the other two genotypes. Since
genotype is also a significant factor, it was concluded from Tukey post-hoc tests that the
population group means for product formation differ significantly (for all levels). These results
are presently being prepared for publication in an accredited international journal.
Finally, perturbations in 23 biochemical parameters measured in the PURE study were
analyzed as a function of the SULT1A1 SNP and CNP were evaluated. No group separation in
this regard could be found. It could be shown however, that sulfonation of the iodothyronines,
which are endogenous substrates for the SULTs, was influenced by the SULT1A1 genotype.
The relative concentrations in plasma of the sulphonated iodothyronines may be expressed as
T2S > T3S >> T4S, which coincides with the substrate preference of the SULT1A1 enzymes.
This observation may, however, only be qualitatively interpreted as (1) the targeted
metabolomics mass spectrometric method used for the quantitative analysis of these
substances needs further development, and (2) the influence of deiodonation was not taken into
account in these studies. In conclusion, three perspectives are given at the end of the thesis
which might be considered for further investigations. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2010.
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Physicochemical and crystallographic investigations into the salt formation of two heterocyclic drugsElder, David January 1992 (has links)
Salt formation provides a means of altering the physicochemical and resultant biological characteristics of a drug entity without modifying its molecular structure. Many published reviews have indicated the importance of the selection of the most appropriate salt form. This work is an investigation into the salt formation of two heterocyclic drugs. This is done by the physicochemical and the crystallographic studies of 19 high resolution single crystal diffraction studies. The particular targets of the work are the selection of the most appropriate salt forms, investigations into the tautomerism and polymorphism (or pseudopolymorphism) and an understanding of the interactions most likely between these heterocyclic drugs and their specific receptor sites. Section 1 describes the effect of protonation on the absorption of drugs, the rationale for using various salt forms and the resultant effect this has on a number of physicochemical properties of the parent compound. Section 2 is a description of the experimental techniques used in the physicochemical investigations and in crystal structure determination. In Sections 3 and 7, the preparation and characterisation of the salts and modifications of the two heterocyclic drugs, GU and IM is described. In Sections 4 and 8, the physicochemical investigations into the hygroscopicity and solid-state stabilities of the salts of GU and IM is described. Van't Hoff solubility studies are used to determine the enthalpies of solution and where appropriate the relative thermodynamic stabilities of the various phases produced. The structures of 19 of the salts or modifications of GU and IM, together with their packing and hydrogen bonding interactions is described in Sections 5 and 9. Sections 6 and 10 describe the ionisation properties of these molecules. Both the guanidine and imidazole moieties of GU and IM, respectively, are tautomeric, the particular form(s) found in these investigations and the effect of protonation is discussed. The conformations of these structures are discussed and the effect of protonation, especially on the puckering of the piperazine ring, is described.
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No evidence of a death-like function for species B1 human adenovirus type 3 E3-9K during A549 cell line infectionFrietze, Kathryn, Campos, Samuel, Kajon, Adriana January 2012 (has links)
BACKGROUND:Subspecies B1 human adenoviruses (HAdV-B1) are prevalent respiratory pathogens. Compared to their species C (HAdV-C) counterparts, relatively little work has been devoted to the characterization of their unique molecular biology. The early region 3 (E3) transcription unit is an interesting target for future efforts because of its species-specific diversity in genetic content among adenoviruses. This diversity is particularly significant for the subset of E3-encoded products that are membrane glycoproteins and may account for the distinct pathobiology of the different human adenovirus species. In order to understand the role of HAdV-B-specific genes in viral pathogenesis, we initiated the characterization of unique E3 genes. As a continuation of our efforts to define the function encoded in the highly polymorphic ORF E3-10.9K and testing the hypothesis that the E3-10.9K protein orthologs with a hydrophobic domain contribute to the efficient release of viral progeny, we generated HAdV-3 mutant viruses unable to express E3-10.9K ortholog E3-9K and examined their ability to grow, disseminate, and egress in cell culture.RESULTS:No differences were observed in the kinetics of infected cell death, and virus progeny release or in the plaque size and dissemination phenotypes between cells infected with HAdV-3 E3-9K mutants or the parental virus. The ectopic expression of E3-10.9K orthologs with a hydrophobic domain did not compromise cell viability.CONCLUSIONS:Our data show that despite the remarkable similarities with HAdV-C E3-11.6K, HAdV-B1 ORF E3-10.9K does not encode a product with a "death-like" biological activity.
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The genetic basis of flesh quality traits in farmed Atlantic salmonAshton, Thomas James January 2011 (has links)
The aim was to develop new methods for measuring texture of Atlantic salmon (Salmo salar L.) fillets and investigate the genetic basis of flesh quality traits. Firstly, a new tensile strength method was developed to quantify the force required to tear a standardized block of salmon muscle with the aim of identifying those samples more prone to factory downgrading as a result of gaping. The repeatability, sensitivity and predictability of the new technique was evaluated against other common instrumental texture measurement methods. Data from the new method were shown to have the strongest correlations with gaping severity r=-0.514, P<0.001) and the highest level of repeatability of data when analysing cold-smoked samples. The Warner Bratzler shear method gave the most repeatable data from fresh samples and had the highest correlations between fresh and smoked product from the same fish (r=0.811, P<0.001). It is therefore recommended that the new method be adopted for measuring gaping potential and the Warner Bratzler method become the standard for firmness assessment. Genes associated with post mortem softening in mammals were characterised in Atlantic salmon. A previously unknown ancient paralogue of calpastatin (here named CAST2) was identified. Evidence was provided for the existence of highly homologous recent paralogues of CAST2 and CTSD1. Evidence for the ancestral history of these paralogues was provided by phylogenetic analysis. Recent gene duplicates of 6 further genes were identified. In all cases, homology between recent paralogues was greater than 94%. Analysis of synonymous vs non-synonymous nucleotide substitution between the observed paralogue pairs shows a significant purifying selection in most cases. The CTSD1 gene shows significant purifying selection in a pairwise analysis between 12 teleost species (all cases P<0.0001) but a similar analysis of CTSD2 revealed no significant occurrence of purifying selection. The present study provides further support for the idea of asymmetrical selective pressure on paralogues. Genetic markers were developed that can distinguish individuals with above average fillet yield and texture. A database of firmness, tensile strength and fillet yield was made from 254 individuals from 5 batches of farmed salmon and these fish were genotyped at 7 novel SNP loci. Individuals with the combined favourable genotype at CAPN1a and MYOD1b were associated with an average increase in fillet yield of 2.7% above batch average. A combined genotype of CAPN1a, MYOD1b and MYF5 was significantly associated with an average increase in tensile strength of 9.8% above batch average (P=0.015). In both cases individuals with the combined favourable genotype occurred with a frequency of c. 6% across all batches. The favourable genotypes had no unfavourable effects on other traits. Highly polymorphic microsatellite loci were used to perform tests of assignment, which revealed an overall correct assignment rate of 92.7% to batch of origin and a minimum reference sample number of 25 was empirically determined. A phylogenetic analysis supported the results of the assignment tests. Given that 7 microsatellites is a relatively small number for a study of this nature, these results suggest that reliable assignment of unknown fish to the true batch of origin is potentially rapid and cost effective. Overall, the thesis presents molecular markers for broodstock selection, new genes of relevance to flesh quality, a new method of texture analysis and a proposal for an escapee traceability project.
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Characterisation of the mechanism of human serum resistance in Trypanosoma brucei gambiense.Felu, Cécile 15 September 2006 (has links)
The two human pathogenic sub-species T.b.gambiense and T.b.rhodesiense can be distinguished from the morphologically identical T.b.brucei by their ability to infect humans, enabling them to cause sleeping sickness. This is because they are resistant to lysis by the lytic factor (APOL-I) present in normal human serum (NHS). In T.b.rhodesiense resistance to this lytic factor is due to a truncated VSG gene termed SRA which blocks lysis by interacting with APOL-I in the lysosome. SRA does not exist in T.b.gambiense. The search for a similar truncated VSG gene lead to the identification of a T.b.gambiense specific glycoprotein termed TGSGP. TGSGP transfected alone into the sensitive T.b.brucei is unable to confer resistance to this sub-species. This is either due to incorrect processing of this gene is this sub-species or because TGSGP requires a partner to confer resistance.
In the search for a partner, the genomic locus of TGSGP was cloned and sequenced. We found that TGSGP is linked to a truncated gene homologous to the S.cerevisiae AUT1 gene, a gene implicated in autophagy and more specifically in membrane expansion. Southern blot hybridization and PCR analysis on genomic DNA from several isolates demonstrated that this feature was a specific to T.b.gambiense. In addition, we observed a correlation between the aut1 allele size and the geographical origin of the isolate.
Since in trypanosomes lysis by NHS is due to an uncontrolled expansion of the lysosome, we speculated that the truncation of the aut1 allele could be implication in the resistance to human serum. We characterized the genomic organisation of the AUT1 locus. T.b.brucei possesses two native AUT1 alleles whilst T.b.gambiense possesses a truncated aut1 allele, as well as a native AUT1 allele. We showed that in the T.b.gambiense LiTAR isolate (aut1/AUT1), despite the presence of a wild-type allele this gene is no longer expressed at the mRNA and protein level. Our complimentary results by run-on transcription assay showed that the AUT1 region is transcribed but that the messenger is unstable. LiTAR is a functional knock-out for AUT1, but Northern blot analysis on several T.b.gambiense isolates showed that this is not a generalised T.b.gambiense characteristic.
We explored the role of AUT1 in trypanosomes by invalidation of the AUT1 gene in T.b.brucei and by the over-expression of the AUT1 and aut1 alleles in T.b.brucei. By functional analysis of AUT1 knocked-down cells we showed that AUT1 is not essential in trypanosomes. By recreating in T.b.brucei the T.b.gambiense AUT1/aut1 genotype we were able to show that the expression of the aut1 UTR down-regulated the expression of the wild-type AUT1 allele. We speculated that this may be due to a natural RNAi mechanism. Par northern blot, using probes covering the potential target region of AUT1, we detected a 50nt small RNA specific to T.b.gambiense. In addition, we showed that in a LiTAR strain in which the RNAi pathway was abolished AUT1 expression is restored.
We continued to investigate TGSGP’s role in the resistance to human serum by invalidation of TGSGP in T.b.gambiense and by expressing TGSGP in the NHS-sensitive T.b.brucei. Because T.b.gambiense cannot be cultured in vitro we established a new in vivo transfection technique and as the knock-out of TGSGP is most probably lethal, we created an inducible RNAi T.b.gambiense cell strain. These indispensable tools will be used to test whether invalidation TGSGP is sufficient to confer resistance to NHS. Many strategies were tested in order to correctly expressing TGSGP in T.b.brucei; in none of these transfectants was TGSGP correctly located in the flagellar pocket as is the case in T.b.gambiense and only partial resistance was ever obtained. In order to identify the factors in human serum that could interacts with TGSGP, we subjected NHS to affinity chromatography using TGSGP as bait. We showed that TGSGP interacts with APOA-I, a major component of HDLs.
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