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1. Abstrakt:Bakgrund: Malaria är en av världens viktigaste infektionssjukdomar och det beräknas vara ca300 miljoner drabbade varje år, därför är metabolismen av läkemedel som används för attbehandla malaria såsom kinin och artemisinin värt att lägga fokus på. Cytokrom P450enzymerna har en viktig roll i metabolism av malarialäkemedel och dessa uppvisar variationinterindividuellt samt mellan olika populationer på grund av polymorfism.Syfte: Denna studie har fokuserat på att undersöka tre polymorfa gener (CYP3A4*1B,CYP3A5*3 och CYP2B6*6) hos både friska och malariasmittade barn i Uganda för attjämföra resultaten med en population i Mwanza, Tanzania. Dessa polymorfa alleler påverkarmetabolismen av artemisinin och kinin, vilket i sin tur kan förorsaka minskad/ökad ellermisslyckad klinisk effekt.Metod: Genotypning av individernas blodprov gällande dessa genvarianter undersöktesgenom laboratoriestudier med PCR som huvudsaklig metod. DNA sekvensering utfördes vidUppsala Genome Center.Resultat: Resultaten visade att allelfrekvensen i Mwanza för CYP3A4*1B var 78%respektive 16% för CYP3A5*3, medan de var 72 % respektive 50% hos populationen iUganda. Allelfrekvensen för CYP2B6*6 i Uganda var 72 % och 36 % i Mwanza, Tanzania.Sammanfattning: De flesta malariamediciner uppvisar skillnader i kinetik och dynamikvilket kräver terapeutisk läkemedelsmonitorering. Sammanfattningsvis finns det skillnaderoch variationer bland de studerade polymorfa CYP enzymer interindividuellt och mellanolikaetniska grupper.Resultatet av denna studie visade både små skillnader mellan de två studerade populationernamen även skillnader mellan individer i den studerade gruppen i Uganda.

The use of molecular chaperones can increase the yield of correctly folded proteins. This is especially needed in the expression of proteins non-native to the host organism. This study set out to investigate the function of the chaperone GroES; a component in the GroE-system. The function of this chaperone has only been studied alone in vitro. Here we lay ground to further studies on GroES and its ability to act alone in vivo. GroES was expressed from a plasmid and characterized through its potential to increase the amount of correctly folded proteins. Characterization was mainly done by fluorescence spectroscopy with hard-to-fold proteins linked to fluorescent probes. Results show a very clear increase in fluorescence for most of the substrate proteins tested, indicating that GroES has a significant role in the GroE-system and perhaps outside of it.

Approximately 30 million years ago, ancestors of fungus-growing termites started an obligate mutualistic relationship with a Basidiomycete fungus Termitomyces. The success of this obligate relation is the division of labour and reliance on termite caste gut microbial symbionts. Termites workers maintain Termitomyces fungal garden with their workforce and dual gut passage while the soldier caste protects the colony from predators. The fungal garden concurrently provides enough food for the colony members. Royal pair (a king and a queen) is the centralised caste in the colony, and they control the colony population by their massive reproduction, but their gut community composition remains unexplored. This project aimed to characterise the gut microbes associated with royal pairs of a fungus‐growing termite species Macrotermes natalensis. Four colonies were explored using high throughput sequencing of 16S rRNA gene amplicon dataset. The high-throughput sequence result showed that royal gut microbiotas were comprised of a lower number of bacterial taxa than sterile caste (workers and soldiers). This less number of bacterial taxa suggested that the royal pair gut was completely decoupled from the sterile castes gut, which indicates that the royal pair were possibly provided with a unique diet. The study also showed diversity in bacterial genus-level OTUs of royal pairs in all four colonies which indicated that there is a diet variation between the king and queen. The media predicting strategy could facilitate future cultivation efforts for targeted royal pair gut bacterial strains.

<p>Hair is important for thermoregulation, physical protection, sensory activity, seasonal camouflage and social interactions. Hair is produced in hair follicles (HFs), complex mini-organs in the skin devoted to this task. HFs are formed during embryonic development (morphogenesis) and new hair is continuously generated throughout life since the postnatal HF goes through cycles of regression (catagen), quiescence (telogen) and growth (anagen). The transcriptional regulation of this process is not well understood. The LIM-homeodomain transcription factor Lhx2 has previously been shown to be critically involved in epithelial-mesenchymal interactions during development of various organs and a potent regulator of stem cell function. We therefore elucidated the expression pattern and function of <em>Lhx2</em> during hair formation.</p><p><em>Lhx2</em> is expressed during both morphogenesis and anagen in cells scattered in the outer root sheath and in a subpopulation of the matrix cells in the proximal part of the hair bulb. Matrix cells are proliferating progenitor cells that differentiate into the components of the HF including the hair shaft. Expression is turned off during telogen, however <em>Lhx2</em> expression reappears in the secondary hair germ immediately prior to initiation of the anagen stage. In contrast to previously published results <em>Lhx2</em> appears to be expressed by progenitor cells distinct from those in the stem cell niche in the bulge region. The developmental-, stage- and cell-specific expression pattern of <em>Lhx2</em> suggests that Lhx2 is involved in the generation and regeneration of hair.</p><p>To test our hypothesis we used different genetically modified mouse strains. First we studied the effect of over-expression of <em>Lhx2</em> in the HFs using a mouse model where transgenic Lhx2 expression could be induced in dorsal skin. Using this model we could show that <em>Lhx2</em> expression is sufficient to induce anagen. To analyze the consequence of lack-of-function of Lhx2 we developed a mouse model where it is possible to conditionally inactivate <em>Lhx2</em> and a mouse strain harbouring a hypomorphic allele of Lhx2. Mice where <em>Lhx2</em> was conditionally inactivated in postnatal HFs were unable to regrow hair on a shaved area whereas all controls did regrow their hair. The mutant HFs initiated anagen but were unable to produce normal hair shafts. Thus Lhx2 is required for postnatal hair formation. We used the mouse strain carrying a hypomorphic allele of <em>Lhx2</em> to study the role of Lhx2 during HF morphogenesis. Embryos homozygous for the hypomorphic allele form significantly less HFs compared to control embryos, and the HFs that do form in the mutant embryos appear to be developmentally arrested. These results suggest that Lhx2 is also important during HF morphogenesis. Thus, Lhx2 is an essential positive regulator of hair generation and regeneration.</p>

In bacteria, tRNA molecules are produced as precursors with additional nucleotides both upstream and downstream of the tRNA coding sequence. To generate a mature tRNA, the endoribonuclease RNase P removes the upstream sequence, while a number of enzymes can remove the downstream sequence. In this thesis, the influence of such upstream and downstream sequences on the expression of mature functional tRNA was studied. It was found that in Escherichia coli, the presence of an upstream sequence positively influences tRNA expression. Furthermore, it was shown that the identity of the nucleotide immediatedly 5' of the canonical RNase P cleavage site in a tRNA precursor influenced RNase P cleavage site selection in vivo in E. coli, but not in Pseudomonas aeruginosa. Additionally, a stem-loop in the precursor just downstream of the tRNA increased this "miscleavage". This stem-loop resembled a rho-independent transcription terminator and overlapped the trpT gene in P. aeruginosa, but it only marginally influenced the expression of the downstream secE gene. The trpT and secE genes were found to be cotranscribed. The trpT-secE gene order was also conserved in the majority of bacteria investigated. The expression of tRNA genes during development in Streptomyces coelicolor was also studied. Here, the expression of most tRNA genes tested increased with time during development. Similarly, the expression of the RNase P RNA increased. The relative increase was quantified and correlated with different criteria related to gene structure and gene organisation, but no significant differences could be found. Moreover, a tRNALeuCAA UUA codon suppressor as well as the cognate bldA tRNA could restore differentiation to a developmentally blocked bldA deletion strain. For both tRNAs, the efficiency of restoration depended on the 5'- and 3'-flanks. In conclusion, both 5'- and 3'-flanking sequences influence tRNA expression, and bacteria respond differently to changes in their tRNA gene structures.

Protein synthesis is a highly regulated and energy consuming process, during which a large ribonucleoprotein particle called the ribosome, synthesizes new proteins. The eukaryotic ribosome consists of two unequal subunits called: small and large subunits. Both subunits are composed of ribosomal RNA (rRNA) and ribosomal proteins (r-proteins). Although rRNAs build the matrix of the ribosome and carries out catalysing of the peptide-bond formation between amino acids, r-proteins also appear to play important structural and functional roles. The primary role of r-proteins is to initiate the correct tertiary fold of rRNA and to organize the overall structure of the ribosome. In this thesis, I focus on two proteins from the large subunit of the eukaryotic ribosome: r-proteins L5 and L15 from bakers yeast S. cerevisiae. Both r-proteins are essential for ribosome function. Their life cycle is primarily associated with rRNA interactions. As a consequence, the proteins show high sequence homology across the species borders. Furthermore, both L5 and L15 are connected to human diseases, which makes the study their role in ribosome biogenesis and ribosome function important. By applying random- and site-directed mutagenesis, coupled with functional complementation tests, I aimed to elucidate functionally regions in both proteins, implicated in transport to the cell nucleus, protein-protein interactions and/or rRNA binding. The importance of individual and multiple amino acid exchanges in the primary sequence of rpL5 and rpL15 were studied in vivo. The obtained results show that S. cerevisiae rpL15 was tolerant to amino acid exchanges in the primary sequence, whereas rpL5 was not. Consequently, A. thaliana rpL15 could substitute for the function of wild type rpL15, whereas none of the tested orthologous proteins to rpL5 could substitute yeast rpL5 in vivo. These observations further emphasize the importance of studying r-proteins as separate entities in the ribosome context.

This thesis presents structural studies on the plant virus Ryegrass mottle virus (RGMoV), the bacteriophage φCb5, and the icosahedral particles and octahedral crystal assembly of a bacteriophage MS2 coat protein mutant. In contrast to other sobemoviruses, the RGMoV coat protein is missing several residues in two of the loop regions important for capsid assembly. The first loop contributes to contacts between subunits around the quasi-three fold symmetry axis. The altered contact interface results in tilting of the subunits towards the quasi-threefold axis. The assembly of the T=3 capsid of sobemoviruses is controlled by the N-termini of the C subunits. The second and smaller RGMoV loop does not interact with the N-terminus of the C subunit as do the corresponding loops of other sobemoviruses. The loss of interaction has been compensated for by additional interactions between the N-terminal arms of RGMoV C subunits. The bacteriophage MS2 belongs to the Leviviridae family of small RNA phages. Covalent dimers of the coat protein with insertions in the surface loops are known to be highly immunogenic epitope carriers. We crystallized the icosahedral particle assembled from covalent coat protein dimers in space group P213. At 4.7Å resolution the structure resembles the wildtype MS2 virion except for the intersubunit linker regions. The covalent dimer also crystallized in the cubic space group F432. The organization of the asymmetric unit in combination with the F432 symmetry results in an arrangement of subunits that corresponds to T=3 octahedral particles. Our crystal structure of the bacteriophage φCb5 capsid showed that it is stabilized by four calcium ions per icosahedral asymmetric unit. One ion is located between the quasi-threefold related subunits and is important for formation of a network of hydrogen bonds stabilizing the interface. The remaining calcium ions stabilized the contacts within the coat protein dimer. There was electron density of three putative RNA nucleotides per icosahedral asymmetric unit in the φCb5 structure. The nucleotides mediated contacts between two subunits forming a dimer and a third subunit in another dimer. On the basis of these findings, we have proposed a model for φCb5 capsid assembly in which addition of coat protein dimers to the forming capsid is facilitated by interaction with the RNA genome. The structure of RGMoV increases our understanding of mechanisms controlling sobemovirus assembly. This knowledge could be used to create genetically modified plants resistant to sobemovirus infection. The modified capsids of leviviruses can be used in immunization and as vehicles for gene or therapeutic compound delivery.

<!--[if !mso]> <object classid="clsid:38481807-CA0E-42D2-BF39-B33AF135CC4D" id=ieooui></object><mce:style><! st1\:*{behavior:url(#ieooui) } --> <!--[if !mso]> <object classid="clsid:38481807-CA0E-42D2-BF39-B33AF135CC4D" id=ieooui></object><mce:style><! st1\:*{behavior:url(#ieooui) } --> <!--[if !mso]> <object classid="clsid:38481807-CA0E-42D2-BF39-B33AF135CC4D" id=ieooui></object><mce:style><! st1\:*{behavior:url(#ieooui) } --> <!--[if !mso]> <object classid="clsid:38481807-CA0E-42D2-BF39-B33AF135CC4D" id=ieooui></object><mce:style><! st1\:*{behavior:url(#ieooui) } --> <!--[if !mso]> <object classid="clsid:38481807-CA0E-42D2-BF39-B33AF135CC4D" id=ieooui></object><mce:style><! st1\:*{behavior:url(#ieooui) } --> Regulation of mRNA degradation is a powerful way for the cell to regulate gene expression. A critical step in eukaryotic mRNA degradation is the removal of the poly(A) tail at the 3'-end of the mRNA. Poly(A)-specific ribonuclease (PARN) is an oligomeric, processive and cap-interacting 3'-5' exoribonuclease that efficiently degrades eukaryotic mRNA poly(A) tails. In addition to the exonuclease domain, PARN harbors two RNA-binding domains, a classical RNA recognition motif (RRM) and an R3H-domain. In this project we have studied mechanisms by which PARN specifically recognizes and degrades poly(A). We investigated the RNA binding properties of PARN by using electrophoretic mobility shift assays and filter-binding analysis and we could show that PARN binds poly(A) with high affinity and specificity. Furthermore, we showed that the RRM and R3H domains of PARN separately could bind to poly(A). To investigate specificity for and recognition of poly(A) in the active site of PARN, we performed a kinetic analysis on a repertoire of trinucleotide substrates in vitro. We showed that PARN harbors affinity for adenosines in the active site and that both the penultimate and the 3' end located nucleotide play an important role for providing adenosine-specificity in the active site of PARN. Moreover, we solved a crystal structure of PARN in complex with m7GpppG cap analogue and showed that the cap binding and active sites overlap both structurally and functionally. By mutational analysis we identified residues in the active site that specifically recognize adenosines. Furthermore, biochemical data showed that the adenosine specificity in the active site is lost when Mn2+ is used instead of Mg2+ as divalent metal ion. Taken together, these results demonstrate that both RNA-binding properties of the RRM and R3H-domains in addition to base recognition in the active site contributes to PARN poly(A)-specificity.

Promoter elements prediction with bioinformatics means is very promising direction in molecular biology. Such predictions allow to clarify the mechanisms of gene regulation and to increase the success of the “wet” experiments. The study was aimed to predict motifs for the 20 genes expressed in the thermosensory AFD neurons of the C.elegans and ortholog species. As theoretical basis of investigation was accepted theory about conservation of functionally important non-coding regions between ortholog genes. Some of such conserved regions can serve as the putative promoter elements. For the investigation the set of ortholog genes of the C.elegans, C.briggsae and C.remani was used. During the work suitable bioinformatics programs and the set of promoter criteria were worked out. Predicted promoters with the different level of conservation were compared to the each other and with the known regulatory elements. Some of the promoter elements (CAGCTG, CAGGTG) represent sequences are highly conserved from worms to the human others are similar to the known worm motifs (skn-1). Genes with the similar putative elements were combined in the groups with the possible common mechanisms of the gene regulation. The investigation of the putative promoter elements requires the development of new programs,methods and criteria of the evaluation. This would be appropriate strategy for the further motifs and non-coding DNA studying.