Mutations as molecular tools : The metabolic-rate dependent molecular clock and DNA barcoding of allied species

Mutation and recombination are the fundamental processes leading to genetic variation in natural populations. This variation forms the raw material for evolution through natural selection and drift. Therefore, studying mutation rates may reveal information about evolutionary histories as well as phylogenetic interrelationships of organisms. In this thesis two molecular tools, DNA barcoding and the molecular clock were examined. In the first part, the efficiency of mutations to delineate closely related species was tested and the implications for conservation practices were assessed. The second part investigated the proposition that a constant mutation rate exists within invertebrates, in form of a metabolic-rate dependent molecular clock, which can be applied to accurately date speciation events. DNA barcoding aspires to be an efficient technique to not only distinguish between species but also reveal population-level variation solely relying on mutations found on a short stretch of a single gene. In this thesis barcoding was applied to discriminate between Hylochares populations from Russian Karelia and new Hylochares findings from the greater Helsinki region in Finland. Although barcoding failed to delineate the two reproductively isolated groups, their distinct morphological features and differing life-history traits led to their classification as two closely related, although separate species. The lack of genetic differentiation appears to be due to a recent divergence event not yet reflected in the beetles molecular make-up. Thus, the Russian Hylochares was described as a new species. The Finnish species, previously considered as locally extinct, was recognized as endangered. Even if, due to their identical genetic make-up, the populations had been regarded as conspecific, conservation strategies based on prior knowledge from Russia would not have guaranteed the survival of the Finnish beetle. Therefore, new conservation actions based on detailed studies of the biology and life-history of the Finnish Hylochares were conducted to protect this endemic rarity in Finland. The idea behind the strict molecular clock is that mutation rates are constant over evolutionary time and may thus be used to infer species divergence dates. However, one of the most recent theories argues that a strict clock does not tick per unit of time but that it has a constant substitution rate per unit of mass-specific metabolic energy. Therefore, according to this hypothesis, molecular clocks have to be recalibrated taking body size and temperature into account. This thesis tested the temperature effect on mutation rates in equally sized invertebrates. For the first dataset (family Eucnemidae, Coleoptera) the phylogenetic interrelationships and evolutionary history of the genus Arrhipis had to be inferred before the influence of temperature on substitution rates could be studied. Further, a second, larger invertebrate dataset (family Syrphidae, Diptera) was employed. Several methodological approaches, a number of genes and multiple molecular clock models revealed that there was no consistent relationship between temperature and mutation rate for the taxa under study. Thus, the body size effect, observed in vertebrates but controversial for invertebrates, rather than temperature may be the underlying driving force behind the metabolic-rate dependent molecular clock. Therefore, the metabolic-rate dependent molecular clock does not hold for the here studied invertebrate groups. This thesis emphasizes that molecular techniques relying on mutation rates have to be applied with caution. Whereas they may work satisfactorily under certain conditions for specific taxa, they may fail for others. The molecular clock as well as DNA barcoding should incorporate all the information and data available to obtain comprehensive estimations of the existing biodiversity and its evolutionary history.

Total hip arthroplasty in young patients : with special references to patients under 55 years of age and to patients with developmental dysplasia of the hip

There is an ongoing controversy as to which methods in total hip arthroplasty (THA) could provide young patients with best long-term results. THA is an especially demanding operation in patients with severely dysplastic hips. The optimal surgical treatment for these patients also remains controversial. The aim of this study was to evaluate the long-term survival of THA in young patients (<55 years at the time of the primary operation) on a nation-wide level, and to analyze the long-term clinical and radio-graphical outcome of uncemented THA in patients with severely dysplastic joints. Survival of 4661 primary THAs performed for primary osteoarthritis (OA), 2557 primary THAs per-formed for rheumatoid arthritis (RA), and modern uncemented THA designs performed for primary OA in young patients, were analysed from the Finnish Arthroplasty Register. A total of 68 THAs were per-formed in 56 consecutive patients with high congenital hip dislocation between 1989-1994, and 68 THAs were performed in 59 consecutive patients with severely dysplastic hips and a previous Schanz osteotomy of the femur between 1988-1995 at the Orton Orthopaedic Hospital, Helsinki, Finland. These patients underwent a detailed physical and radiographical evaluation at a mean of 12.3 years and 13.0 years postoperatively, respectively. The risk of stem revision due to aseptic loosening in young patients with primary OA was higher for cemented stems than for proximally porous-coated or HA-coated uncemented stems implanted over the 1991-2001 period. There was no difference in the risk of revision between all-poly cemented-cups and press-fit porous-coated uncemented cups implanted during the same period, when the end point was defined as any revision (including exchange of liner). All uncemented stem designs studied in young patients with primary OA had >90% survival rates at 10 years. The Biomet Bi-Metric stem had a 95% (95% CI 93-97) survival rate even at 15 years. When the end point was defined as any revision, 10 year survival rates of all uncemented cup designs except the Harris-Galante II decreased to <80%. In young patients with RA, the risk of stem revision due to aseptic loosening was higher with cemented stems than with proximally porous-coated uncemented stems. In contrast, the risk of cup revision was higher for all uncemented cup concepts than for all-poly cemented cups with any type of cup revision as the end point. The Harris hip score increased significantly (p<0.001) both in patients with high con-genital hip dislocation and in patients with severely dysplastic hips and a previous Schanz osteotomy, treated with uncemented THA. There was a negative Trendelenburg sign in 92% and in 88% of hips, respectively. There were 12 (18%) and 15 (22%) perioperative complications. The rate of survival for the CDH femoral components, with revision due to aseptic loosening as the end point, was 98% (95% CI 97-100) at 10 years in patients with high hip dislocation and 92% (95% CI, 86-99) at 14 years in patients with a previous Schanz osteotomy. The rate of survival for press-fit, porous-coated acetabular components, with revision due to aseptic loosening as the end point, was 95% (95% CI 89-100) at 10 years in patients with high hip dislocation, and 98% (95% CI 89-100) in patients with a previous Schanz osteotomy. When revision of the cup for any reason was defined as the end point, 10 year sur-vival rates declined to 88% (95% CI 81-95) and to 69% (95% CI, 56-82), respectively. For young patients with primary OA, uncemented proximally circumferentially porous- and HA-coated stems are the implants of choice. However, survival rates of modern uncemented cups are no better than that of all-poly cemented cups. Uncemented proximally circumferentially porous-coated stems and cemented all-poly cups are currently the implants of choice for young patients with RA. Uncemented THA, with placement of the cup at the level of the true acetabulum, distal advancement of the greater trochanter and femoral shortening osteotomy provided patients with high congenital hip dislocation good long-term outcomes. Most of the patients with severely dysplastic hips and a previous Schanz osteotomy can be successfully treated with the same method. However, the subtrochanteric segmental shortening with angular correction gives better leg length correction for the patients with a previous low-seated unilateral Schanz osteotomy.