No / BACKGROUND: Vitamin D deficiency rickets is associated with skeletal deformities including swollen rib junctions, bowing of the legs, and the flaring and fraying of the wrist and long-bone metaphyses. There is, however, scarce information on the direct effect of rickets on skeletal growth in either present or past populations. AIM: The study investigated the effect of vitamin D deficiency rickets on long-bone growth in two post-medieval skeletal populations from East London (Broadgate and Christ Church Spitalfields). Subsequently, inter-population growth variations in relation to non-specific environmental stress (dental enamel defects), industrialization, urbanization and socio-economic status during infancy (birth to 3 years) and early childhood (3-7 years) were examined. SUBJECTS AND METHODS: Data on long-bone diaphyseal length dimensions and stress indicators of 234 subadults from Anglo-Saxon, late medieval and post-medieval archaeological skeletal samples were analysed using both linear and non-linear growth models. RESULTS: Rickets had no effect on the growth curves for any of the long bones studied. However, pronounced variations in growth between the four populations were noted, mainly during infancy. The diaphyseal length of long bones of Broadgate were significantly smaller-per-age than those of Spitalfields and the other samples up to the age of 4 years, and were associated with a high prevalence of enamel defects during early infancy. CONCLUSION: Socio-economic status, rather than urbanization, industrialization or rickets, was the central factor behind the observed differences in growth among the post-medieval populations. The observed inter-population growth variations were only significant during infancy.

Height is a complex physical trait that displays strong heritability. Adult height is related to length of the long bones, which is determined by growth at the epiphyseal growth plate. Longitudinal bone growth occurs via the process of endochondral ossification, where bone forms over the differentiating cartilage template at the growth plate. Estrogen plays a major role in regulating longitudinal bone growth and is responsible for inducing the pubertal growth spurt and fusion of the epiphyseal growth plate. However, the mechanism by which estrogen promotes epiphyseal fusion is poorly understood. It has been hypothesised that estrogen functions to regulate growth plate fusion by stimulating chondrocyte apoptosis, angiogenesis and bone cell invasion in the growth plate. Another theory has suggested that estrogen exposure exhausts the proliferative capacity of growth plate chondrocytes, which accelerates the process of chondrocyte senescence, leading to growth plate fusion. The overall objective of this study was to gain a greater understanding of the molecular mechanisms behind estrogen-mediated growth and height attainment by examining gene regulation in chondrocytes and the role of some of these genes in normal height inheritance. With the heritability of height so well established, the initial hypothesis was that genetic variation in candidate genes associated with longitudinal bone growth would be involved in normal adult height variation. The height-related genes FGFR3, CBFA1, ER and CBFA1 were screened for novel polymorphisms using denaturing HPLC and RFLP analysis. In total, 24 polymorphisms were identified. Two SNPs in ER (rs3757323 C>T and rs1801132 G>C) were strongly associated with adult male height and displayed an 8 cm and 9 cm height difference between homozygous genotypes, respectively. The TC haplotype of these SNPs was associated with a 6 cm decrease in height and remarkably, no homozygous carriers of the TC haplotype were identified in tall subjects. No significant associations with height were found for polymorphisms in the FGFR3, CBFA1 or VDR genes. In the epiphyseal growth plate, chondrocyte proliferation, matrix synthesis and chondrocyte hypertrophy are all major contributors to long bone growth. As estrogen plays such a significant role in both growth and final height attainment, another hypothesis of this study was that estrogen exerted its effects in the growth plate by influencing chondrocyte proliferation and mediating the expression of chondrocyte marker genes. The examination of genes regulated by estrogen in chondrocyte-like cells aimed to identify potential regulators of growth plate fusion, which may further elucidate mechanisms involved in the cessation of linear growth. While estrogen did not dramatically alter the proliferation of the SW1353 cell line, gene expression experiments identified several estrogen regulated genes. Sixteen chondrocyte marker genes were examined in response to estrogen concentrations ranging from 10-12 M to 10-8 M over varying time points. Of the genes analysed, IHH, FGFR3, collagen II and collagen X were not readily detectable and PTHrP, GHR, ER, BMP6, SOX9 and TGF1 mRNAs showed no significant response to estrogen treatments. However, the expression of MMP13, CBFA1, BCL-2 and BAX genes were significantly decreased. Interestingly, the majority of estrogen regulated genes in SW1353 cells are expressed in the hypertrophic zone of the growth plate. Estrogen is also known to regulate systemic GH secretion and local GH action. At the molecular level, estrogen functions to inhibit GH action by negatively regulating GH signalling. GH treated SW1353 cells displayed increases in MMP9 mRNA expression (4.4-fold) and MMP13 mRNA expression (64-fold) in SW1353 cells. Increases were also detected in their respective proteins. Treatment with AG490, an established JAK2 inhibitor, blocked the GH mediated stimulation of both MMP9 and MMP13 mRNA expression. The application of estrogen and GH to SW1353 cells attenuated GH-stimulated MMP13 levels, but did not affect MMP9 levels. Investigation of GH signalling revealed that SW1353 cells have high levels of activated JAK2 and exposure to GH, estrogen, AG490 and other signalling inhibitors did not affect JAK2 phosphorylation. Interestingly, AG490 treatment dramatically decreased ERK2 signalling, although GH did stimulate ERK2 phosphorylation above control levels. AG490 also decreased CBFA1 expression, a transcription factor known to activate MMP9 and MMP13. Finally, GH and estrogen treatment increased expression of SOCS3 mRNA, suggesting that SOCS3 may regulate JAK/STAT signalling in SW1353 cells. The modulation of GH-mediated MMP expression by estrogen in SW1353 cells represents a potentially novel mechanism by which estrogen may regulate longitudinal bone growth. However, further investigation is required in order to elucidate the precise mechanisms behind estrogen and GH regulation of MMP13 expression in SW1353 cells. This study has provided additional evidence that estrogen and the ER gene are major factors in the regulation of growth and the determination of adult height. Newly identified polymorphisms in the ER gene not only contribute to our understanding of the genetic basis of human height, but may also be useful in association studies examining other complex traits. This study also identified several estrogen regulated genes and indicated that estrogen modifies the expression of genes which are primarily expressed in the hypertrophic region of the epiphyseal growth plate. Furthermore, synergistic studies incorporating GH and estrogen have revealed the ability of estrogen to attenuate the effects of GH on MMP13 expression, revealing potential pathways by which estrogen may modulate growth plate fusion, longitudinal bone growth and even arthritis.

Abstract Persistent organic pollutants (POPs), especially dioxin-like chemicals, have been shown to have adverse effects on skeleton and these effects are likely to be mediated via the aryl hydrocarbon receptor (AHR). In spite of the extensive research, the characteristics of developmental effects of POPs are poorly known and the role of AHR in POP bone toxicity and skeletal development in general. In this project changes in bone morphology and strength as well as tissue matrix mechanics are studied by applying state of the art biomedical engineering methods. This allows understanding of the effects of dioxins exposure and AHR activity on the development and maturation of extracellular matrix in musculoskeletal tissues from a completely new perspective, and thereby improving the health risk assessment of POPs. In the present study skeletal properties of rats exposed maternally to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), Northern Contaminant Mixture (NCM) and Aroclor1254 (A1254) were studied for cross-sectional morphometric and biomechanical properties, and data were analysed with benchmark dose modelling. In addition, extracellular matrix properties were analysed using nanoindentation. Similar measurements were performed for adult wild-type and AHR-null mice after TCDD exposure. The same animals were also analysed for microstructural changes using micro-computed tomography and their bone cell activity was estimated from serum markers and gene expression. Analyses show decreased bone length and cross-sectional properties with consequently decreased bone strength. On the other hand, an increased trabecular BMD in response to NCM and A1254 was observed. In addition, bone matrix properties indicated delayed maturation or early senescence after maternal or adult exposure, respectively. The AHR is mainly responsible for bone toxicity of dioxin-like compounds and plays a role in bone development. This is likely due to disturbed bone remodeling as indicated by altered serum markers and gene expression. Overall these results indicate that POPs decrease bone strength, but the interpretation is difficult as there is more trabecular bone within cortical bone with compromised quality and increased porosity. / Tiivistelmä Altistumisen pysyville orgaanisille ympäristökemikaaleille on todettu heikentävän luustoa. Dioksiinien ja dioksiininkaltaisten yhdisteiden vaikutusten on havaittu välittyvän aryylihiilivetyreseptorin (AHR) välityksellä. Huolimatta pitkään kestäneestä tutkimuksesta POP-yhdisteiden sikiönkehityksen aikaisen altistuksen vaikutukset ja etenkin niiden mekanismit ovat edelleen huonosti tunnettuja, samoin kuin AHR:n osuus POP-yhdisteiden luutoksisuudessa ja luuston kehityksessä ylipäätään. Tässä työssä tutkittiin luuston rakenteellisia ja mekaanisia ominaisuuksia niin perinteisillä kuin uusimmilla biolääketieteen tekniikan menetelmillä. Tutkimuksen tavoitteena on saada uutta tietoa POP-altistuksen ja AHR-aktiivisuuden vaikutuksista luuston kehitykseen ja luukudoksen ikääntymisprosesseihin, mikä edesauttaa kyseisten yhdisteiden riskinarviointia. Tutkimuksissa altistettiin kantavia rottaemoja 2,3,7,8-tetraklooridibenzo-p-dioksiinille (TCDD), pohjoiselle saasteseokselle ja kaupalliselle Arokloori 1254 PCB-seokselle. Sikiönkehityksen aikana altistuneiden jälkeläisten luuston poikkileikkausen morfologia ja biomekaaniset ominaisuudet mitattiin ja tulokset mallinnettiin vertailuannoksen määrittämiseksi. Lisäksi TCDD-altistettujen rottien luustomatriisin ominaisuuksia selvitettiin nanoindentaatiomenetelmällä. Samaa menetelmää käytettiin myös aikuisiässä TCDD:lle altistettujen villityypin hiirten ja AHR-poistogeenisiten hiirten tutkimiseen. Näiden hiirten luuston hienorakennetta mitattiin myös korkean resoluution mikro-tietokonetomografialla ja niiden luusolujen aktiivisuutta tutkittiin seerumin biomarkkerien ja luun muodostumiseen osallistuvien geenien ekspressiotasojen avulla. Sikiönkehityksen aikainen altistuminen pohjoiselle saasteseokselle ja Arokloori 1254:lle hidasti luiden pituuskasvua. Lisäksi luiden poikkileikkauspinta-alat olivat pienentyneet ja mekaaniset ominaisuudet heikentyneet. Toisaalta hohkaluun määrä oli lisääntynyt altistumisen seurauksena. Myös sikiönkehityksen aikainen altistuminen TCDD:lle hidasti luukudoksen kypsymistä ja johti aikuisiällä luukudoksen ennenaikaiseen vanhenemiseen. AHR:llä oli päärooli ainakin aikuisiän vaikutusten ilmenemiselle ja reseptorilla vaikutti olevan rooli luuston kehityksessä ylipäätään. Seerumin biomarkkereiden ja geeniekspression muutosten perusteella nämä vaikutukset johtuvat todennäköisesti luuston uusiutumisen häiriöistä. Yhteenvetona voidaan todeta, että POP-yhdisteet heikentävät luustoa, mutta tämän ilmiön diagnosoiminen on hankalaa, koska huonolaatuisen kuoriluun sisällä hohkaluun määrä on lisääntynyt.

La Dysplasie Ectodermique Hypohidrotique liée à l'X (DEX) est une maladie génétique liée à une mutation du gène EDA. Le phénotype exprimé par le modèle murin Tabby est l'équivalent de celui observé dans l'espèce humaine et présente des anomalies dentaires, cranio-faciales, vertébrales et des défauts de trabéculation osseuse. Dans ce contexte, une cartographie de ces anomalies chez le mutant Tabby était nécessaire et l'analyse de l’impact de la mutation Eda/Ta sur la croissance du squelette crânien et post-crânien a été étudiée. Un suivi longitudinal d'une cohorte d'individus murin Tabby (5 mâles hémizygotes EdaTa/Y, 6 femelles hétérozygotes EdaTa/+) et sauvages (n=12) a été réalisé à partir d’une succession d’acquisitions TDM pendant plus de 2 ans. L'observation des profils de croissance et de leurs paramètres a montré des anomalies de croissance du complexe crânio-facial, de la base du crâne (hypo-développement crânien) et un déficit de croissance relatif des os longs (fémur et humérus) chez les souris hémizygotes EdaTa/Y. Ces résultats mettent pour la première fois en évidence des anomalies de développement des os longs et confirment le rôle d’EDA-A dans la formation normale du squelette. Ces données constituent un pré-requis essentiel pour tester l’efficacité de tentatives de réversion phénotypique à partir de protéines recombinantes. / X-linked Hypohidrotic Ectodermal Dysplasia (XLHED) is a genetic disorder due to a mutation of the EDA gene. The phenotype expressed by Tabby mice, murine model of XLHED, is equivalent to that observed in humans including dental anomalies, craniofacial and vertebral trabecular bone defects. In this context, a mapping of these anomalies in Tabby mice was necessary and the impact of the EdaTa mutation on cranial and post -cranial skeletal growth was studied. A 2 years (112 weeks) μCT follow-up of Tabby mice (5 hemizygous males EdaTa/Y, 6 heterozygous females EdaTa/+) and Wild Type group (n = 12) hasbeen performed. The observation of growth patterns and parameters showed a relative cranial hypodevelopment, abnormal growth of the craniofacial complex and a relative hypo-development of appendicular skeleton (femur and humerus) in Tabby mice. These results allowed for the first time to highlight appendicular developmental abnormalities, confirming the role of EDA-A in the normal formation of the skeleton. While enriching the phenotypic picture of this syndrome, in a therapeuticperspective, all of these data are an essential prerequisite to test the effectiveness of attempts to phenotypic reversion from recombinant proteins.

<p dir="ltr">Porcine reproductive and respiratory syndrome virus (PRRSV) is a devastating virus that is endemic to the swine industry. This virus has little direct effect on the dam but results in abortions, stillborn, and delivery of viremic piglets. PRRSV is unable to cross the swine placenta in early gestation but as gestation progresses, the placenta becomes permissible during late gestation. The mechanisms that allow the virus to cross the late gestation placenta are not well understood, but several theories have been presented regarding changes in placental morphology or enzymatic changes. Additionally, piglets who experience IUGR due to uterine crowding have been found to be more resistant to PRRSV infection, having lower viral levels than their normal litter mates. When vertical transmission from the dam to fetus occurs not only can the effects previously mentioned occur, but PRRSV is also known to cause suppression of maternal and fetal thyroid hormone. Thyroid hormone plays numerous roles in fetal development such as accretion of fetal mass, appetite regulation, and coincidently follows a similar increase trajectory to that of fetal growth during mid- to late-gestation. Consequently, any dysregulation of thyroid hormone has the potential to cause severe side-effects and may alter fetal growth. The relationship between thyroid hormone and fetal growth and development is not well understood. Chapter 2 investigates the potential cause-and-effect relationship between fetal growth and thyroid hormone through the induction of fetal hypothyroidism. Pregnant gilts (n=24) were given a sham treatment (CON; n=12) or treated with methimazole (MMI; n=12), a goitrogen capable of crossing the placenta. These gilts were then further subdivided across four gestational timepoints spanning mid- to late-gestation, these being days 55, 66, 76, and 86. Treatment started 21 days prior to these desired dates. Upon completion of treatment the gilts were humanely euthanized, and fetuses were extracted (resulting populations of n=174 MMI and n=166 CON) and fetal body and organ weights were recorded. Collected tissues included heart, liver, lung, kidneys, spleen, brain and thyroid. Fetuses were imaged in the left and right lateral recumbency for phenotypic analysis, including novel head measurements. Placental sample were also taken. Additionally fore- and hind limbs were taken from the centermost male and female from each litter so that radiographs could be taken to analyze bone growth. Statistical analysis of all phenotypic differences was carried out using a linear mixed effect model including gestational age and treatment as fixed effects and gilt as a random effect. The data revealed that the left and right phenotypic parameters are highly correlated (R2>0.9). Upon extraction, goiters were present in the MMI fetuses and there was a significant increase in both absolute and relative thyroid weights. Thus, the use of MMI during this period of gestation was successful in inducing hypothyroidism. Additionally, the MMI treated thyroids had a significant treatment by time interaction with 0.014g and 0.21g increase at day 55 and 66 respectively indicating reduced compensatory action within the fetal hypothalamic-pituitary-thyroid axis during this earliest period. Liver weight as a percentage of body weight decreased from 6.06% to 2.56% between days 55 and 86 in the CON group but, was significantly increased at all time points in response to MMI induced hypothyroidism (P<0.01). Thus, the in brain to liver weight ratio decreases over time, in MMI fetuses (P<0.05). While all other phenotypic parameters were significantly altered by gestation age, there was no significant impact of fetal hypothyroidism. This indicates that fetal thyroid hormone is not the driving factor for the exponential fetal growth seen in mid- to late-gestation. PRRSV virus is a complex and devastating virus to the swine industry, especially when it infects pregnant gilts and sows. PRRSV is unable to cross the swine placenta during mid-gestation but as gestation progresses the virus readily crosses the placenta and is able to infect piglets during this late gestation period. The mechanisms by which PRRSV crosses the highly restrictive porcine placenta are not clear. Additionally, piglets who experience intrauterine growth retardation experience lower virus levels than their normal counterparts. Chapter 3 investigates the changes in three genes of interest that we hypothesized, had the potential to fluctuate throughout gestation and facilitate PRRSV transfer, as well as the morphological changes that occur in the maternal-fetal interface through mid- to late-gestation and how these aspects may vary between IUGR and normal piglets. Placental samples were collected from pregnant gilts (n=12) equally divided across days 55, 66, 76, and 86 of gestation. Samples were taken of each fetus’s placenta adjacent to the umbilical cord. A portion of the sample was cut into 1 cm2 and placed into a mold with optimal cutting temperature media (OCT) for later cryo-sectioning and histology. The remaining portion had the fetal placenta peeled from the endometrium and flash frozen in liquid nitrogen for RNA extraction. A subset of samples was chosen based on fetus’s brain to liver weight ratios (n=96). From each litter two males and two females with the most extreme case of IUGR, based on z-scores, were chosen and the same was done for the two males and females with lowest brain to liver weight ratios, the later were classified as large for gestational age (LGA). 56 of the original 64 had acceptable levels of placental RNA for analysis. A total of 3 genes were chosen for analysis based on their function and previous literature. These included CD163, SIGLEC1 and IL-10. No significant up or down regulation was seen in any of the selected genes and there was no variation between IUGR and LGA fetuses. Additionally, placenta histology was conducted to evaluate populations of CD163 positive macrophages throughout the maternal fetal interface across mid- to late-gestation. Populations of CD163 positive macrophages were found on both the maternal and fetal sides of the maternal fetal interface at all timepoints. Collectively these results show there is no fluctuation in CD163, SIGLEC1, or IL-10 among timepoints or between IUGR and LGA fetuses. Additionally, the histology samples confirm the presence of resident populations of CD163 positive macrophages on maternal and fetal sides of the MFI. Collectively these results indicate that more research needs to be done to determine the underlying mechanisms of PRRSV transmission during late gestation.</p>

The dissertation deals with the image processing in digital holographic microscopy and X-ray computed tomography. The focus of the work lies in the proposal of data processing techniques to meet the needs of the biological experiments. Transmitted light holographic microscopy is particularly used for quantitative phase imaging of transparent microscopic objects such as living cells. The phase images are affected by the phase aberrations that make the analysis particularly difficult. Here, we present a novel algorithm for dynamical processing of living cells phase images in a time-lapse sequence. The algorithm compensates for the deformation of a phase image using weighted least squares surface fitting. Moreover, it identifies and segments the individual cells in the phase image. This property of the algorithm is important for real-time cell quantitative phase imaging and instantaneous control of the course of the experiment. The efficiency of the propounded algorithm is demonstrated on images of rat fibrosarcoma cells using an off-axis holographic microscope. High resolution X-ray computed tomography is increasingly used technique for the study of the small rodent bones micro-structure. In this part of the work, the trabecular and cortical bone morphology is assessed in the distal half of rat femur. We developed new method for mapping the cortical position and dimensions from a central longitudinal axis with one degree angular resolution. This method was used to examine differences between experimental groups. The bone position in tomographic slices is aligned before the mapping using the propound standardization procedure. The activity of remodelling process of the long bone is studied on the system of cortical canals.