Development of zonal cartilage constructs : effects of chondrocyte subpopulation, compressive stimulation, and culture models

Articular cartilage is a highly resilient tissue located at the ends of long bones. It has a zonal structure, which has functional significance in load-bearing. Cartilage does not spontaneously heal itself when damaged, and untreated cartilage lesions or age-related wear often lead to osteoarthritis (OA). OA is a degenerative condition that is highly prevalent, age-associated, and significantly affects patient mobility and quality of life. There is no cure for OA, and patients usually resort to replacing the biological joint with an artificial prosthesis. An alternative approach is to dynamically regenerate damaged or diseased cartilage through cartilage tissue engineering, where cells, materials, and stimuli are combined to form new cartilage. However, despite extensive research, major limitations remain that have prevented the wide-spread application of tissue-engineered cartilage. Critically, there is a dearth of information on whether autologous chondrocytes obtained from OA patients can be used to successfully generate cartilage tissues with structural hierarchy typically found in normal articular cartilage. I aim to address these limitations in this thesis by showing that chondrocyte subpopulations isolated from macroscopically normal areas of the cartilage can be used to engineer stratified cartilage tissues and that compressive loading plays an important role in zone-dependent biosynthesis of these chondrocytes. I first demonstrate that chondrocyte subpopulations from the superficial (S) and middle/deep (MD) zones of OA cartilage are responsive to compressive stimulation in vitro, and that the effect of compression on construct quality is zone-dependent. I also show that compressive stimulation can influence pericelluar matrix production, matrix metalloproteinase secretion, and cytokine expression in zonal chondrocytes in an alginate hydrogel model. Subsequently, I focus on recreating the zonal structure by forming layered constructs using the alginate-released chondrocyte (ARC) method either with or without polymeric scaffolds. Resulting zonal ARC constructs had hyaline morphology, and expressed cartilage matrix molecules such as proteoglycans and collagen type II in both scaffold-free and scaffold-based approaches. Overall, my findings demonstrate that chondrocyte subpopulations obtained from OA joints respond sensitively to compressive stimulation, and are able to form cartilaginous constructs with stratified organization similar to native cartilage using the scaffold-free and scaffold-based ARC technique. The ultimate goal in tissue engineering is to help provide improved treatment options for patients suffering from debilitating conditions such as OA. Further investigations in developing functional cartilage replacement tissues using autologous chondrocytes will bring us a step closer to improving the quality of life for millions of OA patients worldwide.

Insights into historical drainage evolution based on the phylogeography of the chevron snakehead fish (Channa striata) in the Mekong Basin

1. The phylogeography of freshwater taxa is often integrally linked with landscape changes such as drainage re-alignments that may present the only avenue for historical dispersal for these taxa. Classical models of gene flow do not account for landscape changes and so are of little use in predicting phylogeography in geologically young freshwater landscapes. When the history of drainage formation is unknown, phylogeographical predictions can be based on current freshwater landscape structure, proposed historical drainage geomorphology, or from phylogeographical patterns of co-distributed taxa. 2. This study describes the population structure of a sedentary freshwater fish, the chevron snakehead (Channa striata), across two river drainages on the Indochinese Peninsula. The phylogeographical pattern recovered for C. striata was tested against seven hypotheses based on contemporary landscape structure, proposed history and phylogeographical patterns of codistributed taxa. 3. Consistent with the species ecology, analysis of mitochondrial and microsatellite loci revealed very high differentiation among all sampled sites. A strong signature of historical population subdivision was also revealed within the contemporary Mekong River Basin (MRB). Of the seven phylogeographical hypotheses tested, patterns of co-distributed taxa proved to be the most adequate for describing the phylogeography of C. striata. 4. Results shed new light on SE Asian drainage evolution, indicating that the Middle MRB probably evolved via amalgamation of at least three historically independent drainage sections and in particular that the Mekong River section centred around the northern Khorat Plateau in NE Thailand was probably isolated from the greater Mekong for an extensive period of evolutionary time. In contrast, C. striata populations in the Lower MRB do not show a phylogeographical signature of evolution in historically isolated drainage lines, suggesting drainage amalgamation has been less important for river landscape formation in this region.

A population genomics overview of the neuronal nitric oxide synthase (nNOS) gene and its relationship to migraine susceptibility

The ubiquitous chemical messenger molecule nitric oxide (NO) has been implicated in a diverse range of biological activities including neurotransmission, smooth muscle motility and mediation of nociception. Endogenous synthesis of NO by the neuronal isoform of the nitric oxide synthase gene family has an essential role within the central and peripheral nervous systems in addition to the autonomic innervation of cerebral blood vessels. To investigate the potential role of NO and more specifically the neuronal nitric oxide synthase (nNOS) gene in migraine susceptibility, we investigated two microsatellite repeat variants residing within the 5′ and 3′ regions of the nNOS gene. Population genomic evaluation of the two nNOS repeat variants indicated significant linkage disequilibrium between the two loci. Z-DNA conformational sequence structures within the 5′ region of the nNOS gene have the potential to enhance or repress gene promoter activity. We suggest that genetic analysis of this 5′ repeat variant is the more functional variant expressing gene wide information that could affect endogenous NO synthesis and potentially result in diseased states. However, no association with migraine (with or without aura) was seen in our extensive case-control cohort (n = 579 affected with matched controls), when both the 5′ and 3′ genetic variants were investigated.

Microwave gas discharge produced and sustained by a surface wave propagating along a cylindrical metal antenna with a dielectric coating

The structure of a microwave gas discharge produced and sustained by a surface wave (SW) propagating along a cylindrical metal antenna with a dielectric coating is studied. The SW that produces and sustains the microwave gas discharge propagates along an external magnetic field and has an eigenfrequency in the range between the electron cyclotron and electron plasma frequencies. The presence of a dielectric (vacuum) sheath region separating the antenna from the plasma is assumed. The spatial distributions of the produced plasma density, electromagnetic fields, energy flow density, phase velocity and reverse skin depth of the SW are obtained analytically and numerically.

One and the same: Integrative taxonomic evidence that Bactrocera invadens (Diptera: Tephritidae) is the same species as the Oriental fruit fly Bactrocera dorsalis

The invasive fruit fly Bactrocera invadens Drew, Tsuruta & White, and the Oriental fruit fly Bactrocera dorsalis (Hendel) are highly destructive horticultural pests of global significance. Bactrocera invadens originates from the Indian subcontinent and has recently invaded all of sub-Saharan Africa, while B. dorsalis principally occurs from the Indian subcontinent towards southern China and South-east Asia. High morphological and genetic similarity has cast doubt over whether B. invadens is a distinct species from B. dorsalis. Addressing this issue within an integrative taxonomic framework, we sampled from across the geographic distribution of both taxa and: (i) analysed morphological variation, including those characters considered diagnostic (scutum colour, length of aedeagus, width of postsutural lateral vittae, wing size, and wing shape); (ii) sequenced four loci (ITS1, ITS2, cox1 and nad4) for phylogenetic inference, and; (iii) generated a cox1 haplotype network to examine population structure. Molecular analyses included the closely related species, Bactrocera kandiensis Drew & Hancock. Scutum colour varies from red-brown to fully black for individuals from Africa and the Indian subcontinent. All individuals east of the Indian subcontinent are black except for a few red-brown individuals from China. The postsutural lateral vittae width of B. invadens is narrower than B. dorsalis from eastern Asia, but the variation is clinal, with subcontinent B. dorsalis populations intermediate in size. Aedeagus length, wing shape and wing size cannot discriminate between the two taxa. Phylogenetic analyses failed to resolve B. invadens from B. dorsalis, but did resolve B. kandiensis. Bactrocera dorsalis and B. invadens shared cox1 haplotypes, yet the haplotype network pattern does not reflect current taxonomy or patterns in thoracic colour. Some individuals of B. dorsalis/B. invadens possessed haplotypes more closely related to B. kandiensis than to conspecifics, suggestive of mitochondrial introgression between these species. The combined evidence fails to support the delimitation of B. dorsalis and B. invadens as separate biological species. Consequently, existing biological data for B. dorsalis may be applied to the invasive population in Africa. Our recommendation, in line with other recent publications, is that B. invadens be synonymized with B. dorsalis.

Comparison of fractional and geodesic anisotropy in diffusion tensor images of 90 monozygotic and dizygotic twins

We used diffusion tensor magnetic resonance imaging (DTI) to reveal the extent of genetic effects on brain fiber microstructure, based on tensor-derived measures, in 22 pairs of monozygotic (MZ) twins and 23 pairs of dizygotic (DZ) twins (90 scans). After Log-Euclidean denoising to remove rank-deficient tensors, DTI volumes were fluidly registered by high-dimensional mapping of co-registered MP-RAGE scans to a geometrically-centered mean neuroanatomical template. After tensor reorientation using the strain of the 3D fluid transformation, we computed two widely used scalar measures of fiber integrity: fractional anisotropy (FA), and geodesic anisotropy (GA), which measures the geodesic distance between tensors in the symmetric positive-definite tensor manifold. Spatial maps of intraclass correlations (r) between MZ and DZ twins were compared to compute maps of Falconer's heritability statistics, i.e. the proportion of population variance explainable by genetic differences among individuals. Cumulative distribution plots (CDF) of effect sizes showed that the manifold measure, GA, comparably the Euclidean measure, FA, in detecting genetic correlations. While maps were relatively noisy, the CDFs showed promise for detecting genetic influences on brain fiber integrity as the current sample expands.

Population structure, mortality and growth of Pinna nobilis Linnaeus, 1758 (Mollusca, Bivalvia) at different depths in Moraira bay (Alicante, Western Mediterranean)

An investigation to characterize the causes of Pinna nobilis population structure in Moraira bay (Western Mediterranean) was developed. Individuals of two areas of the same Posidonia meadow, located at different depths (A1, -13 and A2, -6 m), were inventoried, tagged, their positions accurately recorded and monitored from July 1997 to July 2002. On each area, different aspects of population demography were studied (i.e. spatial distribution, size structure, displacement evidences, mortality, growth and shell orientation). A comparison between both groups of individuals was carried out, finding important differences between them. In A1, the individuals were more aggregated and mean and maximum size were higher (A1, 10.3 and A2, 6 individuals/100 m(2); A1, x = 47.2 +/- 9.9; A2, x = 29.8 +/- 7.4 cm, P < 0.001, respectively). In A2, growth rate and mortality were higher, the latter concentrated on the largest individuals, in contrast to A1, where the smallest individuals had the higher mortality rate [A1, L = 56.03(1 - e(-0.17t)); A2, L = 37.59(1 - e(-0.40t)), P < 0.001; mean annual mortality A1: 32 dead individuals out of 135, 23.7% and A2: 16 dead individuals out of 36, 44.4%, and total mortality coefficients (z), z(A1(-30)) = 0.28, z(A1(31-45)) = 0.05, z(A1(46-)) = 0.08; z(A2(-30)) = 0.15, z(A2(31-45)) = 0.25]. A common shell orientation N-S, coincident with the maximum shore exposure, was observed in A2. Spatial distribution in both areas showed not enough evidence to discard a random distribution of the individuals, despite the greater aggregation on the deeper area (A1) (A1, chi(2) = 0.41, df = 3, P > 0.5, A2, chi(2)= 0.98, df = 2 and 0.3 < P < 0.5). The obtained results have demonstrated that the depth-related size segregation usually shown by P. nobilis is mainly caused by differences in mortality and growth among individuals located at different depths, rather than by the active displacement of individuals previously reported in the literature. Furthermore, dwarf individuals are observed in shallower levels and as a consequence, the relationship between size and age are not comparable even among groups of individuals inhabiting the same meadow at different depths. The final causes of the differences on mortality and growth are also discussed.

Genomic inflation factors under polygenic inheritance

Population structure, including population stratification and cryptic relatedness, can cause spurious associations in genome-wide association studies (GWAS). Usually, the scaled median or mean test statistic for association calculated from multiple single-nucleotide-polymorphisms across the genome is used to assess such effects, and 'genomic control' can be applied subsequently to adjust test statistics at individual loci by a genomic inflation factor. Published GWAS have clearly shown that there are many loci underlying genetic variation for a wide range of complex diseases and traits, implying that a substantial proportion of the genome should show inflation of the test statistic. Here, we show by theory, simulation and analysis of data that in the absence of population structure and other technical artefacts, but in the presence of polygenic inheritance, substantial genomic inflation is expected. Its magnitude depends on sample size, heritability, linkage disequilibrium structure and the number of causal variants. Our predictions are consistent with empirical observations on height in independent samples of ~4000 and ~133,000 individuals.

Randomized controlled trial of cognitive-behavioural therapy for coexisting depression and alcohol problems : short-term outcome

AIMS: Alcohol use disorders and depression co-occur frequently and are associated with poorer outcomes than when either condition occurs alone. The present study (Depression and Alcohol Integrated and Single-focused Interventions; DAISI) aimed to compare the effectiveness of brief intervention, single-focused and integrated psychological interventions for treatment of coexisting depression and alcohol use problems. METHODS: Participants (n = 284) with current depressive symptoms and hazardous alcohol use were assessed and randomly allocated to one of four individually delivered interventions: (i) a brief intervention only (single 90-minute session) with an integrated focus on depression and alcohol, or followed by a further nine 1-hour sessions with (ii) an alcohol focus; (iii) a depression focus; or (iv) an integrated focus. Follow-up assessments occurred 18 weeks after baseline. RESULTS: Compared with the brief intervention, 10 sessions were associated with greater reductions in average drinks per week, average drinking days per week and maximum consumption on 1 day. No difference in duration of treatment was found for depression outcomes. Compared with single-focused interventions, integrated treatment was associated with a greater reduction in drinking days and level of depression. For men, the alcohol-focused rather than depression-focused intervention was associated with a greater reduction in average drinks per day and drinks per week and an increased level of general functioning. Women showed greater improvements on each of these variables when they received depression-focused rather than alcohol-focused treatment. CONCLUSIONS: Integrated treatment may be superior to single-focused treatment for coexisting depression and alcohol problems, at least in the short term. Gender differences between single-focused depression and alcohol treatments warrant further study.

Depression, anxiety and body image after treatment for invasive stage one epithelial ovarian cancer

Background: Diagnosis of epithelial ovarian cancer (EOC) in young women has major implications including those to their reproductive potential. We evaluated depression, anxiety and body image in patients with stage I EOC treated with fertility sparing surgery (FSS) or radical surgery (RS). We also investigated fertility outcomes after FSS.----- Methods: A retrospective study was undertaken in which 62 patients completed questionnaires related to anxiety, depression, body image and fertility outcomes. Additional information on adjuvant therapy after FSS and RS and demographic details were abstracted from medical records. Both bi and multivariate regression models were used to assess the relationship between demographic, clinical and pathological results and scores for anxiety, depression and body image.----- Results: Thirty-nine patients underwent RS and the rest, FSS. The percentage of patients reporting elevated anxiety and depression (subscores ≥ 11) were 27 % and 5% respectively. The median (inter quartile range) score for body image scale (BIS) was 6 (3-15). None of the demographic or clinical factors examined showed significant association with anxiety and BIS with the exception of ‘time since diagnosis’. For depression, post-menopausal status was the only independent predictor. Among those 23 patients treated by FSS, 14 patients tried to conceive (7 successful), resulting in 7 live births, one termination of pregnancy and one miscarriage.----- Conclusion: This study shows that psychological issues are common in women treated for stage I EOC. Reproduction after FSS is feasible and lead to the birth of healthy babies in about half of patients who wished to have another child. Further prospective studies with standardised instruments are required.

Cognitive behaviour therapy (CBT) for the treatment of co-occurring depression and substance use : current evidence and directions for future research

Issues and Approach: The high rates of co-occurring depression and substance use, and the negative impact of this on illness course and outcomes have been well established. Despite this, few clinical trials have examined the efficacy of cognitive behaviour therapy (CBT). This paper systematically reviews these clinical trials, with an aim of providing recommendations for how future research can develop a more robust evidence base for the treatment of these common comorbidities. Leading electronic databases, including PubMed (ISI) and PsychINFO (CSA), were searched for peer-reviewed journal articles using CBT for the treatment of co-occurring depression and substance use. Of the 55 articles identified, 12 met inclusion criteria and were included in the review. ---------- Key Findings: There is only a limited evidence for the effectiveness of CBT either alone or in combination with antidepressant medication for the treatment of co-occurring depression and substance use. While there is support for the efficacy of CBT over no treatment control conditions, there is little evidence that CBT is more efficacious than other psychotherapies. There is, however, consistent evidence of improvements in both depression and substance use outcomes, regardless of the type of treatment provided and there is growing evidence that that the effects of CBT are durable and increase over time during follow up. ---------- Conclusions. Rather than declaring the ‘dodo bird verdict’ that CBT and all other psychotherapies are equally efficacious, it would be more beneficial to develop more potent forms of CBT by identifying variables that mediate treatment outcomes.

Brief motivational interviewing for depression and anxiety

Motivational interviewing (MI)can be applied as a brief, low intensity (LI) intervention of 1-4 individualised sessions (typically 45-60 minutes in duration), including screening, assessment feedback, and psycho-education. MI is a client-centred, directive therapeutic style that enhances readiness for change by helping clients explore and resolve ambivalence (Miller and Rollnick 2002). A summary of the key components of brief MI interventions is provided in Table 16.1. There is a well-established evidence base for MI in the treatment of substance misuse (particularly alcohol misuse; Moyer et al. 2002), as well as a growing evidence for the use of MI in the treatment of other mental disorders (e.g. depression, PTSD, OCD), as well as suicidality and physical health problems (Hettema et al. 2005). Brief MI intervention can be delivered as a standalone treatment or as a motivational prelude to pharmacological and/or other psychological treatments (Hettema et al. 2005). MI has been used as an accompaniment to cognitive behavioural therapy (CBT) in the treatment of both depression and anxiety for resolving ambivalence about change and developing strategies for responding to resistance (e.g. treatment attendance, homework/medication compliance; Arkowitz et al. 2008a, 2008b). This chapter will describe how to apply brief MI interventions to the treatment of depression and anxiety as applied to the case of Megan (see Box 16.1) along with some of the challenges and potential solutions to applying MI in practice.