Phenotype and genotype correlation of the microconversion from the CYP21A1P to the CYP21A2 gene in congenital adrenal hyperplasia

Deficiency of 21-hydroxylase is the most common form of congenital adrenal hyperplasia (CAH-21OH). We determined by allele-specific PCR the frequency of microconversion in the CYP21A2 gene in 50 Brazilian patients with the classical (salt wasting: SW and simple virilizing: SV) forms and nonclassical (NC) form of CAH-21OH and correlated genotype with phenotype. Genotypes were classified into three mutation groups (A, B, and C) based on the amount of enzymatic activity in in vitro studies using adrenal cells. In 94 unrelated alleles, we diagnosed 76% of the affected alleles after screening for 7 microconversions. The most frequent point mutations observed in this series were I172N (19%), V281L (18%), and IVS2,A/C>G,-12 (15%). In the SW form, the most frequent mutation was IVS2,A/C>G,-12 (38%), in the SV form it was I172N (53%), and in the NC form it was V281L (57.7%). We observed a good correlation between genotype and phenotype. Discordance between genotype and phenotype was found in one SV patient with a mild mutation in one of the alleles (R356W/V281L). However, we cannot rule out the presence of an additional mutation in these alleles. We also observed a good correlation of genotype with 17alpha-hydroxyprogesterone, testosterone, and androstenedione levels. The severity of external genitalia virilization correlated with the severity of mutation. In conclusion, the frequencies described in the present study did not differ from worldwide studies, including the Brazilian population. The few differences observed may reflect individual sample variations. This new Brazilian cohort study suggests the presence of new mutations in Brazilian patients with different forms of CAH-21OH.

Thr(118)Met amino acid substitution in the peripheral myelin protein 22 does not influence the clinical phenotype of Charcot-Marie-Tooth disease type 1A due to the 17p11.2-p12 duplication

The Thr(118)Met substitution in the peripheral myelin protein 22 (PMP22) gene has been detected in a number of families with demyelinating Charcot-Marie-Tooth (CMT1) neuropathy or with the hereditary neuropathy with liability to pressure palsy, but in none of them has it consistently segregated with the peripheral neuropathy. We describe here a CMT1 family (a 63-year-old man, his brother and his niece) in which two mutations on different chromosomes were found in the PMP22 gene, the 17p duplication, detected by fluorescent semiquantitative polymerase chain reaction (PCR) of microsatellite markers localized within the duplicated region on chromosome 17p11.2-p12, and the Thr(118)Met substitution, detected by direct sequencing the four coding exons of the PMP22 gene. A genotype/phenotype correlation study showed that the neuropathy segregates with the duplication and that the amino acid substitution does not seem to modify the clinical characteristics or the severity of the peripheral neuropathy. We did not find any evidence to characterize this substitution as a polymorphism in the population studied and we propose that the high frequency reported for this point mutation in the literature suggests that the Thr(118)Met substitution may be a hotspot for mutations in the PMP22 gene.

From genotype to phenotype : diversity and population structure of Flavobacterium psychrophilum

Flavobacterium psychrophilum is the etiological agent of bacterial cold-water disease (BCWD) causing high fish mortalities and significant economic losses to the freshwater salmonid aquaculture industry around the world. Today BCWD outbreaks are mainly treated with environmentally hazardous antimicrobial agents and alternative preventative measures are urgently needed in order to ensure the well-being of animals and the sustainability of aquaculture. The diversity of pathogenic bacteria challenges the development of universal control strategies and in many cases the pathogen population structure, i.e. the total genetic diversity of the species must be taken into account. This work integrates the tools of modern molecular biology and conventional phenotypic microbiology to gain knowledge about the diversity and population structure of F. psychrophilum. The present work includes genetic characterization of a large collection of isolates collected from diverse origins and years, from aquaculture in a whole region including different countries, and provides the first international validation of a universal multilocus sequence typing (MLST) approach for unambiguous genetic typing of F. psychrophilum. Population structure analyses showed that the global F. psychrophilum population is subdivided into pathogenic species-specific clones, of which one particular genetic lineage, clonal complex CC-ST2, has been responsible for the majority of BCWD outbreaks in rainbow trout (Oncorhynchus mykiss) in European aquaculture facilities over several decades. Genotypic and phenotypic population heterogeneity affecting antimicrobial resistance in F. psychrophilum within BCWD outbreaks was discovered. Specific genotypes were associated with severe infections in farmed rainbow trout and Atlantic salmon (Salmo salar), and in addition to high adherence, antimicrobial resistance was strongly associated with outbreak strains. The study brought additional support for the hypothesis of an epidemic F. psychrophilum population structure, where recombination is an important force for the generation and maintenance of genetic diversity, and a significant contribution towards mapping the genetic diversity of this important fish pathogen. Evidence indicating dissemination of virulent strains with commercial movement of fish and fish products was strengthened.

Assessment of techno-economic use cases for small-scale island grids

The main objective of the study was to define the methodology for assessing the limits for application island grids instead of interconnecting with existing grid infrastructure. The model for simulation of grid extension distance and levelised cost of electricity has been developed and validated by the case study in Finland. Thereafter, sensitivities of the application limits were examined with the respect to operational environment, load conditions, supply security and geographical location. Finally, recommendations for the small-scale rural electrification projects in the market economy environment have been proposed.

Lack of evidence for monosomy 1p36 in patients with Prader-Willi-like phenotype

Monosomy 1p36 is the most common subtelomeric microdeletion syndrome with an incidence rate estimated to be 1 in 5000 births. A hypothesis of a similarity between patients with 1p36 deletion and those with Prader-Willi syndrome and the existence of two different phenotypes for 1p36 microdeletion has been suggested. The main objective of the present study was to determine the existence of 1p36 microdeletion in a sample of patients with mental retardation, obesity and hyperphagia who tested negative by the methylation test for Prader-Willi syndrome. Sixteen patients (7 females, 9 males), 16-26 years old, were evaluated with high-resolution cytogenetic analysis at 550-850 band levels and with 11 polymorphic microsatellite markers located in the 1p36 region. All patients had normal cytogenetic and molecular results. The results obtained by high-resolution cytogenetic methodology were confirmed by the molecular analyses. We did not detect a 1p36 microdeletion in 16 subjects with the Prader-Willi-like phenotype, which reinforces that no correlation seems to exist between Prader-Willi-like phenotype and the 1p36 microdeletion syndrome.

Trailing end-point phenotype antibiotic-sensitive strains of Candida albicans produce different amounts of aspartyl peptidases

Candida albicans is an opportunistic fungal pathogen that causes severe systemic infections in immunosuppressed individuals. C. albicans resistance to antifungal drugs is a severe problem in patients receiving prolonged therapy. Moreover, trailing yeast growth, which is defined as a resistant MIC after 48 h of incubation with triazole antifungal agents but a susceptible MIC after 24 h, has been noted in tests of antifungal susceptibility against some C. albicans isolates. In this context, we recently noticed this phenomenon in our routine susceptibility tests with fluconazole/itraconazole and C. albicans clinical isolates. In the present study, we investigated the production of cell-associated and secreted aspartyl peptidases (Saps) in six trailing clinical isolates of C. albicans, since this class of hydrolytic enzymes is a well-known virulence factor expressed by this fungal pathogen. Sap2, which is the best-studied member of the Sap family, was detected by flow cytometry on the cell surface of yeasts and as a 43-kDa polypeptide in the culture supernatant, as demonstrated by Western blotting assay using an anti-Sap1-3 polyclonal antibody. Released aspartyl peptidase activity was measured with BSA hydrolysis and inhibited by pepstatin A, showing distinct amounts of proteolytic activity ranging from 5.7 (strain 44B) to 133.2 (strain 11) arbitrary units. Taken together, our results showed that trailing clinical isolates of C. albicans produced different amounts of both cellular and secreted aspartyl-type peptidases, suggesting that this phenotypic feature did not generate a regular pattern regarding the expression of Sap.

Effect of JJYMD-C, a novel synthetic derivative of gallic acid, on proliferation and phenotype maintenance in rabbit articular chondrocytes in vitro

Tissue engineering encapsulated cells such as chondrocytes in the carrier matrix have been widely used to repair cartilage defects. However, chondrocyte phenotype is easily lost when chondrocytes are expanded in vitro by a process defined as “dedifferentiation”. To ensure successful therapy, an effective pro-chondrogenic agent is necessary to overcome the obstacle of limited cell numbers in the restoration process, and dedifferentiation is a prerequisite. Gallic acid (GA) has been used in the treatment of arthritis, but its biocompatibility is inferior to that of other compounds. In this study, we modified GA by incorporating sulfamonomethoxine sodium and synthesized a sulfonamido-based gallate, JJYMD-C, and evaluated its effect on chondrocyte metabolism. Our results showed that JJYMD-C could effectively increase the levels of the collagen II, Sox9, and aggrecan genes, promote chondrocyte growth, and enhance secretion and synthesis of cartilage extracellular matrix. On the other hand, expression of the collagen I gene was effectively down-regulated, demonstrating inhibition of chondrocyte dedifferentiation by JJYMD-C. Hypertrophy, as a characteristic of chondrocyte ossification, was undetectable in the JJYMD-C groups. We used JJYMD-C at doses of 0.125, 0.25, and 0.5 µg/mL, and the strongest response was observed with 0.25 µg/mL. This study provides a basis for further studies on a novel agent in the treatment of articular cartilage defects.

Ocurrence of Vibrio spp., positive coagulase staphylococci and enteric bacteria in oysters (Crassostrea gigas) harvested in the south bay of Santa Catarina island, Brazil

The aim of this study was to assess the contamination of oysters (Crassostrea gigas), harvested in six different regions of the South Bay of Santa Catarina Island, with Coliforms at 45 ºC, Escherichia coli, Vibrio spp., positive coagulase staphylococci, and Salmonella sp. over a period of one year. One hundred eighty oyster samples were collected directly from their culture sites and analyzed. Each sample consisted of a pool of 12 oysters. All of the samples analyzed showed absence of Salmonella, 18 (10%) samples showed presence of Escherichia coli, 15 (8.3%) samples were positive for V. alginolyticus, and Vibriocholerae was detected in 4 samples (2.2%). The counts of positive-coagulase staphylococci varied from <10 to 1.9 x 102 CFU.g-1, whereas the counts of Coliforms at 45 ºC and E. coli ranged from <3 to 1.5 x 102 MPN.g-1 and <3 and 4.3 x 10 MPN.g-1, respectively. Counts of V. parahaemolyticus and V. vulnificus ranged between <3 and 7 MPN.g-1, for both microorganisms. This suggests the need for monitoring these Vibrios contamination in oysters. Based on the results of the microbiological assays, the samples analyzed showed acceptable bacteriological quality, i.e., they were within the parameters established by Brazilian Legislation.

Energy Management System for LVDC Island Networks

Recent developments in power electronics technology have made it possible to develop competitive and reliable low-voltage DC (LVDC) distribution networks. Further, islanded microgrids—isolated small-scale localized distribution networks— have been proposed to reliably supply power using distributed generations. However, islanded operations face many issues such as power quality, voltage regulation, network stability, and protection. In this thesis, an energy management system (EMS) that ensures efficient energy and power balancing and voltage regulation has been proposed for an LVDC island network utilizing solar panels for electricity production and lead-acid batteries for energy storage. The EMS uses the master/slave method with robust communication infrastructure to control the production, storage, and loads. The logical basis for the EMS operations has been established by proposing functionalities of the network components as well as by defining appropriate operation modes that encompass all situations. During loss-of-powersupply periods, load prioritizations and disconnections are employed to maintain the power supply to at least some loads. The proposed EMS ensures optimal energy balance in the network. A sizing method based on discrete-event simulations has also been proposed to obtain reliable capacities of the photovoltaic array and battery. In addition, an algorithm to determine the number of hours of electric power supply that can be guaranteed to the customers at any given location has been developed. The successful performances of all the proposed algorithms have been demonstrated by simulations.

Electrical Safety of Island Operated Low Voltage DC Network

Today, renewable energy technologies and modern power electronics have made it feasible to implement low voltage direct current (LVDC) microgrids (MGs) ca-pable to island operation. Such LVDC networks are particularly useful in remote areas. However, there are still pending issues in island operated LVDC MGs like electrical safety and controlled operation, which should be addressed before wide-scale implementation. This thesis is focused on the overall protection of an island operated LVDC network concept, including protection against electrical shocks, mains equipment protection and protection of photovoltaic (PV) power sources and battery energy storage systems (BESSs). The topic is approached through ex-amination of the safety hazards and the appropriate methods to protect against them, comprising considerations for earthing system selection and realisation of the protection system.

Reducing auxiliary power of a fluid bed boiler island

The aim of this thesis is to find and analyze different methods which reduce fluid bed boilers’ auxiliary power consumption. The objective is to examine the effects and feasibility of these methods. The literature part explains how fluid bed boilers work and what are the main sources of auxiliary power consumption. Designs and operation of these equipment are presented. The literature part also discusses the basics of auxiliary power consumption reduction and introduces four low pressure drop constructions. The experimental part inspects six different methods. Effects of these methods on the auxiliary power consumption are calculated and their impacts on the operation of the boiler are modeled. Calculations show that reasonable changes can reduce fluid bed boiler’s auxiliary power consumption by 2,1-10,2 %. Biggest reductions come from lower air coefficients, smaller bed a-level pressures and lower primary/secondary air –ratios. Models showed no problems with the smaller bed a-level pressures. With the lower air coefficients and smaller primary/secondary air –ratios the models showed a significant increase in the carbon monoxide levels.

Quaternary geology of the Campbellford, Trenton, Consecon, Tweed, Belleville, Wellington, Sydenham, Bath, and Yorkshire Island map-areas, Ontario /

Sediment relationships observed during geological mapping in southeastern Ontario indicate a relatively simple deglaciation history for the area during late Wisconsin time. The ice from the north (part of the Lake Simcoe lobe) and the Lake Ontario ice lobe, which were coalesced during most of late Wisconsin time, initially separated along the crest of the Oak Ridges Moraine. Available data indicate that the Oak Ridges Moraine is composed primarily of sediments pre-late Wisconsin in age capped by late Wisconsin till and interlobate deposits. Retreat of the northern ice was relatively steady and resulted in the deposition of the Dummer Moraines, a facies of the drumlinized till to the south. Retreat of the Lake Ontario ice lobe into the Lake Ontario basin was interrupted by a re-advance which covered the southeastern half of the map area. The northern ice had already retreated from the area by this time. The Lake Ontario lobe was fed through the St. Lawrence Valley, indicating that the Ottawa Valley was ice filled at this time. High level glacial lakes fronted the ice during deglaciation. These waters quickly fell to low levels as the ice retreated from the St. Lawrence Valley, opening lower outlets.

A reconnaissance study of the metamorphic petrology and volcanic geochemistry of a portion of the Island Lake greenstone belt, Manitoba /

The Island Lake greenstone belt is one of the major Archean supracrustal exposures in the northwestern part of the Superior Province of the Canadian Shield. This belt is subdivided into two units: 1) a lower sequence characterised by pillowed to massive, locally pyroclastic, basalt to andesite with a thin central zone of felsic derivatives, all of which are interbedded with and overlain by thick sequences of turbidite facies rock; 2) the upper unit which consists of thick stratified conglomerate overlain by thickly bedded arkose and feldspathic greywacke. Reconnaissance sampling traverses were completed across both the strike of the belt and along its margins with adjacent granitoids. Most of the belt is within the greenschist metamorphic f acies with amphibolite facies occurring in certain areas near t he margins. A post-tectonic, low pressure thermal event may be responsible for the development of a unit of cordierite schi s t which stretches southeastwards from the east end of Cochrane Bay. Volcanism is cyclical in nature changing from tholeiitic to calc-alkaline. There is a general progression in the character of the lavas from mafic t o felsic with stratigraphic height. Chemica l d a ta sugges t that h i gh level fractionation of a mantle- derived ' dry' magma i s t he s ource of the thole i iti c lavas. Contamination of this magma with 'we t' sia l and subsequent fractionation may be r esponsi b l e for the calcalkaline phases .Observations of stratigraphic relationships (in particular the contact between the supracrustals and the granitoids) coupled with the metamorphic and chemical studies, allow the construction of a preliminary model for the evolution of the Island Lake greenstone belt. The following sequential development is suggested: 1) a platform stage characterised by the subaqueous effusion of mafic to intermediate lavas of alternating tholeiitic and calc-alkaline affinities; 2) an edifice stage marked by the eruption of felsic calc-alkaline rocks; 3) an erosional stage characterised by the deposit~on of thick sequences of turbidite facies rocks; 4) the impingement of granitic masses into the margins of the greenstone belt, which was probably related to a downward warping of the supracrustal pilei 5) the erosion of sialic massifs surrounding and within the greenstone belt and of early supracrustal piles, to give the clastic upper unit.

Breeding parameters of great cormorants (Phalacrocorax carbo carbo) at mixed species colonies on Prince Edward Island, Canada

Breeding parameters of Great Cormorants (PkaZac/iOCOfiCLX CCUibo dCUtbo) and Double-crested Cormorants (P. CLU/uXuA CMJhLtllb) were examined at two mixed species colonies at Cape Tryon and Durell Point, Prince Edward Island from 1976 to 1978. Differential access to nests at the two colony sites resulted in more complete demographic data for P. CCUibo than for P. CLUJiituA. In 1911j P. CCtfibo was present at both colonies by 21 March, whereas P. auAAJtuA did not return until 1 April and 16 April at Cape Tryon and Durell Point, respectively. Differences in the arrival chronology by individuals of each species and differences in the time of nest site occupation according to age, are suggested as factors influencing the nest site distribution of P. CXUtbo and P. aiVtituA at Cape Tryon. Forty-eight P. dOJtbo chicks banded at the Durell Point colony between 19 74 and 19 76 returned there to nest as two- to four-year olds in 19 77 and 19 78. Unmarked individuals with clutch-starts in April were likely greater than four years old as all marked two to four-year olds (with one possible exception) in 19 77 and 1978 had clutch-starts in May and June. Seasonal variation in the breeding success of P. dOJibo individuals was examined at Durell Point in 1977. Mean clutch-size, hatching success and fledging success exhibited a seasonal decline. Four- and 5-egg clutches represented the majority (75%) of all P. CCUibo clutches at Durell Point in 1977 and had the highest reproductive success (0.48 and 0.43 chicks fledged per egg laid respectively). Smaller clutches produced small broods with significantly higher chick mortality while larger clutches suffered high egg loss prior to clutch completion.