Friedreich ataxia (FRDA) is an autosomal recessive, neurodegenerative disease caused by the excessive pathological expansion of an unstable GAA trinucleotide repeat within intron 1 of the FXN gene. FRDA occurs due to a pathological GAA repeat ranging from 70-1200 repeats, whereas normal individuals have up to 40 repeats. The prevalence of FRDA in Caucasians is 1:50,000 and it is the most common inherited ataxia. The presence of this large GAA repeat leads to silencing of the FXN gene, resulting in severely diminished levels of the essential mitochondrial protein, frataxin. Frataxin is required throughout development. Therefore, decreased frataxin levels affect the onset and progression of FRDA due to mitochondrial iron accumulation and an increase in oxidative damage, leading to detrimental cellular defects. The pathology of FRDA predominantly affects the cerebellum. Various FRDA cellular and mouse models have been developed to represent the characteristics of this debilitating disease. Data has revealed a positive correlation to exist between an increase in the number of GAA repeats and the severity of the disease. The analysis of these models has shown the expansion of the GAA repeat to be unstable, resulting in both somatic and intergenerational instability. In recent times, a single nucleotide polymorphism (SNP), Asn/Ser46, in the Sirt6 protein of FRDA patients has been associated with a better outcome of the disease. Therefore, Sirt6 heterozygote knockout mice obtained from the Jackson Laboratory were bred with YG8sR FRDA mice, to observe the effects of Sirt6 status on expanded GAA repeat instability, compared to Sirt6 wild type YG8sR FRDA mice. Densitometry analysis showed decreased levels of somatic GAA repeat instability and an increase in FXN protein in 6-month old heterozygous Sirt6 KO mice. However, increased levels of GAA repeat instability and decreased levels of FXN protein was seen in 12-month heterozygous Sirt6 KO mice. The SNP and decreased Sirt6 function may be beneficial to begin with, but not in the long run. A major aim of scientific research is to pave way for the generation of effective therapy. Therefore, attention turned to therapy to increase frataxin protein levels in YG8sR transgenic mice. The effects of the Src kinase inhibitor dasatinib, which had previously been shown to increase frataxin in cell culture, were investigated in YG8sR mice. Dasatinib was administered over a 3-month period, during which time motor coordination ability and frataxin protein levels were investigated. No effect on motor coordination was seen over the course of the treatment. Although, there was a non-significant decrease in frataxin protein levels in the brain in comparison to the vehicle-treated mice. In addition, further analysis by immunohistochemistry showed a decrease of frataxin protein expression in the dorsal root ganglia (DRG). Furthermore, the mean body weight of the dasatinib-treated mice was shown to decrease over time in comparison to vehicle-treated mice. Overall, these studies do not support the use of dasatinib for FRDA therapy. Further studies investigated a new line of GAA repeat expansion-containing FRDA transgenic mice, designated 'YG8LR'. These mice contain approximately 410 GAA repeats, which is significantly more than the previous 'YG8sR' line of mice which contained approximately 220 GAA repeats. As result of this larger GAA repeat, a further decrease of frataxin mRNA and protein was detected in the cerebellum and heart of YG8LR mice compared to YG8sR mice. Further investigations were undertaken to study the epigenetic status and the effects of lowered frataxin protein on the phenotype of the YG8LR model. Increased levels of histone deacetylation and methylation, together with DNA methylation, were detected at the FXN transgenic locus. The YG8LR mice also showed somatic and intergenerational GAA repeat instability as previously detected in earlier FRDA mouse models. Immunohistochemistry analysis of DRG sections showed a decreased level of frataxin protein in YG8LR mice. However, there was an absence of vacuoles in the DRG of the YG8LR mice as previously seen in YG8sR mice, although this particular phenotype is not actually seen in FRDA patients. Motor coordination ability and locomotor activity were significantly decreased as assessed by accelerating rotarod, beam-walk and beam-breaker open-field locomotor analysis. YG8LR mice showed increased glucose intolerance and insulin hypersensitivity in comparison to YG8sR and Y47R mice, investigated by glucose and insulin tolerance tests. Moreover, the most common cause of death in FRDA patients is caused by cardiomyopathy, therefore heart weights were obtained from B6, Y47R, YG8sR and YG8LR mice and a slight increase in mean heart weight was observed in YG8LR mice compared to the other models. This could suggest the lower levels of frataxin leads to the pathology of the heart as seen in FRDA patients. These investigations may help to provide an insight into and confirm molecular mechanisms of the disease, as well as providing a great mouse model for testing future FRDA therapies.

In this thesis, compared to the conventional pseudo gate all around SOI MOSFET, a similar device is successfully implemented already. This three dimension structure illustrate a pseudo gate all around SOI MOSFET which contains at least four advantages as listed in the following: (1). Using nearly pseudo gate all around to substitute a conventional single gate structure; in this new design, except the poly-silicon gate formed above the silicon film layer, we further implement a tetragonal-like gate in the beneath, front and back sides of the aforementioned silicon film layer. Such tetragonal-like gate can reach some goals obviously, like better gate controllability and stronger current drive. (2). The simulation of pseudo gate all around will be compared to a conventional pseudo gate all around by many different electric features to verify that this type of structure should have possibly higher manufacture yield and reliable characteristic. (3). We can separate the gate into the above gate and the beneath gate to form a four terminals device. By applying different touching electrodes to the two gates, we can bring the gate controllability up, and strengthen the current drive, and suppress some negative effects of the device. (4). Pseudo gate all around device will be simpler compared to a conventional pseudo gate all around device in the manufacture effort, so it is easier for devices miniaturization in the future, and it can also improve some shortages, like hard-etching and hole-fulfillment, the technical issues in the conventional pseudo gate all arounds device, thus the devices manufacture yield will be brought up greatly, even a conventional pseudo gate all arounds device is applied to thin-film transistor made way, it still needs more one mask for holes plight compared to a pseudo gate all arounds device, it will reflect to the investment. The simulation tool ISE TCAD is being courtesy of the simulation for pseudo gate all around device structure to verify the feasibility of manufacture and process. And TCAD Dessis is applied to simulate I-V characteristic and other electric analysis. Based on the different curves simulated, we reach the conclusion: put this pseudo gate all around device on the comparison with a conventional pseudo gate all around device, in the following items, threshold voltage, sub-threshold factor are drawn. Incredibly, our design has a flattered input curve and less leakage Ioff, respectively. In the output curve, our work has harvester saturated current and finally, by different structure of simulated result to verify the active region depth may eliminate ¡§kink effect¡¨ under 50nm. Hence, pseudo gate all around SOI MOSFET will be more potentially applied to ULSI (ultra large schematic integrated circuit) in the future, compared to other previous SOI devices.

Neste trabalho, é apresentada uma análise da operação analógica de transistores de múltiplas portas, avaliando a tensão Early, o ganho de tensão em malha aberta, a razão da transcondutância pela corrente de dreno (gm/IDS), a condutância de dreno e, em especial, a distorção harmônica, exibida por estes dispositivos. Ao longo deste trabalho, foram estudados FinFETs, dispositivos de porta circundante (Gate-All-Around GAA) com estrutura de canal gradual (Graded-Channel GC) e transistores MOS sem junções (Junctionless - JL). Inicialmente, foi efetuada a análise da distorção harmônica apresentada por FinFETs com e sem a presença de tensão mecânica biaxial, com diversas larguras de fin (Wfin) e comprimentos de canal (L), quando estes operavam em saturação, como amplificadores de um único transistor. Nesta análise, as não-linearidades foram avaliadas através da extração das distorções harmônicas de segunda e terceira ordens (HD2 e HD3, respectivamente), mostrando que a presença de tensão mecânica tem pouca influência em HD2, mas altera levemente a HD3. Quando os ganhos de tensão em malha aberta dos dispositivos são levados em conta, transistores sem tensão, também chamados de convencionais, mais estreitos apresentam grande vantagem em termos de HD2 em relação aos tensionados. Ainda nesta análise, percebeu-se que HD2 e HD3 de transistores tensionados pioram com a redução da temperatura, especialmente em inversão mais forte. Na seqüência, foi efetuada uma análise de HD3 em FinFETs com e sem tensão mecânica de vários comprimentos e larguras de canal, operando em região triodo e aplicados a estruturas balanceadas 2-MOS, mostrando que presença de tensão mecânica traz pouca influência em HD3, mas reduz a resistência do canal dos dispositivos (RON), o que não é bom em estruturas resistivas, como as avaliadas. Nesta análise, ainda, pode-se perceber uma melhora em HD3 superior a 30 dB ao se incrementar VGT de zero a 1,0 V, em cuja tensão dispositivos mais estreitos apresentam curvas mais lineares que os mais largos. Então, foi estudada a distorção apresentada por transistores GAA e GC GAA operando em regime triodo, aplicados a estruturas 2-MOS, onde se pôde perceber que GC GAAs com maiores comprimentos da região fracamente dopada apresentam vantagem em HD3 em relação aos demais, para valores de VGT superiores a 2 V. Na avaliação destas estruturas em função da temperatura, percebeu-se que, para VGT superiores a 1,1 V, HD3 depende fortemente da temperatura e piora conforme a temperatura diminui. O estudo envolvendo transistores sem junções foi mais focado em seus parâmetros analógicos, comparando-os aos apresentados por dispositivos de porta tripla ou FinFETs. Em inversões moderada e forte, transistores sem junção apresentaram menores valores para gm/IDS em relação a dispositivos de FinFETs polarizados em um mesmo nível de corrente, entretanto, a dependência de gm/IDS com a temperatura em transistores sem junção também foi menor que a apresentada por FinFETs. JL e FinFETs apresentaram comportamentos distintos para a tensão Early e o ganho de tensão em malha aberta em função da temperatura. Estes parâmetros sempre melhoram com o aumento da temperatura em dispositivos JL, enquanto que exibem seu máximo valor em temperatura ambiente em FinFETs. Nas proximidades da tensão de limiar, transistores sem junção com largura de fin de 30 nm exibiram tensão Early e ganho superiores a 80 V a 57 dB, respectivamente, enquanto que FinFETs mostraram Tensão Early de 35 V e ganho de 50 dB. Em todos os estudos efetuados ao longo do trabalho, procurou-se apontar as causas das não-linearidades apresentadas pelos dispositivos, a partir de modelos analíticos que pudessem relacionar a física de funcionamento dos transistores com os resultados experimentalmente obtidos. / In this work it is presented an analysis of the analog operation of multiple gate transistors, evaluating the Early Voltage, the open-loop voltage gain, the transconductance over the drain current ratio (gm/IDS), the drain conductance and, especially, the harmonic distortion exhibited by these devices. Along the work, FinFETs, Gate-All-Around (GAA) devices with the Graded-Channel (GC) structure and MOS transistors without junctions (Junctionless - JL) were studied. Initially, an analysis of the harmonic distortion presented by conventional and biaxially strained FinFETs with several fin widths (Wfin) and channel lengths (L) was performed, when these devices were operating in saturation as single transistor amplifiers. In this analysis, the non-linearities were evaluated through the extraction of the second and the third order harmonic distortions (HD2 and HD3, respectively), and it was shown that the presence of strain has negligible influence in HD2, but slightly changes HD3. When the open loop voltage gain of the devices is taken into consideration, narrower conventional transistors present a huge advantage with respect to the strained ones in terms of HD2. Also, it was perceived that both HD2 and HD3 of strained FinFETs worsen with the temperature decrease, especially in stronger inversion. In the sequence, an analysis of the HD3 presented by conventional and strained FinFETs of several fin widths and channel lengths operating in the triode regime was performed. These devices were applied to 2-MOS balanced structures, showing that the presence of the strain does not influence significantly the HD3, but reduces the resistance in the channel of the transistors (RON), which is not good for resistive structures as the ones evaluated. In this analysis, it can also be observed an HD3 improvement of 30 dB when VGT is increased from zero up to 1,0 V, where narrower devices present transfer characteristics more linear than the wider ones. Then, it was studied the distortion presented by GAA and GC GAA devices operating in the triode regime, applied to 2-MOS structures. In this case, it could be perceived that GC GAAs with longer lightly doped regions present better HD3 in comparison to the other devices for VGT higher than 2.0 V. In the evaluation of these structures as a function of the temperature, it could be seen that for VGT higher than 1.1 V, HD3 strongly depends on the temperature and worsens as the temperature decreases. The study involving JL transistors was focused on their analog parameters, comparing them to the ones presented by triple gate devices or FinFETs. In moderate and strong inversions, Junctionless showed lower values for gm/IDS with respect to triple gate devices biased at a similar current level. However, the dependence of gm/IDS from Junctionless with the temperature was also smaller than the one presented by FinFETs. Junctionless and FinFETs exhibited distinct behaviors for the Early voltage and the open-loop voltage gain as a function of the temperature. These parameters always improve with the temperature raise in JL devices whereas they exhibit their maximum values around room temperatures for FinFETs. In the proximity of the threshold voltage, Junctionless with fin width of 30 nm presented Early voltage and intrinsic gain larger than 80 V and 57 dB, respectively, whereas FinFETs exhibited Early voltage of 35 V and gain of 50 dB. For all the studies performed in this work, the probable causes of the non-linearities were pointed out, from analytic models that could correlate the physical work of the devices with the experimental results.

Neste trabalho, é apresentada uma análise da operação analógica de transistores de múltiplas portas, avaliando a tensão Early, o ganho de tensão em malha aberta, a razão da transcondutância pela corrente de dreno (gm/IDS), a condutância de dreno e, em especial, a distorção harmônica, exibida por estes dispositivos. Ao longo deste trabalho, foram estudados FinFETs, dispositivos de porta circundante (Gate-All-Around GAA) com estrutura de canal gradual (Graded-Channel GC) e transistores MOS sem junções (Junctionless - JL). Inicialmente, foi efetuada a análise da distorção harmônica apresentada por FinFETs com e sem a presença de tensão mecânica biaxial, com diversas larguras de fin (Wfin) e comprimentos de canal (L), quando estes operavam em saturação, como amplificadores de um único transistor. Nesta análise, as não-linearidades foram avaliadas através da extração das distorções harmônicas de segunda e terceira ordens (HD2 e HD3, respectivamente), mostrando que a presença de tensão mecânica tem pouca influência em HD2, mas altera levemente a HD3. Quando os ganhos de tensão em malha aberta dos dispositivos são levados em conta, transistores sem tensão, também chamados de convencionais, mais estreitos apresentam grande vantagem em termos de HD2 em relação aos tensionados. Ainda nesta análise, percebeu-se que HD2 e HD3 de transistores tensionados pioram com a redução da temperatura, especialmente em inversão mais forte. Na seqüência, foi efetuada uma análise de HD3 em FinFETs com e sem tensão mecânica de vários comprimentos e larguras de canal, operando em região triodo e aplicados a estruturas balanceadas 2-MOS, mostrando que presença de tensão mecânica traz pouca influência em HD3, mas reduz a resistência do canal dos dispositivos (RON), o que não é bom em estruturas resistivas, como as avaliadas. Nesta análise, ainda, pode-se perceber uma melhora em HD3 superior a 30 dB ao se incrementar VGT de zero a 1,0 V, em cuja tensão dispositivos mais estreitos apresentam curvas mais lineares que os mais largos. Então, foi estudada a distorção apresentada por transistores GAA e GC GAA operando em regime triodo, aplicados a estruturas 2-MOS, onde se pôde perceber que GC GAAs com maiores comprimentos da região fracamente dopada apresentam vantagem em HD3 em relação aos demais, para valores de VGT superiores a 2 V. Na avaliação destas estruturas em função da temperatura, percebeu-se que, para VGT superiores a 1,1 V, HD3 depende fortemente da temperatura e piora conforme a temperatura diminui. O estudo envolvendo transistores sem junções foi mais focado em seus parâmetros analógicos, comparando-os aos apresentados por dispositivos de porta tripla ou FinFETs. Em inversões moderada e forte, transistores sem junção apresentaram menores valores para gm/IDS em relação a dispositivos de FinFETs polarizados em um mesmo nível de corrente, entretanto, a dependência de gm/IDS com a temperatura em transistores sem junção também foi menor que a apresentada por FinFETs. JL e FinFETs apresentaram comportamentos distintos para a tensão Early e o ganho de tensão em malha aberta em função da temperatura. Estes parâmetros sempre melhoram com o aumento da temperatura em dispositivos JL, enquanto que exibem seu máximo valor em temperatura ambiente em FinFETs. Nas proximidades da tensão de limiar, transistores sem junção com largura de fin de 30 nm exibiram tensão Early e ganho superiores a 80 V a 57 dB, respectivamente, enquanto que FinFETs mostraram Tensão Early de 35 V e ganho de 50 dB. Em todos os estudos efetuados ao longo do trabalho, procurou-se apontar as causas das não-linearidades apresentadas pelos dispositivos, a partir de modelos analíticos que pudessem relacionar a física de funcionamento dos transistores com os resultados experimentalmente obtidos. / In this work it is presented an analysis of the analog operation of multiple gate transistors, evaluating the Early Voltage, the open-loop voltage gain, the transconductance over the drain current ratio (gm/IDS), the drain conductance and, especially, the harmonic distortion exhibited by these devices. Along the work, FinFETs, Gate-All-Around (GAA) devices with the Graded-Channel (GC) structure and MOS transistors without junctions (Junctionless - JL) were studied. Initially, an analysis of the harmonic distortion presented by conventional and biaxially strained FinFETs with several fin widths (Wfin) and channel lengths (L) was performed, when these devices were operating in saturation as single transistor amplifiers. In this analysis, the non-linearities were evaluated through the extraction of the second and the third order harmonic distortions (HD2 and HD3, respectively), and it was shown that the presence of strain has negligible influence in HD2, but slightly changes HD3. When the open loop voltage gain of the devices is taken into consideration, narrower conventional transistors present a huge advantage with respect to the strained ones in terms of HD2. Also, it was perceived that both HD2 and HD3 of strained FinFETs worsen with the temperature decrease, especially in stronger inversion. In the sequence, an analysis of the HD3 presented by conventional and strained FinFETs of several fin widths and channel lengths operating in the triode regime was performed. These devices were applied to 2-MOS balanced structures, showing that the presence of the strain does not influence significantly the HD3, but reduces the resistance in the channel of the transistors (RON), which is not good for resistive structures as the ones evaluated. In this analysis, it can also be observed an HD3 improvement of 30 dB when VGT is increased from zero up to 1,0 V, where narrower devices present transfer characteristics more linear than the wider ones. Then, it was studied the distortion presented by GAA and GC GAA devices operating in the triode regime, applied to 2-MOS structures. In this case, it could be perceived that GC GAAs with longer lightly doped regions present better HD3 in comparison to the other devices for VGT higher than 2.0 V. In the evaluation of these structures as a function of the temperature, it could be seen that for VGT higher than 1.1 V, HD3 strongly depends on the temperature and worsens as the temperature decreases. The study involving JL transistors was focused on their analog parameters, comparing them to the ones presented by triple gate devices or FinFETs. In moderate and strong inversions, Junctionless showed lower values for gm/IDS with respect to triple gate devices biased at a similar current level. However, the dependence of gm/IDS from Junctionless with the temperature was also smaller than the one presented by FinFETs. Junctionless and FinFETs exhibited distinct behaviors for the Early voltage and the open-loop voltage gain as a function of the temperature. These parameters always improve with the temperature raise in JL devices whereas they exhibit their maximum values around room temperatures for FinFETs. In the proximity of the threshold voltage, Junctionless with fin width of 30 nm presented Early voltage and intrinsic gain larger than 80 V and 57 dB, respectively, whereas FinFETs exhibited Early voltage of 35 V and gain of 50 dB. For all the studies performed in this work, the probable causes of the non-linearities were pointed out, from analytic models that could correlate the physical work of the devices with the experimental results.

Increased mutagenesis is a hallmark of cancers. On the other hand, this can trigger the generation of polymorphisms and lead to evolution. Lately, it has become clear that one of the major sources of increased mutation rates in the genome is chromosomal break formation and repair. A variety of factors can contribute to the generation of breaks in the genome. A paradoxical source of breaks is the sequence composition of the genomic DNA itself. Eukaryotic and prokaryotic genomes contain sequence motifs capable of adopting secondary structures often found to be potent inducers of double strand breaks culminating into rearrangements. These regions are therefore termed fragile sequence motifs. Here, we demonstrate that in addition to being responsible for triggering chromosomal rearrangements, inverted repeats and GAA/TTC repeats are also potent sources of mutagenesis. Repeat-induced mutagenesis extends up to 8 kb on either side of the break point. Remarkably, error-prone repair of the break by Polζ reconstitutes the repeats making them a long term source of mutagenesis. Despite its negative connotations for genome stability, the mechanisms underlying the unstable nature of double strand break repair pathways are not known. Previous studies have demonstrated that break induced replication (BIR), a mechanism employed to repair broken chromosomes with only one repairable end, is highly mutagenic, undergoes frequent template switching and often yields half-crossovers. In the work presented here, we show that the instabilities inherent to BIR can be attributed to its unusual mode of synthesis. We determined that BIR proceeds via a migrating bubble with long stretches of single-stranded DNA and culminates with conservative inheritance of the newly synthesized DNA. We propose that the mechanisms described here might be important for generation of repair-associated mutagenesis in higher organisms. Secondary structure forming repeats like inverted repeats have been found to be enriched in cancer cells. These motifs often constitute chromosomal rearrangement hot-spots and demonstrate the phenomenon of kataegis. This study provides a mechanistic insight into how such breakage-prone motifs contribute to hypermutability of cancer genomes.

The main goal of this research is to understand molecular mechanisms of GAA/TTC-associated genetic instability in a model eukaryotic organism, S. cerevisiae. We demonstrate that expanded GAA/TTC repeats represent a threat to eukaryotic genome integrity by triggering double-strand breaks and gross chromosomal rearrangements. The fragility potential strongly depends on the length of the tracts and orientation of the repeats relative to the replication origin and to block replication fork progression. MutSbeta complex and endonuclease activity of MutLalpha play an important role in facilitation of fragility. In addition to GAA/TTC triplex forming repeats, non-GAA polypurine polypyrimidine mirror repeats that are prone to the formation of similar structures were found to be hotspots for rearrangements in humans and other model organisms. These include H-DNA forming sequences located in the major breakpoint cluster region at BCL2, intron 21 of PKD1, and promoter region of C-MYC. Lastly, we have investigated the effect of the triplex-binding small molecules, azacyanines, on GAA-mediated fragility using the chromosomal arm loss assay. We have found that in vivo, azacyanines stimulate (GAA/TTC)-mediated arm loss in a dose dependent manner in actively dividing cells. Azacyanines treatment enhances the GAA-induced replication arrest. We discovered that also, azacyanines at concentrations that induce fragility also inhibit cell growth. Over 60% of yeast cells are arrested at G2/M stage of the cell cycle. This implies an activation of DNA-damage checkpoint response.

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder, caused by a GAA repeat expansion mutation within intron 1 of the FXN gene, resulting in reduced level of frataxin protein. Normal individuals have 5 to 40 GAA repeat sequences, whereas affected individuals have approximately 70 to more than 1000 GAA triplets. Frataxin is a mitochondrial protein involved in iron-sulphur cluster and heme biosynthesis. The reduction in frataxin expression leads to oxidative stress, mitochondrial iron accumulation and consequential cell death with the primary sites of neurons of the dorsal root ganglia and the dentate nucleus of the cerebellum. FRDA, which is the most common inherited ataxia, affecting 1:50,000 Caucasians, is characterised by neurodegeneration, cardiomyopathy, diabetes mellitus and skeletal deformities. To investigate FRDA molecular disease mechanisms and therapy, several human FXN YAC transgenic mouse models have been established: Y47R, containing normal-sized (GAA)9 repeats; YG8R and YG22R, which initially contained expanded GAA repeats of 90-190 units and 190 units, respectively, but which have subsequently been bred to now contain expanded GAA repeats of 120-220 units and 170-260 units, respectively, and YG8sR (YG8R with a small GAA band) that was recently generated from YG8R breeding. To determine the FXN transgene copy number in the enhanced GAA repeat expansion-based FRDA mouse lines, a TaqMan qPCR assay was developed. The results demonstrated that the YG22R and Y47R lines had a single copy of the FXN transgene while the YG8R line had two copies. The YG8s lines showed less than one copy of the target gene, suggesting potential deletion of the FXN gene. Single integration sites of all transgenes were confirmed by fluorescence in situ hybridisation (FISH) analysis of metaphase and interphase chromosomes. However, in the YG8s line, at least 25% of the YG8s cells had no signals, while the remaining cells showed one signal corresponding to the transgenic FXN gene. In addition, the analysis of FXN exons in YG8s rescue mice by PCR confirmed the presence of all FXN exons in these lines, suggesting the incidence of somatic mosaicism in these lines. Extended functional analysis was carried out on these mice from 4 to 12 months of age. Coordination ability of YG8R, YG8sR and YG22R ‘FRDA-like’ mice, together with Y47R and C57BL6/J wild-type control mice, was assessed using accelerating rotarod analysis. The results indicated a progressive decrease in the motor coordination of YG8R, YG22R and YG8sR mice compared to Y47R or C57BL6/J controls. Locomotor activity was also assessed using an open field beam-breaker apparatus followed by four additional functional analyses including beam-walk, hang wire, grip strength and foot print tests. The results indicated significant functional deficits in the FRDA mouse models. Glucose and insulin tolerance tests were also conducted in the FRDA mouse models, indicating glucose intolerance and insulin hypersensitivity in the aforementioned lines. To investigate the correlation between the FRDA-like pathological phenotype and frataxin deficiency in the FRDA mouse models, frataxin mRNA and protein levels as well as somatic GAA repeat instability were examined. The results indicated that somatic GAA repeats increased in the cerebellum and brain of YG22R, YG8R and YG8sR mice, together with significantly reduced levels of FXN mRNA and protein in the liver of YG8R and YG22R compared to Y47R. However, YG8sR lines showed a significant decrease in FXN mRNA in all of the examined tissues compared to Y47R human FXN and C57BL6/J mouse Fxn mRNA. Protein expression levels were also considerably reduced in all the tissues of YG8sR mice compared to Y47R. Subsequently, the telomere length of human and mouse FRDA and control fibroblasts was assessed using qPCR and Q-FISH. The results indicated that the FRDA cells had chromosomes with relatively longer telomeric repeats in comparison to the controls. FRDA cells were screened for expression of telomerase activity using the TRAP assay and a quantitative assay for hTERT mRNA expression using TaqMan qRT-PCR. The results indicated that telomerase activity was not present in the FRDA cells. To investigate whether FRDA cells maintained their telomeres by ALT associated PML bodies (APBs), co-localisation of PML bodies with telomeres was assessed in these cells using combined immunofluorescence to PML and Q-FISH for telomere detection. The results demonstrated that the FRDA cells had significantly higher co-localised PML foci with telomeric DNA compared to the normal cells. Moreover, telomere sister chromatid exchange (T-SCE) frequencies were analysed in the human FRDA cell lines using chromosome orientation FISH (CO-FISH). The results indicated a significant increase in T-SCE levels of the FRDA cell lines relative to the controls. Furthermore, growth curve and population doubling analysis of the human FRDA and control fibroblasts was carried out. The results showed that the FRDA fibroblast cell cultures underwent growth arrest with higher cumulative population doubling compared to the controls. Though, further analysis of telomere length at different passage numbers revealed that the FRDA cells lost telomeres faster than the controls. Finally, the telomere dysfunction-induced foci (TIF) assay was performed to detect DNA damage in the human FRDA fibroblast cells using an antibody against DNA damage marker γ-H2AX and a synthetic PNA probe for telomeres. The frequency of γ-H2AX foci was significantly higher in the FRDA cells compared to the controls. Similarly, the FRDA cells had greater frequencies of TIFs in comparison to the controls, suggesting induced telomere dysfunction in the FRDA cells.

DNA repetitive sequences capable of adopting non-B DNA structures are a potent source of instability in eukaryotic genomes. They are strong inducers of chromosomal fragility and genome rearrangements that cause various hereditary diseases and cancers. In addition, a subset of repeats also has an ability to expand, which leads to more than 20 human genetic diseases that are collectively known as repeat expansion diseases. However, the mechanisms underlying the potential of these structure-prone motifs to break and expand are largely unknown. In this study, a systematic genome-wide screen was employed in yeast Saccharomyces cerevisiae to investigate the contributing factors of the instability of two representative non-B DNA-forming repeats: the triplex-adopting GAA/TTC tracts and the inverted repeats that can form hairpin and cruciform structures. The GAA/TTC screen revealed that DNA replication and transcription initiation are the two major pathways governing the GAA/TTC stability in yeast, as corresponding mutants strongly induce both fragility and large-scale expansions of the repeats. The inverted repeats screen and follow-up experiments revealed that both replication-dependent and -independent pathways are involved in maintaining the stability of palindromic sequences. We propose that similar mechanisms could operate in the human cells to mediate the deleterious metabolism of GAA and inverted repeats.

Evolutionary relationships within and among three Astragalus sections (Incani DC., Hypoglottidei DC., and Dissitiflori DC.) that were native to Turkey were inferred from variations of nucleotide sequences of both chloroplast and nuclear genome regions. In the current study, Fifty-six species included in the three Astragalus sections were utilized to figure out phylogenetic relationships and estimate evolutionary divergence time based on DNA sequence of trnL intron (trnL5&rsquo / -L3&rsquo / ) , trnL3&rsquo / -F(GAA) (trnL-F intergenic spacer), trnV intron, matK (maturase kinase) cpDNA (chloroplast) and ITS (internal transcribed spacer) nDNA (nuclear) regions. Fifty-six Astragalus species with their replicas and one Cicer species as outgroup were analyzed by polymerase chain reaction amplification and DNA sequencing methods. Eleven unknown samples were also used in the current study to understand their section and species name. The results of the study indicated that unknown A35 and A52 samples could be named as A. dasycarpus, while unknown A65 and A66 samples as A. ovatus and lastly unknown A2 sample as A. nitens or A. aucheri. Section of unknown A3, A16, A20, A108, A109 and A110 samples were determined as Incani, but the exact species identification of these samples were not possible because of their close phylogenetic associations with more than one species. Highest genetic diversity was observed when the DNA sequences of ITS nrDNA (nuclear ribosomal) region comprising three subregions as ITS1, 5.8S and ITS2 was used, while the lowest one was calculated when DNA sequence of trnL-F cpDNA region was analyzed. The genetic divergence between Incani and Dissitiflori sections was highest whereas between Hypoglottidei and Dissitiflori was lowest based on all used regions. To figure out phylogenetic relationships among Astragalus species distributed in Turkey and in other regions of the World, DNA sequences of studied regions of foreign samples were collected from the NCBI database and were evaluated with DNA sequence of Turkish species used in the curent study. The Iranian samples either scattered in the phylogenetic tree or attached to our samples externally. South and North American samples (New World Astragalus or Neo Astragalus group) were nested within a different subcluster, which was located in the main cluster produced by samples of Old World Astragalus group (Turkish samples). With these results, we can say that New World Astragalus group is monophyletic and diverged from Old World Astragalus group. Evolutionary divergence time for Astragalus genus was estimated as about 12.5 - 14.5 million years (Ma), and that of New World Astragalus group as 5.0 - 4.0 Ma when rates of nucleotide substitutions of trnL intron and matK cpDNA regions were analyzed. In addition to evolutionary divergence time estimation for Astragalus and New World Astragalus group, divergence times among used three sections of the genus were also calculated by using DNA sequences of trnL, trnV intron and matK cpDNA regions and results indicated that Hypoglottidei and Dissitiflori sections diverged about 5.0-7.0 million years later than Incani section.

<div>In this work, we investigate the trade-off between scalability and reliability for next generation logic-transistors i.e. Gate-All-Around (GAA)-FET, Multi-Bridge-Channel (MBC)-FET. First, we analyze the electronic properties (i.e. bandgap and</div><div>quantum conductance) of ultra-thin silicon (Si) channel i.e. nano-wire and nano-sheet based on first principle simulation. In addition, we study the influence of interface</div><div>states (or dangling bonds) at Si-SiO₂ interface. Second, we investigate the impact of bandgap change and interface states on GAA-FETs and MBC-FETs characteristics by</div><div>employing Non-equilibrium Green's Function based device simulation. In addition to that, we calculate the activation energy of Si-H bond dissociation at Si-SiO₂ interface for different Si nano-wire/sheet thickness and different oxide electric-field. Utilizing these thickness dependent activation energies for corresponding oxide electric-field, in conjunction with reaction-diffusion model, we compute the characteristics shift and analyze the negative bias temperature instability in GAA-FET and MBC-FET. Based on our analysis, we estimate the operational voltage of these transistors for a life-time of 10 years and the ON current of the device at iso-OFF-current condition. For example, for channel length of 5 nm and thickness < 5 nm the safe operating voltage needs to be < 0.55V. Furthermore, our analysis suggests that the benefit of Si thickness scaling can potentially be suppressed for obtaining a desired life-time of GAA-FET and MBC-FET.</div>