*2.4. Data Extraction, Analysis, Synthesis and Quality/Critical Appraisal*

Data were extracted on study details (author/s and country of study), methods (population, sample and sample size, collection and analysis methods), key results and conclusions. Studies were organised and tabulated into classifications by area of specialised fields. The data were analy sed using inductive and descriptive synthesis [43]. The review question was used as the basis for searching the data for relevant expressions, which were then further tabulated. To synthesise the data, the author used tabulation to search the data for similarities and differences and further organise the data into categories named according to the content. The author critically evaluated the final articles using the Joanna Briggs Institute Critical Appraisal tools for qualitative and quantitative studies. The JBI tool with ten items was applied to rate the quality of qualitative studies [44], whereas another JBI tool with eight items was used to evaluate the quantitative (cross-sectional) studies [45]. Finally, 15 studies that met the quality appraisal criteria were retained (n = 15). Table 1 shows the JBI for quantitative and mixed method studies included (n = 12). The included qualitative articles, presented in Table 2, scored yes (n = 2), and the single literature review is presented in Table 3.


*2.3. Yield Components and Other Important Agronomic Traits of BILs and BILF1s*

To assess the distribution pattern of yield-related traits between BILs and BILF1s, we surveyed the yield components of BILs and BILF1s in the paddy field of Shenyang Agricultural University in 2018. The normal distribution and transgressive segregation were observed in all of the traits surveyed in both BIL and BILF1s. These results indicate that all of these agronomic traits were controlled by multiple genes. The plant height of BILF1s mainly ranged from 105 to 120 cm, whereas that of BILs was from 120 to 135 cm. These results indicate that the heterozygous genotype of the F1 plants reduces the plant height, based on the fact that the F1 plants were shorter than Sasanishiki and Habataki (Figure 1A). In addition, the grain number per panicle of BILs ranged from 80 to 180, whereas that of the BILF1s ranged from 130 to 260. These results suggest that the heterozygous genotype of the F1 plants increases the grain number per panicle, which also coincides with the result that the F1 plants have significantly more grains per panicle than Sasanishiki and Habataki (Figure 2). Similar distributions of days to heading, panicle number, and 1000-grain weight were observed in both BILs and BILF1s. However, the variation range of setting rate in BILs was larger than in the BILF1s (Figure 3).

**Figure 3.** The distribution of yield-related traits in BILs and BILs. (**A**–**F**) The distribution of days to heading, number of panicles, plant height, grain number per panicle, setting rate, and 1000-grain weight in BILs and BILF1s.


1.3 Structured laser beam and atom

The development of structured laser beams such as Laguerre-Gaussian (LG) beams has enabled the coherent production of large number of identical spiral photons [44–47], which opens a new set of research in quantum optics in atomic level. LG beams are characterized by three quantum numbers: (i) the wave number k, (ii) the intertwined helical wave fronts l (an integer number) of azimuthal phasedependent that features a screw dislocation, and (iii) the radial nodes p [48, 50]. Each spiral photon in the LG beams carries lℏ of intrinsic orbital angular momentum (OAM) along the direction of propagation [51], which is arising from their nonuniform spatial intensity distribution. The characteristic shape of the intensity distribution of LG beams as well as their intrinsic orbital angular momentum can be observed as a result of this interaction. The exchange of angular momentum induces a torque and an azimuthal shift in the resonant frequency besides the usual axial Doppler shift and recoil shift which tailored the control of the motion of the center of mass of the atoms to rotate about the beam axis [52, 53]. In higher order quadrupole transition processes, the internal motion of the atom participates in orbital angular momentum exchange between structured light and atom [54–57]. However, in dipole transition, the interaction of LG beam with atom and therefore

transferring of OAM from LG beam to the external degree of freedom of the atom presents in the quantization of the center of mass motion of the atom [58].

A Rydberg atom with near classical size comparable to the wavelength of photon is big enough to feel phase differences of the helical wave front of LG beams. The LG beam has an advantage of control and narrows the resonances in electromagnetically induced transparency. The orbital angular momentum of LG beams has emerged as a popular choice for experiments on high-dimensional quantum information [59]. The dark center of blue-detuned Laguerre-Gaussian beam is emerged as an important feature to trap neutral atoms to decrease the atomic heating and decoherence rates and minimize the AC-Stark shift [60, 61]. In quantum information processing and data transmission, optical orbital angular momentum of spiral photons can provide an extra degree of entanglement [48] to carry quantum information in the different degrees of freedom by the higher dimensionality of the Hilbert space. In other words, the information carried by each photon can be increased significantly, from a qubit to a qudit, where d is the number of orthogonal basis vectors of the Hilbert space in which the photon lives.


*Edited by Aldemaro Romero and Edward O. Keith*

The highly specialized nature of marine mammals when compared with their terrestrial counterparts, the environment in which they live, and the impact of humans on them throughout history and at the present, have made of the scholarship on these creatures something unique in itself. Therefore, it is not surprising that many researchers have also taken a distinctive approach to their study. This volume is aimed at providing a glimpse at such diversity of views and approaches while delivering valuable information on marine mammalogy. Given the increasing concerns regarding issues of anthropogenic factors affecting these animals, it is not surprising that the majority of chapters in this book deal with this subject.

ISBN 978-953-51-0844-3

New Approaches to the Study of Marine Mammals

Photo by Velvetfish / iStock


**Acknowledgements**

Parts of chapter two, three and four was first published in the author's thesis [96]. Thanks to my collegues in the ebp-group: Lena Nilsson, Maria Nolemo, Barbara Eberhart, Jessica Forsberg, Ruth Breivik and Anna Fäldt.


*5.2. Effect of Basicity on the Occurrence State of Sulfur in Slag*

The micro-area distribution of the sulfur in the S1 and S3 slag samples in Figure 8 were analyzed by scanning electron microscopy (SEM-EDS), as shown. Through the surface scanning analysis data, it was found that the matrix phase around the CaS grains contained dispersed sulfur elements; thus, it can be determined that the slag matrix phase in the solidification process contained unprecipitated amorphous sulfur. The sulfur of the synthetic slag existed in two different states, namely, CaS crystal and amorphous sulfur. The sulfur elements distributed in the aggregated state of the molten slag were CaS crystals, while the sulfur elements distributed in the dispersed state occurred in the matrix phase as an amorphous structure. Comparing the microzone distribution of the sulfur elements in Figure 8a,b, it can be discovered that the amorphous structure density of the sulfur elements in the S3 slag sample was higher than that in the S1 slag sample, and the content of amorphous sulfur in the slag increased with an increase in basicity. The key reason of such a phenomenon was that the crystallization temperature of the CaS in the molten slag decreased with the increase in basicity and the supercooling zone of the CaS crystallization decreased during solidification in the same cooling system (air cooling), which reduced the amount of precipitation of the CaS phase and increased the amorphous sulfur content in matrix phase. Moreover, the thermodynamic calculation of sulfur precipitation during solidification in Figure 2 indicated that the sulfur in the slag was completely precipitated in the form of CaS, but the surface scanning distribution analytical results showed that the amorphous sulfur content of matrix phase increased with the increase in basicity. This was mainly because the kinetic condition of S2− diffusion in the matrix phase deteriorated with the decrease in slag temperature during solidification, while certain S2− in the matrix phase failed to diffuse to the surface of the CaS crystal nucleus to form CaS crystals and occurred in the form of an amorphous structure in matrix phase. *Minerals* **2021**, *11*, x FOR PEER REVIEW 11 of 12

**Figure 8.** Surface scanning analysis of the sulfur elements in molten slag. (**a**)-S1 with a basicity of 2.5, (**b**)-S3 with a basicity of 3.5. **Figure 8.** Surface scanning analysis of the sulfur elements in molten slag. (**a**) S1 with a basicity of 2.5, (**b**) S3 with a basicity of 3.5.

increase of the slag basicity.

State Key Laboratory of Refractories and Metallurgy.

**Conflicts of Interest:** The authors declare no conflicts of interest.

perature of CaS in slag decreased with the increase of basicity;

of sulfur in matrix phase was mainly homogeneous nucleation;

(1) According to the calculated results of molten slag solidification process based on thermodynamic database FactSage8.1, it was concluded that sulfur in KR desulfurization slag was mainly precipitated in the form of CaS, and the crystallization tem-

(2) CaS grains mainly precipitated along silicate grain boundaries in low-basicity (*R* = 2.5 and 3.0) slags, and the precipitation behavior of CaS was mainly heterogeneous nucleation. There were fewer CaS grains precipitated along silicate grain boundaries in molten slags with high basicity (*R* = 3.5, 4.0 and 4.5), and the precipitation behavior

(3) The number and the size of CaS grains decreased and increased respectively with the

**Author Contributions:** R.Z. drafted the manuscript and conducted the experiments, J.J. helped to conduct the experimental work, and J.L. modified and polished the draft. Y.Y. and H.Z. helped to read the manuscript. All authors have read and agreed to the published version of the manuscript. **Funding:** The work was supported by the National Natural Science Foundation of China (No. 51974210, 52074197), the Hubei Provincial Natural Science Foundation (No. 2019CFB697), and the

**Acknowledgments:** The authors would like to thank the National Natural Science Foundation of

China and the Hubei Provincial Natural Science Foundation for their financial supports.

**6. Conclusions**

Based on the above, research status of KR desulfurization slag and the experimental results are discussed. At present, the comprehensive utilization process of KR desulfurization slag in China is not considered to remove sulfur from KR desulfurization slag and to reuse it in converter smelting processes. Therefore, the results are beneficial to provide a theoretical basis for the subsequent removal of sulfur from slag through oxidizing atmosphere.

## **6. Conclusions**

**Author Contributions:** R.Z. drafted the manuscript and conducted the experiments, J.J. helped to conduct the experimental work, and J.L. modified and polished the draft. Y.Y. and H.Z. helped to read the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** The work was supported by the National Natural Science Foundation of China (No. 51974210, 52074197), the Hubei Provincial Natural Science Foundation (No. 2019CFB697), and the State Key Laboratory of Refractories and Metallurgy.

**Acknowledgments:** The authors would like to thank the National Natural Science Foundation of China and the Hubei Provincial Natural Science Foundation for their financial supports.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


**4. Results**

A first attempt at quantifying the expected amplitude of fixed-point SST fluctuations induced by barotropic and baroclinic tidal flows is presented here, by means of two case studies (Sections 4.1 and 4.2) using infrared SST observations, and at a global scale using clymatological analysis of SST gradients (Section 4.3). The two case studies represent a trade-off between clear sky conditions (see Figure A1) and the intensity of tidal currents (Figure 1).


**3.3.3 New activity and background**

With only two people remaining in the community, the cooperative cannot move ownership to a residents' association. As two people can do very little, the cooperative, a large part of the membership of which are now living in a neighboring community, is attempting to revive local interest in exploiting the forest for a wide range of activities. In the annual meeting of the cooperative in 2005, various important problems were discussed, including how to improve degraded forest resources, protect the natural environment, address a problem of illegal dumping, enhance the low morale of mountain climbers, spark community regeneration and activation, and further utilize forest resources (Kuniyoshi, 2008). The group ultimately decided to develop ecotourism in their forest.

The cooperative set up an organization, together with community members, which included previous community members, the staff and students in an architectural course at a local private college, a local private railway company, a non-profit organization related to the mountains, and individuals, and they are actively recruiting sponsors and volunteers (Kuniyoshi, 2008). Various ecoprojects are now in progress (Kuniyoshi, 2008). The organization helps to maintain trails in Hakusan National Park, with the aim of running ecotourism project there as well. Many tours are already available, and some hunting is allowed. Some old traditional houses are being renovated with help from college staff and students. These renovated houses are expected to become major ecotourism draws. The renovations are also viewed as a starting point for community regeneration. Restoration of the local waterway is also being conducted as part of landscape management. A teagrowing area has been introduced into an abandoned cultivation area, which is also expected to be a major attraction for ecotourists in the future.

It is interesting that the cooperative clearly identified the development of ecotourism as their main forest-management goal, given that ecotourism has never before been a major objective of a forest producer cooperative34.


**4. Hypoxia response system in HSCs**

Cells sense, respond, and adapt to hypoxia using hypoxia-responsive regulatory pathways. HSCs utilize the same hypoxia response pathways as a number of other cell types. A central component of these pathways is hypoxia-inducible factor-1 (HIF-1), a transcription factor that is essential for cellular and systemic responses to a low oxygen microenvironment (Semenza, 2010) (Figure 3). HIF-1 is a heterodimeric transcription factor consisting of the oxygen-dependent HIF-1 subunit and an oxygen-independent HIF-1 subunit (Wang and Semenza, 1995). HIF-1 is hydroxylated at proline (Pro) 402 and/or 564 in the oxygendependent degradation (ODD) domain under normoxic conditions (Kaelin and Ratcliffe, 2008). HIF-1 is hydroxylated by three prolyl hydroxylases (PHD1–3) which require molecular oxygen, Fe2+, 2-oxoglutarate, and ascorbic acid for their full enzymatic activity (Epstein et al, 2001). Prolyl-hydroxylated HIF-1 protein is recognized by the von Hippel-Lindau (VHL) tumor suppressor protein, which recruits the Elongin C/Elongin B/Cullin2/E3 ubiquitin ligase complex. As a result, prolyl-hydroxylated HIF-1 protein is ubiquitinated and degraded by the proteasome. Under a hypoxic environment, prolyl hydroxylases lose their enzymatic activity. Thus, prolyl hydroxylation of HIF-1 is suppressed, and HIF-1 protein is stabilized without degradation (Kaelin and Ratcliffe, 2008). HIF-1 heterodimers (HIF-1:HIF-1) are recruited and bind to hypoxia response elements (HREs) in various target genes and activate transcription programs (Semenza, 2010).

lapse and three-dimensional observation with a multi-photon microscope) of single LT-HSCs in the BM has shed light on this paradox. Real-time tracking of murine BM revealed that HSCs gradually move away from bone marrow blood vessels and then detach from them and translocate to the osteoblastic zone of the BM after transplantation (Lo Celso et al, 2009; Xie et al, 2009). Based on these observations, it is possible that subpopulations of HSCs residing in different specific locations have different

In parallel with the hypoxic microenvironment for HSCs *in vivo*, hypoxic culture phenotypically and functionally sustained HSCs more effectively than normoxic culture (20% oxygen). Also, hypoxic culture enhances the colony-forming ability (progenitor ability) and transplantation capacity (HSC capacity) of cultured BM cells or isolated HSCs (Cipolleschi et al, 1993; Danet et al, 2003; Ivanovic et al, 2004). Hypoxic treatment also induces cell cycle quiescence in cultured HSC (Hermitte et al, 2006; Shima et al, 2010). Quiescent HSCs are defined by a high amount of efflux of the DNA-binding dye Hoechst 33342 from the cytosol (Goodell et al, 1996). These cells are called "side population (SP)" cells due to their specific staining pattern by flow cytometric analysis. Hypoxic treatment also sustains the SP phenotype in HSC *in vitro* (Krishnamurthy et al, 2004). Exclusion of Hoechst dye from the HSC cytosol is supported by Bcrp1/ABCG2, an ATP-dependent transporter, at the plasma membrane. When HSCs were cultured under hypoxic conditions, mRNA expression of Bcrp1/ABCG2 was significantly increased and the number of SP cells was also increased as compared to HSCs cultured at normoxia. Interestingly, because Bcrp1- /- mice show no significant defect in hematopoiesis (Zhou et al, 2001), the functional role of

Cells sense, respond, and adapt to hypoxia using hypoxia-responsive regulatory pathways. HSCs utilize the same hypoxia response pathways as a number of other cell types. A central component of these pathways is hypoxia-inducible factor-1 (HIF-1), a transcription factor that is essential for cellular and systemic responses to a low oxygen microenvironment (Semenza, 2010) (Figure 3). HIF-1 is a heterodimeric transcription factor consisting of the oxygen-dependent HIF-1 subunit and an oxygen-independent HIF-1 subunit (Wang and Semenza, 1995). HIF-1 is hydroxylated at proline (Pro) 402 and/or 564 in the oxygendependent degradation (ODD) domain under normoxic conditions (Kaelin and Ratcliffe, 2008). HIF-1 is hydroxylated by three prolyl hydroxylases (PHD1–3) which require molecular oxygen, Fe2+, 2-oxoglutarate, and ascorbic acid for their full enzymatic activity (Epstein et al, 2001). Prolyl-hydroxylated HIF-1 protein is recognized by the von Hippel-Lindau (VHL) tumor suppressor protein, which recruits the Elongin C/Elongin B/Cullin2/E3 ubiquitin ligase complex. As a result, prolyl-hydroxylated HIF-1 protein is ubiquitinated and degraded by the proteasome. Under a hypoxic environment, prolyl hydroxylases lose their enzymatic activity. Thus, prolyl hydroxylation of HIF-1 is suppressed, and HIF-1 protein is stabilized without degradation (Kaelin and Ratcliffe, 2008). HIF-1 heterodimers (HIF-1:HIF-1) are recruited and bind to hypoxia response elements (HREs) in various target genes and activate transcription programs (Semenza,

oxygenation statuses.

Bcrp1 in HSCs is still uncharacterized.

2010).

**4. Hypoxia response system in HSCs**

The diagrams represent the regulation of HIF-1 protein and interacting factors under different oxygen conditions. HIF-1 is a substrate for both prolyl and asparaginyl hydroxylases. Under normoxia, proline and asparagine residues are hydroxylated. These modifications regulate the stability and transcriptional activity of HIF-1. bHLH, basic-helix-loop-helix domain; PAS, Per-ARNT-Sim domain; TAD-N, transactivation domain N-terminal; ID, inhibitory domain; TAD-C, C-terminal transactivation domain; PHD, prolyl hydroxylase domain-containing protein; and FIH-1, factor-inhibiting HIF-1.

#### Fig. 3. Regulation of hypoxia-inducible factor-1 (HIF-1)

The Hypoxia Regulatory System in Hematopoietic Stem Cells 141

HIF-1 mRNA and protein are highly expressed in LT-HSCs (Takubo et al, 2010; Simsek et al, 2010) (Figure 4). HSCs derived from conditional HIF-1 knockout (HIF-1) mice have a defective capacity for marrow reconstitution during serial BM transplantation (Takubo et al., 2010). HIF-1 LT-HSCs lost cell cycle quiescence, entered the cell cycle from G0 phase, proliferated, and showed reduced tolerance to stresses such as 5-fluorouracil administration or aging. These studies suggest that HIF-1 plays an essential role in the regulation of HSC quiescence and stress resistance *in vivo*. In addition to these HIF-1 loss-of-function studies, conditional deletion of the VHL gene in hematopoietic cells was performed as a HIF-1 gain-of-function experiment. Analysis of VHL mutant hematopoietic cells revealed that the functional properties of LT-HSCs and progenitors are differentially influenced by HIF-1. HIF-1 protein levels are elevated in either biallelic (VHL) or monoallelic (VHL+) conditional knockout hematopoietic cells. For example, only a minor population of normal hematopoietic progenitors (CD34+ LSK cells) are in a quiescent state. In clear contrast, the

higher. At steady state, HIF-1 protein levels are not high in hematopoietic progenitors, and forced stabilization of HIF-1 protein through monoallelic VHL deletion induces VHL+/ CD34+ LSK progenitors to exit the cell cycle and maintains them in the G0 phase. Severe suppression of cell cycling and transplantation capacity is restored in HIF-1:VHL doubly mutated HSCs. The decreased frequency of LT-HSCs seen in VHL mice is rescued by the co-deletion of the HIF-1 gene *in vivo*. Also, long-term *in vitro* exposure of LT-HSCs to a PHD inhibitor (dimethyloxalylglycine; DMOG), which stabilizes HIF-1α even under normoxic conditions, attenuates stem cell ability especially during BM transplantation

Collectively, these results provide evidence that there is an optimal HIF-1 protein level for HSC maintenance. HIF-1 is required for stress resistance and long-term maintenance of HSCs, and within an appropriate range, moderate increases of HIF-1 (to the level caused by VHL heterozygous deletion) are trophic for HSCs through the induction of quiescence. However, aberrantly high HIF-1 levels are also harmful to HSCs and lead to a loss of stem cell capacity and the exhaustion of the HSC pool. Homozygous deletion of VHL results in a severe suppression of the cell cycle and a homing defect during

HIF-1 not only acts in the HSC system but also plays an important role in neural stem cells (NSCs) under hypoxic conditions. In this type of cell, HIF-1 induces the activation of the Wnt/-catenin signalling pathway through the upregulation of -catenin and the expression of the downstream transcription factors lymphoid enhancer-binding factor 1 and T-cell factor 1 (Mazumdar J et al, 2010). Wnt/-catenin activity was closely correlated with hypoxic status in the subgranular zone of the hippocampus, which is one of the niches for NSCs. Loss of HIF-1 in NSCs resulted in a defective Wnt-dependent hippocampal neurogenic niche capacity. As a result, NSC proliferation and differentiation, and the production of new neurons, were attenuated. Interestingly, the biological effects of HIF-1 on NSCs (cell cycle promotion) are clearly different from those seen in HSCs (cell cycle quiescence). It will be important to dissect how these different lineage stem cell systems utilize the same protein (HIF-1) to sustain themselves using different downstream molecular machinery and biological events. It is also of interest to investigate embryonic

hematopoietic progenitors in the quiescent phase is significantly

proportion of VHL+/

(Eliasson P et al, 2010).

transplantation.

Scheme of biological outcomes of different HIF-1 protein levels in HSCs. This is achieved by HIF-1 or VHL deletion in HSCs using knockout mouse models. Normal HSCs (the second from the top) stabilize HIF-1, which maintains cell cycle quiescence at the hypoxic bone marrow niche in the endosteum. Preferential stabilization of HIF-1 was observed in HSCs under hypoxia. HIF-1 HSCs (top) lose cell cycle quiescence and stress resistance against transplantation, chemotherapeutic agents, and aging. In addition, HIF-1 HSCs leave the bone marrow niche and drive extramedullary hematopoiesis in the spleen. Production of ROS is accelerated in HIF-1 HSCs. Heterozygous deletion of VHL results in a slight increase in HIF-1 protein. Under these conditions, cell cycle quiescence in HSCs is enhanced. The VHL+/ HSC (the second from the bottom) is resistant to transplantation and aging. ROS production is also suppressed in VHL+/ HSCs. The homozygous VHL mutant (VHL) HSC has a maximal dose of HIF-1 protein. In contrast to heterozygous VHL mutant HSCs, VHL HSCs completely lost stem cell capacity potentially due to aberrant suppression of the cell cycle and/or homing capacity to the niche. This defect is HIF-1–dependent because the co-deletion of HIF-1 in homozygous VHL-deficient hematopoietic cell rescued the defect. Thus, the precise regulation of HIF-1 levels coordinates stem cell proliferation and differentiation. Recently, it has been reported that vascular endothelial growth factor, heat shock proteins, and GRP78 and its ligand Cripto regulate HSC quiescence and maintain HSCs in hypoxia as downstream factors of HIF-1 (Rehn et al, 2011; Miharada et al, 2011).

Fig. 4. Features of HIF-1 or VHL knockout HSCs

Scheme of biological outcomes of different HIF-1 protein levels in HSCs. This is achieved by HIF-1 or VHL deletion in HSCs using knockout mouse models. Normal HSCs (the second from the top) stabilize HIF-1, which maintains cell cycle quiescence at the hypoxic bone marrow niche in the endosteum. Preferential stabilization of HIF-1 was observed in HSCs under hypoxia. HIF-1 HSCs (top) lose cell cycle quiescence and stress resistance against transplantation, chemotherapeutic agents, and aging. In addition, HIF-1 HSCs leave the bone marrow niche and drive extramedullary hematopoiesis in the spleen. Production of ROS is accelerated in HIF-1 HSCs. Heterozygous deletion of VHL results in a slight increase in HIF-1 protein. Under these conditions, cell cycle quiescence in HSCs is enhanced. The

protein. In contrast to heterozygous VHL mutant HSCs, VHL HSCs completely lost stem cell capacity potentially due to aberrant suppression of the cell cycle and/or homing capacity to the niche. This defect is HIF-1–dependent because the co-deletion of HIF-1 in homozygous VHL-deficient hematopoietic cell rescued the defect. Thus, the precise regulation of HIF-1 levels coordinates stem cell proliferation and differentiation. Recently, it has been reported that vascular endothelial growth factor, heat shock proteins, and GRP78 and its ligand Cripto regulate HSC quiescence and maintain HSCs in hypoxia as downstream

HSC (the second from the bottom) is resistant to transplantation and aging. ROS production is also

HSCs. The homozygous VHL mutant (VHL) HSC has a maximal dose of HIF-1

VHL+/

suppressed in VHL+/

factors of HIF-1 (Rehn et al, 2011; Miharada et al, 2011). Fig. 4. Features of HIF-1 or VHL knockout HSCs

HIF-1 mRNA and protein are highly expressed in LT-HSCs (Takubo et al, 2010; Simsek et al, 2010) (Figure 4). HSCs derived from conditional HIF-1 knockout (HIF-1) mice have a defective capacity for marrow reconstitution during serial BM transplantation (Takubo et al., 2010). HIF-1 LT-HSCs lost cell cycle quiescence, entered the cell cycle from G0 phase, proliferated, and showed reduced tolerance to stresses such as 5-fluorouracil administration or aging. These studies suggest that HIF-1 plays an essential role in the regulation of HSC quiescence and stress resistance *in vivo*. In addition to these HIF-1 loss-of-function studies, conditional deletion of the VHL gene in hematopoietic cells was performed as a HIF-1 gain-of-function experiment. Analysis of VHL mutant hematopoietic cells revealed that the functional properties of LT-HSCs and progenitors are differentially influenced by HIF-1. HIF-1 protein levels are elevated in either biallelic (VHL) or monoallelic (VHL+) conditional knockout hematopoietic cells. For example, only a minor population of normal hematopoietic progenitors (CD34+ LSK cells) are in a quiescent state. In clear contrast, the proportion of VHL+/ hematopoietic progenitors in the quiescent phase is significantly higher. At steady state, HIF-1 protein levels are not high in hematopoietic progenitors, and forced stabilization of HIF-1 protein through monoallelic VHL deletion induces VHL+/ CD34+ LSK progenitors to exit the cell cycle and maintains them in the G0 phase. Severe suppression of cell cycling and transplantation capacity is restored in HIF-1:VHL doubly mutated HSCs. The decreased frequency of LT-HSCs seen in VHL mice is rescued by the co-deletion of the HIF-1 gene *in vivo*. Also, long-term *in vitro* exposure of LT-HSCs to a PHD inhibitor (dimethyloxalylglycine; DMOG), which stabilizes HIF-1α even under normoxic conditions, attenuates stem cell ability especially during BM transplantation (Eliasson P et al, 2010).

Collectively, these results provide evidence that there is an optimal HIF-1 protein level for HSC maintenance. HIF-1 is required for stress resistance and long-term maintenance of HSCs, and within an appropriate range, moderate increases of HIF-1 (to the level caused by VHL heterozygous deletion) are trophic for HSCs through the induction of quiescence. However, aberrantly high HIF-1 levels are also harmful to HSCs and lead to a loss of stem cell capacity and the exhaustion of the HSC pool. Homozygous deletion of VHL results in a severe suppression of the cell cycle and a homing defect during transplantation.

HIF-1 not only acts in the HSC system but also plays an important role in neural stem cells (NSCs) under hypoxic conditions. In this type of cell, HIF-1 induces the activation of the Wnt/-catenin signalling pathway through the upregulation of -catenin and the expression of the downstream transcription factors lymphoid enhancer-binding factor 1 and T-cell factor 1 (Mazumdar J et al, 2010). Wnt/-catenin activity was closely correlated with hypoxic status in the subgranular zone of the hippocampus, which is one of the niches for NSCs. Loss of HIF-1 in NSCs resulted in a defective Wnt-dependent hippocampal neurogenic niche capacity. As a result, NSC proliferation and differentiation, and the production of new neurons, were attenuated. Interestingly, the biological effects of HIF-1 on NSCs (cell cycle promotion) are clearly different from those seen in HSCs (cell cycle quiescence). It will be important to dissect how these different lineage stem cell systems utilize the same protein (HIF-1) to sustain themselves using different downstream molecular machinery and biological events. It is also of interest to investigate embryonic

The Hypoxia Regulatory System in Hematopoietic Stem Cells 143

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113-122.

518.

73.

3056-3063.

HSCs, which actively proliferate in hypoxic conditions, because HIF-1 may support HSC proliferation in that stage. In addition, HIF-1 protein has been reported to inhibit Wnt/ catenin activity in cancer cells (Kaidi A et al, 2007), suggesting that the interaction of the HIF-1 and Wnt/-catenin pathway in stem/progenitor cells may differ from that of more differentiated or transformed cell types.


*7.5. The Temperature of the Molten Metal at the Melt*/*Air Interface*

The hydraulic jump on the inclined refractory plane could substantially degrade the surface quality of the cast product, owing to the generation of free surface waves/discontinuities [14]. As per the numerical simulations, the temperature over of the melt/air interface is above the liquidus for a considerable distance, as shown in Figures 11 and 12, even when considering perfect contact of molten metal with the moving belt, which is held constant at 300 K, by the cooling water under the belt. The experimental casting of the AA6111 alloy strip was in accord with numerical simulation predictions, in which the melt/air interface with the belt was observed to be in a liquid state for approximately the first meter along the moving belt. In this way, any molten metal surface discontinuities had enough time to settle down by the damping forces generated, before final solidification.

**Figure 11.** The predicted temperature distribution along the top and bottom faces of the strip along the casting direction.

**Figure 12.** Contour of molten metal temperature in contact with (**a**) the moving belt and (**b**) the molten metal/air interface.


**Author Contributions**

This project was conceived and designed by MN. AD characterized and discussed the mechanical and tribological properties. MN analyzed and discussed the morphology and thermal properties. Both authors contributed in writing this paper.


**3. Natura 2000 network and diversity of habitats in Spain**

Undoubtedly, the most relevant tool in the promotion of biodiversity conservation in Spain has been the approval of the Directive 92/43/CEE (Habitats Directive) and the implementation of the Natura 2000 network. In fact, Spain has been one of the EU member states that has put more efforts on this figure, since it is the country that holds the largest area occupied by Natura 2000 sites (**Figure 2**), with a total amount of 222,276 km2 and 27.3% of its continental area (**Figure 3**). Natura 2000 network in Spain meets, therefore, the 11th Aichi Biodiversity Targets [5].

it should be noted that 1.9% of Spain is included in the Alpine region, delimited around the Pyrenees mountain range, while the remaining 1.4% corresponds to the Macaronesian region

**Figure 3.** Proportion of the terrestrial and inland water areas occupied by Natura 2000 sites in the EU member states

According to distribution analysis of habitat types in the Iberian Peninsula [17], the areas of the highest diversity are concentrated in the Atlantic portion (**Figure 5**). The 10×10 km2

grids with a higher number of habitats are placed mainly in the northern third of the peninsula, highlighting those that host the maximum (more than 25 types) in the NW coastal area

The Spanish Atlantic biogeographical region includes the most diverse areas in Spain and even several of the most diverse habitat areas of the whole Atlantic biogeographical region or of the entire European Union, according to reports on conservation status and trends of habitat types and species, which are developed every 6 years under Article 17 of Directive 92/43/ EEC and Article 12 of Directive 2009/147/EC. The report regarding the last evaluation period

(Galician region) and along the mountain foothills of the Cantabrian Mountains.

**Figure 2.** Total area occupied by Natura 2000 sites in the EU member states (prepared from [14]).

Biological Conservation and Nature Protection Strategies in Spanish Atlantic Region

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UTM

represented in the Canary Islands.

(prepared from [14]).

Currently, the Spanish Natura 2000 network includes a set of 1,863 sites, consisting of 1,467 SCI/SAC and 644 SPA, which are distributed among the biogeographical regions represented in Spain, i.e., Atlantic, Alpine, Mediterranean, and Macaronesian (**Figure 4**). 85.7% of the Spanish continental area is included in the Mediterranean region, divided between the Iberian Peninsula, the Balearic Islands, Ceuta, and Melilla. The following region by occupation order is the Atlantic region, with 11.0% of the total, present in the NW Spanish mainland. Finally,

**Figure 2.** Total area occupied by Natura 2000 sites in the EU member states (prepared from [14]).

Funding is another key aspect in both models of protected areas. The MAB Programme does not provide any kind of financial aid or credit to the Biosphere Reserves or to the countries that have designated them. On the contrary, the countries are responsible to bear the expenses of the UNESCO and United Nations. Even in most European countries, there is also no spe-

The European Union financing for environment is carried out globally through the ERDF, ESF, and ECF funds. In addition, the EU maintains a successful program for support environmental conservation and restoration activities, the LIFE Programme (acronyms that come from the French l'instrument financier pour l'environnement). This program, mainly focused on the Natura 2000 network, has cofinanced more than 4,600 projects dedicated exclusively to the environment and climate change [13] across the European Union and

Undoubtedly, the most relevant tool in the promotion of biodiversity conservation in Spain has been the approval of the Directive 92/43/CEE (Habitats Directive) and the implementation of the Natura 2000 network. In fact, Spain has been one of the EU member states that has put more efforts on this figure, since it is the country that holds the largest area occupied by Natura 2000

Currently, the Spanish Natura 2000 network includes a set of 1,863 sites, consisting of 1,467 SCI/SAC and 644 SPA, which are distributed among the biogeographical regions represented in Spain, i.e., Atlantic, Alpine, Mediterranean, and Macaronesian (**Figure 4**). 85.7% of the Spanish continental area is included in the Mediterranean region, divided between the Iberian Peninsula, the Balearic Islands, Ceuta, and Melilla. The following region by occupation order is the Atlantic region, with 11.0% of the total, present in the NW Spanish mainland. Finally,

Natura 2000 network in Spain meets, therefore, the 11th Aichi Biodiversity Targets [5].

and 27.3% of its continental area (**Figure 3**).

**3. Natura 2000 network and diversity of habitats in Spain**

**Figure 1.** Number of LIFE projects led by the EU member states (prepared from [13]).

cific financial program for Biosphere Reserves.

sites (**Figure 2**), with a total amount of 222,276 km2

beyond (**Figure 1**).

28 Selected Studies in Biodiversity

**Figure 3.** Proportion of the terrestrial and inland water areas occupied by Natura 2000 sites in the EU member states (prepared from [14]).

it should be noted that 1.9% of Spain is included in the Alpine region, delimited around the Pyrenees mountain range, while the remaining 1.4% corresponds to the Macaronesian region represented in the Canary Islands.

According to distribution analysis of habitat types in the Iberian Peninsula [17], the areas of the highest diversity are concentrated in the Atlantic portion (**Figure 5**). The 10×10 km2 UTM grids with a higher number of habitats are placed mainly in the northern third of the peninsula, highlighting those that host the maximum (more than 25 types) in the NW coastal area (Galician region) and along the mountain foothills of the Cantabrian Mountains.

The Spanish Atlantic biogeographical region includes the most diverse areas in Spain and even several of the most diverse habitat areas of the whole Atlantic biogeographical region or of the entire European Union, according to reports on conservation status and trends of habitat types and species, which are developed every 6 years under Article 17 of Directive 92/43/ EEC and Article 12 of Directive 2009/147/EC. The report regarding the last evaluation period

available [18] includes the information collected for the 2007–2012 period. The list of habitats provided in this report [19] for the Atlantic region includes a total of 121 types, and 30 of them are considered as priority types (those whose conservation supposes a special responsibility for the member states of European Union) in Annex I of Directive 92/43/EEC. In the Spanish Atlantic region, a total of 89 habitat types are considered present, of which 19 are considered priority types [19]. So, Spanish Atlantic biogeographical region plays an important role in biodiversity conservation, since it houses 72% of the variety of habitat types and 63% of the

The analysis of digital cartographic information housed in web servers of [20] and classified

ferent intervals by the number of habitat types (**Figure 6**). The Atlantic region of the Iberian Peninsula concentrates a greater proportion of the top three intervals of the number of habitat

European Union. On the contrary, grids with the lowest number of habitats show a scarcer representation into the Spanish Atlantic region than in the other areas (**Table 2**). The levels of biodiversity of habitats recorded in the grids included into the Spanish Atlantic region are comparable to those that can be identified in the Pyrenees or the Alpine Range (Alpine region), in the Balearic Islands (Mediterranean region), or in small points of the Scandinavian coast (Boreal region). These results are consistent with those obtained by analysis made for

Regarding to the species across the territory, the number of taxa present in the Iberian Peninsula is estimated to amount to a value above 80,000 [22]. In this way, Spain is attributed to be the member state of the European Union responsible for the conservation of the highest species diversity of the continent [23], with more than 60,000 animal and 22,000 plant species. In some of these groups, Spain hosts 80–90% of the total taxa found in the European Union. Within this framework, the portion of the Iberian Peninsula located in the Atlantic biogeographical region plays a key role in the conservation of the main components of biodiversity, since it meets the

The high Spanish biodiversity explains the relevant territorial representation that Natura 2000 sites have achieved within the Spanish national territory. This large area occupied by Spanish Natura 2000 network has allowed the improvement of biodiversity conservation policies and environmental measures at different levels. Inventorying of habitat types and species of community interest

**Table 1.** Comparison of the number of habitats (priority and non-priority) of Annex I of the DC 92/43/EEC present in the

optimum conditions for a significant diversity of different taxon groups [24–28].

**Habitat number**

European Atlantic region 121 30 91 Spanish Atlantic region 89 19 70

European and Spanish Atlantic regions (taken from [19]).

allows to assess the territorial distribution of habitat types in dif-

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with respect to the rest of the Atlantic region or even from the

**Total Priority Non-priority**

priority types present in the Atlantic biogeographical region (**Table 1**).

in LAEA grids 10×10 km2

the period 2001–2006 [21].

types by LAEA grids 10×10 km2

**Figure 4.** Distribution by biogeographic regions of Spanish Natura 2000 network (prepared from [15, 16]).

**Figure 5.** Number of habitats per 10 × 10 km2 UTM grid in Spain (prepared from [17]).

available [18] includes the information collected for the 2007–2012 period. The list of habitats provided in this report [19] for the Atlantic region includes a total of 121 types, and 30 of them are considered as priority types (those whose conservation supposes a special responsibility for the member states of European Union) in Annex I of Directive 92/43/EEC. In the Spanish Atlantic region, a total of 89 habitat types are considered present, of which 19 are considered priority types [19]. So, Spanish Atlantic biogeographical region plays an important role in biodiversity conservation, since it houses 72% of the variety of habitat types and 63% of the priority types present in the Atlantic biogeographical region (**Table 1**).

The analysis of digital cartographic information housed in web servers of [20] and classified in LAEA grids 10×10 km2 allows to assess the territorial distribution of habitat types in different intervals by the number of habitat types (**Figure 6**). The Atlantic region of the Iberian Peninsula concentrates a greater proportion of the top three intervals of the number of habitat types by LAEA grids 10×10 km2 with respect to the rest of the Atlantic region or even from the European Union. On the contrary, grids with the lowest number of habitats show a scarcer representation into the Spanish Atlantic region than in the other areas (**Table 2**). The levels of biodiversity of habitats recorded in the grids included into the Spanish Atlantic region are comparable to those that can be identified in the Pyrenees or the Alpine Range (Alpine region), in the Balearic Islands (Mediterranean region), or in small points of the Scandinavian coast (Boreal region). These results are consistent with those obtained by analysis made for the period 2001–2006 [21].

Regarding to the species across the territory, the number of taxa present in the Iberian Peninsula is estimated to amount to a value above 80,000 [22]. In this way, Spain is attributed to be the member state of the European Union responsible for the conservation of the highest species diversity of the continent [23], with more than 60,000 animal and 22,000 plant species. In some of these groups, Spain hosts 80–90% of the total taxa found in the European Union. Within this framework, the portion of the Iberian Peninsula located in the Atlantic biogeographical region plays a key role in the conservation of the main components of biodiversity, since it meets the optimum conditions for a significant diversity of different taxon groups [24–28].

**Figure 4.** Distribution by biogeographic regions of Spanish Natura 2000 network (prepared from [15, 16]).

UTM grid in Spain (prepared from [17]).

**Figure 5.** Number of habitats per 10 × 10 km2

30 Selected Studies in Biodiversity

The high Spanish biodiversity explains the relevant territorial representation that Natura 2000 sites have achieved within the Spanish national territory. This large area occupied by Spanish Natura 2000 network has allowed the improvement of biodiversity conservation policies and environmental measures at different levels. Inventorying of habitat types and species of community interest

**Table 1.** Comparison of the number of habitats (priority and non-priority) of Annex I of the DC 92/43/EEC present in the European and Spanish Atlantic regions (taken from [19]).

**4. Biosphere Reserves in Spain**

from [15, 16]).

Spain has been one of the countries in the world that has bet more decisively for Biosphere Reserves model, becoming a benchmark on the development of the objectives of the Seville Strategy [31, 32], while the promotion of new designated Biosphere Reserves has placed Spain at the top of the World Network of Biosphere Reserves (WNBR). After the last meeting of the UNESCO International Coordination Council held in Paris in June 2017, WNBR is made up of 669 designated Biosphere Reserves distributed in 120 countries around the world [33]. In Spain, a total of 48 Biosphere Reserves have been designated, 7% of the WNBR, which makes the country with the highest number of Biosphere Reserves, ranking above countries with a

Biological Conservation and Nature Protection Strategies in Spanish Atlantic Region

The numerous and large set (**Figure 7**) of Spanish Network of Biosphere Reserves (SNBR) houses a wide variety of habitats, ecosystems, and socioeconomic and population realities. From a geo-statistical point of view, SNBR has been delimited fundamentally around the Mediterranean biogeographical region. A total of 29 Biosphere Reserves are included totally

in absolute value with respect to the Biosphere Reserves delimited totally or partially in the Atlantic region, which brings together a set of 18 Reserves, including a total area of 12,446 km2

These differences are even greater with respect to the Biosphere Reserves in the Spanish Macaronesian or Alpine regions. However, if the analysis is relativized from a biogeographi-

**Figure 7.** Distribution of the Spanish Network of Biosphere Reserves with respect to biogeographical regions (prepared

. This area is appreciably higher

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33

much larger territorial scope, such as the United States, Russia, or China.

cal point of view, the values indicate a completely different reality (**Table 3**).

or partially within this region, with a total area of 40,139 km2

**Figure 6.** Number of habitats per LAEA grid 10 × 10 km2 in the European Union, with respect to biogeographical regions (prepared from [15, 20]).

**Table 2.** Comparison of the number of habitats per grid 10 × 10 km2 and percentages in the European Union, the Atlantic region, the Spanish Atlantic portion, and the Spanish Atlantic Biosphere Reserves (prepared from [15, 16, 20]).

and delimitation of the Natura 2000 areas have allowed increasing the knowledge on Spanish interesting areas for biodiversity conservation, strengthening, and consolidating the national and regional networks of natural protected areas [29]. On the other hand, the provision of an extensive Natura 2000 network has allowed Spain to lead 821 projects and to participate in 903 projects of the LIFE Programme in 25 years [13]. This means an average participation in 34 LIFE projects per year, making Spain one of the leader countries in terms of the implementation of these initiatives for nature conservation. Finally, it is worth mentioning the establishment of strong synergies with other protection figures, especially the Spanish Network of Biosphere Reserves, whose core areas have been delimited fundamentally around the Spanish Natura 2000 network [30].


*4.3. Burnout and QWL: Contribution to Productivity and Appropriate Salary as Mediators (Model 2)*

Considering model 2, the same set of effects are analyzed (cf. Tables 6–8), evaluating two mediators (i.e., contribution to productivity and appropriate salary). For the first dimension of burnout, i.e., emotional exhaustion, the total effect is −1.054325, which is the effect found if there are no mediators in the model. It is significant, with a z of −6.64. The direct effect for emotional exhaustion is -1.019637, which is still quite significant (z = −5.99), although slightly smaller than the total effect. The indirect effect of emotional exhaustion that passes through the contribution to productivity and appropriate salary is −0.0346888 and is not significant.

The proportion of the total effect mediated is −0.0346888/−1.054325, resulting in −0.0329014. The ratio of the indirect to direct effect is −0.0346888/−1.019637, equaling −0.0340207. The ratio of the total to direct effect corresponds to −1.054325/−1.019637, equaling −1.0340199. The proportion of the total effect that is mediated is almost −0.33, which is a respectable amount. The ratio of the indirect effect to direct effect is about −0.03, and the total effect is about −1.034 times the direct effect.

Concerning the second dimension of burnout, i.e., cynicism, its total effect is -1.011313, which is the effect found if there are no mediators in the model. It is significant, with a z of −6.30. The direct effect for cynicism is −0.9078191, which, while still significant (z = −5.29), is smaller than the total effect. The indirect effect of cynicism that passes through the contribution to productivity is −0.01034937 and is also not significant.

The proportion of the total effect mediated is −0.01034937/−1.011313, resulting in −0.0102335. The ratio of the indirect to direct effect is −0.01034937/−0.9078191, equaling −0.0114001. The ratio of the total to direct effect corresponds to −1.011313/−0.9078191, equaling −1.1140027. The proportion of the total effect that is mediated is almost −0.010, which is a small amount. The ratio of the indirect effect to direct effect is about -0.011, and the total effect is about −1.114 times the direct effect.

**Table 6.** Model 2: Direct effects.

Legend: \*\*\* 1% statistical significance; and \*\* 5% statistical significance.

**Table 7.** Model 2: Indirect effects.

Legend: \*\*\* 1% statistical significance.

Regarding the third dimension of burnout, i.e., the feeling of low effectiveness, its total effect is −1.716576, which is the effect found if there are no mediators in the model. It is significant, with a z of −10.15. The direct effect for low effectiveness is −1.444998, which, while still significant (z = −8.13), is smaller than the total effect. The indirect effect of low effectiveness that passes through the contribution to productivity is −0.2715781, and it is also highly significant.

**Table 8.** Model 2: Total effects.

Legend: \*\*\* 1% statistical significance; and \*\* 5% statistical significance.

The proportion of the total effect mediated is −0.2715781/−1.716576, resulting in −0.1582091. The ratio of the indirect to direct effect is −0.2715781/−1.444998, equaling −0.1879435. The ratio of the total to direct effect corresponds to −1.716576/−1.444998, equaling −1.1879435. The proportion of the total effect that is mediated is almost −0.16, which is quite an important amount. The ratio of the indirect effect to direct effect is about −0.19, and the total effect is about −1.19 times the direct effect.